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Wednesday, August 5, 2026

Cognitive science

From Wikipedia, the free encyclopedia
Figure illustrating the fields that contributed to the birth of cognitive science, including philosophy of mind, linguistics, neuroscience, artificial intelligence, anthropology, and psychology

Cognitive science is the interdisciplinary, scientific study of the mind and its processes. It examines the nature, the tasks, and the functions of cognition (in a broad sense). Mental faculties of concern to cognitive scientists include perception, memory, attention, reasoning, language, and emotion. To understand these faculties, cognitive scientists borrow from fields such as psychology, philosophy, artificial intelligence, neuroscience, linguistics, and anthropology. The typical analysis of cognitive science spans many levels of organization, from learning and decision-making to logic and planning; from neural circuitry to modular brain organization. One of the fundamental concepts of cognitive science is that "thinking can best be understood in terms of representational structures in the mind and computational procedures that operate on those structures."

History

The cognitive sciences began as an intellectual movement in the 1950s, called the cognitive revolution. Cognitive science has a prehistory traceable back to ancient Greek philosophical texts (see Plato's Meno and Aristotle's De Anima).

The modern culture of cognitive science can be traced back to the early cyberneticists in the 1930s and 1940s, such as Warren McCulloch and Walter Pitts, who sought to understand the organizing principles of the mind. McCulloch and Pitts developed the first variants of what are now known as artificial neural networks, models of computation inspired by the structure of biological neural networks.

Another precursor was the early development of the theory of computation and the digital computer in the 1940s and 1950s. Kurt Gödel, Alonzo Church, Claude Shannon, Alan Turing, and John von Neumann were instrumental in these developments. The modern computer, or Von Neumann machine, would play a central role in cognitive science, both as a metaphor for the mind and as a tool for investigation.

The first instance of cognitive science experiments being conducted at an academic institution occurred at MIT Sloan School of Management, where J.C.R. Licklider worked in the psychology department and conducted experiments using computer memory as a model of human cognition. In 1959, Noam Chomsky published a scathing review of B. F. Skinner's book Verbal Behavior. At the time, Skinner's behaviorist paradigm dominated the field of psychology within the United States. Most psychologists focused on functional relations between stimulus and response, without positing internal representations. Chomsky argued that to explain language, we needed a theory like generative grammar, which not only posited internal representations but also characterized their underlying order.

The term cognitive science was coined by Christopher Longuet-Higgins in his 1973 commentary on the Lighthill report, which concerned the then-current state of artificial intelligence research. In the same decade, the journal Cognitive Science and the Cognitive Science Society were founded. The founding meeting of the Cognitive Science Society was held at the University of California, San Diego in 1979, which resulted in cognitive science becoming an internationally visible enterprise. In 1972, Hampshire College started the first undergraduate education program in Cognitive Science, led by Neil Stillings. In 1982, with assistance from Professor Stillings, Vassar College became the first institution in the world to grant an undergraduate degree in Cognitive Science. In 1986, the first Cognitive Science Department in the world was founded at the University of California, San Diego.

In the 1970s and early 1980s, as access to computers increased, artificial intelligence research expanded. Researchers such as Marvin Minsky would write computer programs in languages such as LISP to attempt to formally characterize the steps that human beings went through, for instance, in making decisions and solving problems, in the hope of better understanding human thought, and also in the hope of creating artificial minds. This approach is known as "symbolic AI".

Eventually, the limits of the symbolic AI research program became apparent. For instance, it seemed to be unrealistic to comprehensively list human knowledge in a form usable by a symbolic computer program. The late 80s and 90s saw the rise of neural networks and connectionism as a research paradigm. From this point of view, often attributed to James McClelland and David Rumelhart, the mind can be characterized as a set of complex associations represented as a layered network. Critics argue that symbolic models better capture some phenomena, and that connectionist models are often so complex as to have little explanatory power. Recently, symbolic and connectionist models have been combined, enabling the use of both forms of explanation. While both connectionism and symbolic approaches have proven useful for testing various hypotheses and exploring approaches to understanding aspects of cognition and lower level brain functions, neither are biologically realistic and therefore, both suffer from a lack of neuroscientific plausibility. Connectionism has proven useful for exploring computationally how cognition emerges in development and occurs in the human brain, and has provided alternatives to strictly domain-specific / domain general approaches. For example, scientists such as Jeff Elman, Liz Bates, and Annette Karmiloff-Smith have posited that brain networks emerge from the dynamic interaction between them and environmental input.

Recent developments in quantum computation, including the ability to run quantum circuits on quantum computers such as the IBM Quantum Platform, have accelerated research into cognitive models that incorporate elements of quantum mechanics.

Principles

Levels of analysis

A central tenet of cognitive science is that a complete understanding of the mind/brain cannot be attained by studying only a single level. An example would be the problem of remembering a phone number and recalling it later. One approach to understanding this process would be to study behavior through direct observation, or naturalistic observation. A person could be presented with a phone number and asked to recall it after a delay; the accuracy of the response could then be measured. Another approach to measuring cognitive ability would be to study the firing of individual neurons while a person is trying to remember a phone number. Neither of these experiments on its own would fully explain how the process of remembering a phone number works.

Even if the technology to map every neuron in the brain in real time were available, and it were known when each neuron fired, it would still be impossible to know how a particular firing of neurons translates into the observed behavior. Thus, an understanding of how these two levels relate is imperative. Francisco Varela, in The Embodied Mind: Cognitive Science and Human Experience, argues that "the new sciences of the mind need to enlarge their horizon to encompass both lived human experience and the possibilities for transformation inherent in human experience". On the classic cognitivist view, this can be provided by a functional level account of the process. Studying a phenomenon at multiple levels provides a better understanding of the brain processes that give rise to a particular behavior. Marr gave a famous description of three levels of analysis:

  1. The computational theory, specifying the goals of the computation;
  2. Representation and algorithms, giving a representation of the inputs and outputs and the algorithms which transform one into the other; and
  3. The hardware implementation, or how algorithm and representation may be physically realized.

Interdisciplinary nature

Cognitive science is an interdisciplinary field with contributors from various fields, including psychology, neuroscience, linguistics, philosophy of mind, computer science, anthropology, and biology. Cognitive scientists work collectively in the hope of understanding the mind and its interactions with the surrounding world, much like scientists in other fields. The field regards itself as compatible with the physical sciences. It uses the scientific method as well as simulation or modeling, often comparing model outputs with aspects of human cognition. Similar to the field of psychology, there is some doubt whether there is a unified cognitive science, which has led some researchers to prefer 'cognitive sciences' in plural.

Many, but not all, who consider themselves cognitive scientists hold a functionalist view of the mind—the view that mental states and processes should be explained by their function – what they do. According to the multiple realizability account of functionalism, even non-human systems such as robots and computers can be ascribed as having cognitive states.

Cognitive science, the term

The term "cognitive" in "cognitive science" is used for "any kind of mental operation or structure that can be studied in precise terms" (Lakoff and Johnson, 1999). This conceptualization is very broad and should not be confused with the use of "cognitive" in some traditions of analytic philosophy, where "cognitive" concerns only formal rules and truth-conditional semantics.

The earliest entries for the word "cognitive" in the OED take it to mean roughly "pertaining to the action or process of knowing". The first entry, from 1586, shows that the word was once used in discussions of Platonic theories of knowledge. Most in cognitive science, however, presumably do not believe their field is the study of anything as certain as the knowledge sought by Plato.

Scope

Cognitive science is a large field that covers a wide array of topics in cognition. However, it should be recognized that cognitive science has not always been equally concerned with every topic that might bear relevance to the nature and operation of minds. Classical cognitivists have largely de-emphasized or avoided social and cultural factors, embodiment, emotion, consciousness, animal cognition, and comparative and evolutionary psychologies. However, with the decline of behaviorism, internal states such as affects and emotions, as well as awareness and covert attention, became approachable again. For example, situated and embodied cognition theories take into account the current state of the environment and the body's role in cognition. With the newfound emphasis on information processing, observable behavior was no longer the hallmark of psychological theory, but the modeling or recording of mental states.

Below are some of the main topics that cognitive science is concerned with; see List of cognitive science topics for a more exhaustive list.

Artificial intelligence

Artificial intelligence (AI) involves the study of cognitive phenomena in machines. One of the practical goals of AI is to implement aspects of human intelligence in computers. Computers are also widely used as tools for studying cognitive phenomena. Computational modeling uses simulations to study how human intelligence may be structured. (See § Computational modeling.)

There is some debate in the field as to whether the mind is best viewed as a vast array of small but individually feeble elements (i.e., neurons) or as a collection of higher-level structures such as symbols, schemes, plans, and rules. The former view uses connectionism to study the mind, whereas the latter emphasizes symbolic artificial intelligence. One way to view the issue is whether it is possible to accurately simulate a human brain on a computer without accurately simulating the neurons that make it up.

Attention

Attention is the selection of important information. The human mind is bombarded with millions of stimuli, and it must have a way of deciding which of this information to process. Attention is sometimes seen as a spotlight, meaning one can only shine the light on a particular set of information. Experiments that support this metaphor include the dichotic listening task and studies of inattentional blindness (Mack and Rock, 1998). In the dichotic listening task, subjects are bombarded with two different messages, one in each ear, and are told to focus on only one. At the end of the experiment, when asked about the content of the unattended message, subjects were unable to report it.

The psychological construct of attention is sometimes confused with the concept of intentionality due to some degree of semantic ambiguity in their definitions. At the beginning of experimental research on attention, Wilhelm Wundt defined this term as "that psychical process, which is operative in the clear perception of the narrow region of the content of consciousness." His experiments showed the limits of attention in space and time, which were 3-6 letters during an exposition of 1/10 s. Because this notion develops within the framework of the original meaning during a hundred years of research, the definition of attention would reflect the sense when it accounts for the main features initially attributed to this term – it is a process of controlling thought that continues over time. While intentionality is the power of minds to be about something, attention is the concentration of awareness on some phenomenon during a period of time, which is necessary to elevate the clear perception of the narrow region of the content of consciousness and which is feasible to control this focus in mind.

The significance of knowledge about the scope of attention in the study of cognition is that it defines the cognitive functions of cognition, such as apprehension, judgment, reasoning, and working memory. The development of attention scope increases the set of faculties responsible for the mind, which relies on how it perceives, remembers, considers, and evaluates in making decisions. The ground of this statement is that the more details (associated with an event) the mind may grasp for their comparison, association, and categorization, the closer the apprehension, judgment, and reasoning of the event are in accord with reality.

According to Latvian professor Sandra Mihailova and professor Igor Val Danilov, the more elements of the phenomenon (or phenomena) the mind can keep in the scope of attention simultaneously, the greater the number of reasonable combinations within that event it can achieve, enhancing the probability of better understanding the features and particularity of the phenomenon (phenomena). For example, three items in the focal point of consciousness yield six possible combinations (3 factorial) and four items – 24 (4 factorial) combinations. The number of reasonable combinations becomes significant for a focal point with six items, yielding 720 possible combinations (6 factorial).

Embodied cognition approaches to cognitive science emphasize the role of body and environment in cognition. This includes both neural and extra-neural bodily processes, and factors that range from affective and emotional processes, to posture, motor control, proprioception, and kinaesthesis, to autonomic processes that involve heartbeat and respiration, to the role of the enteric gut microbiome. It also includes accounts of how the body engages with or is coupled to social and physical environments. 4E cognition includes a broad range of views about brain-body-environment interaction, from causal embeddedness to stronger claims about how the mind extends to include tools and instruments, as well as the role of social interactions, action-oriented processes, and affordances. 4E theories range from those closer to classic cognitivism (so-called "weak" embodied cognition) to stronger extended and enactive versions that are sometimes referred to as radical embodied cognitive science.

A hypothesis of pre-perceptual multimodal integration supports embodied cognition approaches and converges two competing naturalist and constructivist viewpoints about cognition and the development of emotions. According to this hypothesis, supported by empirical data, cognition and emotion development are initiated by the association of affective cues with stimuli responsible for triggering the neuronal pathways of simple reflexes. This pre-perceptual multimodal integration can succeed owing to neuronal coherence in mother-child dyads beginning from pregnancy. These cognitive-reflex and emotion-reflex stimuli conjunctions further form simple innate neuronal assemblies, shaping the cognitive and emotional neuronal patterns in statistical learning that are continuously connected with the neuronal pathways of reflexes.

Knowledge and processing of language

A well known example of a phrase structure tree. This is one way to represent human language, showing how its components are organized hierarchically.

The ability to learn and understand language is an extremely complex process. Language is acquired within the first few years of life, and under normal circumstances, all humans can do so proficiently. A major driving force in the theoretical linguistic field is discovering the nature that language must have in the abstract to be learned in such a fashion. Some of the driving research questions in studying how the brain itself processes language include: (1) To what extent is linguistic knowledge innate or learned?, (2) Why is it more difficult for adults to acquire a second language than it is for infants to acquire their first language?, and (3) How are humans able to understand novel sentences?

The study of language processing ranges from the investigation of the sound patterns of speech to the meaning of words and whole sentences. Linguistics often divides language processing into orthography, phonetics, phonology, morphology, syntax, semantics, and pragmatics. Many aspects of language can be studied from each of these components and from their interaction.

The study of language processing in cognitive science is closely tied to the field of linguistics. Linguistics was traditionally studied as part of the humanities, alongside history, art, and literature. In the last fifty years or so, more and more researchers have studied knowledge and use of language as a cognitive phenomenon, with the main problems being how language knowledge can be acquired and used, and what precisely it consists of. Linguists have found that, while humans form sentences in ways apparently governed by very complex systems, they are remarkably unaware of the rules that govern their own speech. Thus, linguists must resort to indirect methods to determine what those rules might be, if indeed rules as such exist. In any event, if rules indeed govern speech, they appear to be opaque to any conscious consideration.

Learning and development

Learning and development are the processes by which we acquire knowledge and information over time. Infants are born with little or no knowledge (depending on how knowledge is defined). Yet, they rapidly acquire the ability to use language, walk, and recognize people and objects. Research in learning and development aims to explain the mechanisms underlying these processes.

A major question in the study of cognitive development is the extent to which certain abilities are innate or learned. This is often framed in terms of the nature and nurture debate. The nativist view emphasizes that certain features are innate to an organism and are determined by its genetic endowment. The empiricist view, on the other hand, emphasizes that certain abilities are learned from the environment. Although clearly both genetic and environmental input is needed for a child to develop normally, considerable debate remains about how genetic information might guide cognitive development. In the area of language acquisition, for example, some (such as Steven Pinker) have argued that specific information containing universal grammatical rules must be contained in the genes, whereas others (such as Jeffrey Elman and colleagues in Rethinking Innateness) have argued that Pinker's claims are biologically unrealistic. They argue that genes determine the architecture of a learning system, but that specific "facts" about how grammar works can only be learned through experience.

Memory

Memory allows us to store information for later retrieval. Memory is often thought of as consisting of both a long-term and short-term store. Long-term memory allows us to store information over prolonged periods (days, weeks, years). We do not yet know the practical limit of long-term memory capacity. Short-term memory allows us to store information over short time scales (seconds or minutes).

Memory is also often grouped into declarative and procedural forms. Declarative memory—grouped into subsets of semantic and episodic forms of memory—refers to our memory for facts and specific knowledge, specific meanings, and specific experiences (e.g. "Are apples food?", or "What did I eat for breakfast four days ago?"). Procedural memory allows us to remember actions and motor sequences (e.g., how to ride a bicycle) and is often referred to as implicit knowledge or memory.

Cognitive scientists study memory just as psychologists do, but tend to focus more on how memory relates to cognitive processes and the interrelationship between cognition and memory. One example of this could be: what mental processes does a person go through to retrieve a long-lost memory? Or, what differentiates between the cognitive process of recognition (seeing hints of something before remembering it, or memory in context) and recall (retrieving a memory, as in "fill-in-the-blank")?

Perception and action

The Necker cube, an example of an optical illusion
An optical illusion. The square A is the same shade of gray as square B. See checker shadow illusion.

Perception is the ability to take in information via the senses and process it in some way. Vision and hearing are two dominant senses that allow us to perceive the environment. Some questions in the study of visual perception, for example, include: (1) How are we able to recognize objects?, (2) Why do we perceive a continuous visual environment, even though we only see small bits of it at any one time? One tool for studying visual perception is by looking at how people process optical illusions. The image on the right, a Necker cube, is an example of a bistable percept; that is, the cube can be interpreted as oriented in two different directions.

The study of haptic (tactile), olfactory, and gustatory stimuli also fall into the domain of perception.

Action is taken to refer to the output of a system. In humans, this is accomplished through motor responses. Spatial planning and movement, speech production, and complex motor movements are all aspects of action.

Consciousness

17th-century representation of consciousness by Robert Fludd, an English Paracelsian physician

Consciousness is being aware of something internal to one's self, or of states or objects in one's external environment. It has been the topic of extensive explanations, analyses, and debate among philosophers, scientists, and theologians for millennia. There is no consensus on what exactly needs to be studied, or whether consciousness can be considered a scientific concept. In some explanations it is synonymous with mind, while in others it is considered an aspect of it.

In the past, consciousness meant one's "inner life": the world of introspection, private thought, imagination, and volition. Today, it often includes various forms of cognition, experience, feeling, or perception. It may be awareness, awareness of awareness, metacognition, or self-awareness, either continuously changing or not. There is also a medical definition that helps, for example, to discern "coma" from other states. The disparate range of research, notions, and speculations raises some curiosity about whether the right questions are being asked.

Examples of the range of descriptions, definitions and explanations are: ordered distinction between self and environment, simple wakefulness, one's sense of selfhood or soul explored by "looking within", being a metaphorical "stream" of contents, or being a mental state, mental event, or mental process of the brain.

Research methods

Many different methodologies are used to study cognitive science. As the field is highly interdisciplinary, research often spans multiple areas of study, drawing on methods from psychology, neuroscience, computer science, and systems theory.

Behavioral experiments

To describe what constitutes intelligent behavior, one must study behavior itself. This type of research is closely tied to that in cognitive psychology and psychophysics. By measuring behavioral responses to different stimuli, one can understand something about how those stimuli are processed. Lewandowski & Strohmetz (2009) reviewed a collection of innovative uses of behavioral measurement in psychology, including behavioral traces, behavioral observations, and behavioral choice. Behavioral traces are pieces of evidence that indicate behavior occurred, but the actor is not present (e.g., litter in a parking lot or readings on an electric meter). Behavioral observations involve directly witnessing the actor engaging in the behavior (e.g., observing how close a person sits to another). Behavioral choices are decisions a person makes among two or more options (e.g., voting behavior, choosing a punishment for another participant).

  • Reaction time. The time between the presentation of a stimulus and an appropriate response can indicate differences between two cognitive processes and reveal aspects of their nature. For example, if, in a search task, reaction times vary proportionally with the number of elements, then it is evident that this cognitive process of searching involves serial rather than parallel processing.
  • Psychophysical responses. Psychophysical experiments are an old psychological technique that has been adopted by cognitive psychology. They typically involve making judgments of some physical property, e.g., the loudness of a sound. Correlations of subjective scales between individuals can reveal cognitive or sensory biases relative to actual physical measurements. Some examples include:
    • sameness judgments for colors, tones, textures, etc.
    • threshold differences for colors, tones, textures, etc.
  • Eye tracking. This methodology is used to study a variety of cognitive processes, most notably visual perception and language processing. The point of fixation of the eyes is linked to an individual's attentional focus. Thus, by monitoring eye movements, we can study what information is being processed at a given time. Eye tracking allows us to study cognitive processes on extremely short time scales. Eye movements reflect online decision-making during a task and provide insight into how those decisions may be processed.

Brain imaging

Image of the human head with the brain. The arrow indicates the position of the hypothalamus.

Brain imaging involves analyzing brain activity while performing various tasks. This allows us to link behavior and brain function to help understand how information is processed. Different types of imaging techniques vary in their temporal (time-based) and spatial (location-based) resolution. Brain imaging is often used in cognitive neuroscience.

  • Single-photon emission computed tomography and positron emission tomography. SPECT and PET use radioactive isotopes, which are injected into the subject's bloodstream and taken up by the brain. By observing which areas of the brain take up the radioactive isotope, we can see which areas are more active than others. PET has a similar spatial resolution to fMRI but extremely poor temporal resolution.
  • Electroencephalography. EEG measures the electrical fields generated by large populations of neurons in the cortex by placing a series of electrodes on the scalp of the subject. This technique has an extremely high temporal resolution, but a relatively poor spatial resolution.
  • Functional magnetic resonance imaging. fMRI measures the relative amount of oxygenated blood flowing to different parts of the brain. More oxygenated blood in a particular region is assumed to correlate with an increase in neural activity in that part of the brain. This allows us to localize particular functions within different brain regions. fMRI has moderate spatial and temporal resolution.
  • Optical imaging. This technique uses infrared transmitters and receivers to measure the amount of light reflectance by blood near different areas of the brain. Since oxygenated and deoxygenated blood reflect light to different extents, we can study which areas are more active (i.e., those with more oxygenated blood). Optical imaging has moderate temporal resolution, but poor spatial resolution. It also has the advantage of being extremely safe and suitable for studying infants' brains.
  • Magnetoencephalography. MEG measures magnetic fields resulting from cortical activity. It is similar to EEG, except that it has improved spatial resolution since the magnetic fields it measures are not as blurred or attenuated by the scalp, meninges, and so forth as the electrical activity measured in EEG is. MEG uses SQUID sensors to detect tiny magnetic fields.

Computational modeling

An artificial neural network with two layers

Computational models require a mathematically and logically formal representation of a problem. Computer models are used to simulate and experimentally verify specific and general properties of intelligence. Computational modeling can help us understand the functional organization of a particular cognitive phenomenon.

Approaches to cognitive modeling can be categorized as: (1) symbolic, on abstract mental functions of an intelligent mind by means of symbols; (2) subsymbolic, on the neural and associative properties of the human brain; and (3) across the symbolic–subsymbolic border, including hybrid.

  • Symbolic modeling evolved from the computer science paradigms using the technologies of knowledge-based systems, as well as a philosophical perspective (e.g., "Good Old-Fashioned Artificial Intelligence" (GOFAI)). The first cognitive researchers developed them, and they were later used in information engineering for expert systems. Since the early 1990s, it was generalized in systemics for the investigation of functional human-like intelligence models, such as personoids, and, in parallel, developed as the SOAR environment. Recently, especially in the context of cognitive decision-making, symbolic cognitive modeling has been extended to the socio-cognitive approach, which encompasses social and organizational cognition and is interrelated with a sub-symbolic, non-conscious layer.
  • Subsymbolic modeling includes connectionist/neural network models. Connectionism relies on the idea that the mind/brain is composed of simple nodes and its problem-solving capacity derives from the connections between them. Neural nets are textbook implementations of this approach. Some critics of this approach feel that while these models represent biological reality as a description of how the system works, they lack explanatory power because, even in systems endowed with simple connection rules, the emergent high complexity makes them less interpretable at the connection level than they appear to be at the macroscopic level.
  • Other approaches gaining in popularity include (1) dynamical systems theory, (2) mapping symbolic models onto connectionist models (Neural-symbolic integration or hybrid intelligent systems), and (3) and Bayesian models, which are often drawn from machine learning.

All the above approaches tend either to be generalized to the form of integrated computational models of a synthetic/abstract intelligence (i.e. cognitive architecture) to be applied to the explanation and improvement of individual and social/organizational decision-making and reasoning[69][70] or to focus on single simulative programs (or microtheories/"middle-range" theories) modeling specific cognitive faculties (e.g., vision, language, categorization, etc.).

Neurobiological methods

Research methods borrowed directly from neuroscience and neuropsychology can also help us to understand aspects of intelligence. These methods allow us to understand how intelligent behavior is implemented in a physical system.

Key findings

Cognitive science has given rise to models of human cognitive bias and risk perception and has been influential in the development of behavioral finance, a subfield of economics. It has also given rise to a new theory of the philosophy of mathematics (related to denotational mathematics), and many theories of artificial intelligence, persuasion, and coercion. It has made its presence known in the philosophy of language and epistemology as well as constituting a substantial wing of modern linguistics.

Fields of cognitive science have been influential in understanding the brain's particular functional systems (and functional deficits), ranging from speech production to auditory processing and visual perception. It has made progress in understanding how damage to particular areas of the brain affects cognition, and has helped uncover the root causes and consequences of specific dysfunctions, such as dyslexia, anopsia, and hemispatial neglect.

Notable researchers

NameYear of birth Year of contributionContribution(s)
David Chalmers1966 1995 Dualism, hard problem of consciousness
Daniel Dennett1942 1987Offered a computational systems perspective (multiple drafts model)
John Searle1932 1980 Chinese room
Douglas Hofstadter1945 1979 Gödel, Escher, Bach
Jerry Fodor1935 1968, 1975 Functionalism
Alan Baddeley1934 1974 Baddeley's model of working memory
Marvin Minsky 1927 1970s, early 1980s Wrote computer programs in languages such as LISP to attempt to formally characterize the steps that human beings go through, such as making decisions and solving problems
Christopher Longuet-Higgins 1923 1973 Coined the term cognitive science
Noam Chomsky 1928 1959 Published a review of B.F. Skinner's book Verbal Behavior which began cognitivism against then-dominant behaviorism
George Miller 1920 1956 Wrote about the capacities of human thinking through mental representations
Herbert Simon 1916 1956 Co-created Logic Theory Machine and General Problem Solver with Allen Newell, EPAM (Elementary Perceiver and Memorizer) theory, and organizational decision-making
John McCarthy 1927 1955 Coined the term artificial intelligence and organized the famous Dartmouth conference in Summer 1956, which started AI as a field
McCulloch and Pitts
1930s–1940s Developed early artificial neural networks
Lila R. Gleitman 1929 1970s-2010s Wide-ranging contributions to understanding the cognition of language acquisition, including syntactic bootstrapping theory
Eleanor Rosch 1938 1976 Development of the Prototype Theory of categorisation
Philip N. Johnson-Laird 1936 1980 Introduced the idea of mental models in cognitive science
Dedre Gentner 1944 1983 Development of the Structure-mapping Theory of analogical reasoning
Allen Newell 1927 1990 Development of the field of Cognitive architecture in cognitive modelling and artificial intelligence
Annette Karmiloff-Smith 1938 1992 Integrating neuroscience and computational modelling into theories of cognitive development
David Marr (neuroscientist) 1945 1990 Proponent of the Three-Level Hypothesis of levels of analysis of computational systems
Peter Gärdenfors 1949 2000 Creator of the conceptual space framework used in cognitive modeling and artificial intelligence.
Linda B. Smith 1951 1993 Together with Esther Thelen, created a dynamical systems approach to understanding cognitive development

Some of the more recognized names in cognitive science are usually either the most controversial or the most cited. Within philosophy, some familiar names include Daniel Dennett, who writes from a computational systems perspective, John Searle, known for his controversial Chinese room argument, and Jerry Fodor, who advocates functionalism.

Others include David Chalmers, who advocates Dualism and is also known for articulating the hard problem of consciousness, and Douglas Hofstadter, famous for writing Gödel, Escher, Bach, which questions the nature of words and thought.

In the realm of linguistics, Noam Chomsky and George Lakoff have been influential (both have also become notable as political commentators). In artificial intelligence, Marvin Minsky, Herbert A. Simon, and Allen Newell are prominent.

Popular names in the discipline of psychology include George A. Miller, James McClelland, Philip Johnson-Laird, Lawrence Barsalou, Vittorio Guidano, Howard Gardner and Steven Pinker. Anthropologists Dan Sperber, Edwin Hutchins, Bradd Shore, James Wertsch, and Scott Atran, have been involved in collaborative projects with cognitive and social psychologists, political scientists and evolutionary biologists in attempts to develop general theories of culture formation, religion, and political association.

Computational theories (with models and simulations) have also been developed by David Rumelhart, James McClelland, and Philip Johnson-Laird.

Epistemics

Epistemics is a term coined in 1969 by the University of Edinburgh with the foundation of its School of Epistemics. Epistemics is to be distinguished from epistemology in that epistemology is the philosophical theory of knowledge, whereas epistemics signifies the scientific study of knowledge.

Christopher Longuet-Higgins has defined it as "the construction of formal models of the processes (perceptual, intellectual, and linguistic) by which knowledge and understanding are achieved and communicated."

In his 1978 essay "Epistemics: The Regulative Theory of Cognition", Alvin I. Goldman claims to have coined the term "epistemics" to describe a reorientation of epistemology. Goldman maintains that his epistemics is continuous with traditional epistemology, and that the new term is only meant to avoid opposition. Epistemics, in Goldman's version, differs only slightly from traditional epistemology in its alliance with the psychology of cognition; epistemics stresses the detailed study of mental processes and information-processing mechanisms that lead to knowledge or beliefs.

In the mid-1980s, the School of Epistemics was renamed as The Centre for Cognitive Science (CCS). In 1998, CCS was incorporated into the University of Edinburgh's School of Informatics.

Binding problem in cognitive science

One of the core aims of cognitive science is to achieve an integrated theory of cognition. This requires integrative mechanisms that explain how the information processing occurring simultaneously in spatially segregated (sub-)cortical areas of the brain is coordinated and bound together to give rise to coherent perceptual and symbolic representations. One approach is to solve this "Binding problem" (that is, the problem of dynamically representing conjunctions of informational elements, from the most basic perceptual representations ("feature binding") to the most complex cognitive representations, like symbol structures ("variable binding")), by means of integrative synchronization mechanisms. In other words, one of the coordinating mechanisms appears to be the temporal (phase) synchronization of neural activity based on dynamical self-organizing processes in neural networks, described by the Binding-by-synchrony (BBS) Hypothesis from neurophysiology. Connectionist cognitive neuroarchitectures have been developed that use integrative synchronization mechanisms to solve this binding problem in perceptual cognition and in language cognition.

In perceptual cognition, the problem is to explain how elementary object properties and object relations, like the object color or the object form, can be dynamically bound together or can be integrated into a representation of this perceptual object by means of a synchronization mechanism ("feature binding", "feature linking"). In language cognition, the problem is to explain how semantic concepts and syntactic roles can be dynamically bound together or can be integrated into complex cognitive representations like systematic and compositional symbol structures and propositions by means of a synchronization mechanism ("variable binding") (see also the "Symbolism vs. connectionism debate" in connectionism).

However, despite significant advances in understanding the integrated theory of cognition (specifically the Binding problem), the debate over the origins of cognition is still ongoing. From the different perspectives noted above, this problem can be reduced to the question of how organisms at the simple-reflex stage of development overcome the threshold of environmental chaos in sensory stimuli: electromagnetic waves, chemical interactions, and pressure fluctuations. The so-called Primary Data Entry (PDE) thesis poses doubts about the ability of such an organism to overcome this cue threshold on its own. In terms of mathematical tools, the PDE thesis underlines the insurmountable high threshold of the cacophony of environmental stimuli (the stimuli noise) for young organisms at the onset of life. It argues that the temporal (phase) synchronization of neural activity based on dynamical self-organizing processes in neural networks, any dynamical bound together or integration to a representation of the perceptual object by means of a synchronization mechanism can not help organisms in distinguishing relevant cue (informative stimulus) for overcome this noise threshold.

Holism

From Wikipedia, the free encyclopedia
https://en.wikipedia.org/wiki/Holism

Holism is the interdisciplinary idea that systems possess properties as wholes apart from the properties of their component parts. The aphorism "The whole is greater than the sum of its parts", is often given as a summary of this proposal. The concept of holism can inform the methodology for a broad array of scientific fields and lifestyle practices. When applications of holism are said to reveal properties of a whole system beyond those of its parts, these qualities are referred to as emergent properties of that system. Holism in all contexts is often placed in opposition to reductionism, a dominant notion in the philosophy of science that systems containing parts contain no unique properties beyond those parts. Proponents of holism consider the search for emergent properties within systems to be demonstrative of their perspective.

Background

Smuts considered natural processes (such as evolution) to contain emergent properties.

The term "holism" was coined by Jan Smuts (1870–1950) in his 1926 book Holism and Evolution. While he never assigned a consistent meaning to the word, Smuts used holism to represent at least three features of reality. First, holism claims that every scientifically measurable thing, either physical or psychological, does possess a nature as a whole beyond its parts. His examples include atoms, cells, or an individual's personality. Smuts discussed this sense of holism in his claim that an individual's body and mind are not completely separated but instead connect and represent the holistic idea of a person. In his second sense, Smuts referred to holism as the cause of evolution. He argued that evolution is neither an accident nor is it brought about by the actions of some transcendent force, such as a God. Smuts criticized writers who emphasized Darwinian concepts of natural selection and genetic variation to support an accidental view of natural processes within the universe. Smuts perceived evolution as the process of nature correcting itself creatively and intentionally. In this way, holism is described as the tendency of a whole system to creatively respond to environmental stressors, a process in which parts naturally work together to bring the whole into more advanced states. Smuts used Pavlovian studies to argue that the inheritance of behavioral changes supports his idea of creative evolution as opposed to purely accidental development in nature. Smuts believed that this creative process was intrinsic within all physical systems of parts and ruled out indirect, transcendent forces. Finally, Smuts used holism to explain the concrete (nontranscendent) nature of the universe in general. In his words, holism is "the ultimate synthetic, ordering, organizing, regulative activity in the universe which accounts for all the structural groupings and syntheses in it." Smuts argued that a holistic view of the universe explains its processes and their evolution more effectively than a reductive view.

Professional philosophers of science and linguistics did not consider Holism and Evolution seriously upon its initial publication in 1926 and the work has received criticism for a lack of theoretical coherence. Some biological scientists, however, did offer favorable assessments shortly after its first print. Over time, the meaning of the word holism became most closely associated with Smuts' first conception of the term, yet without any metaphysical commitments to monism, dualism, or similar concepts which can be inferred from his work.

Scientific applications

Physics

Nonseparability

The advent of holism in the 20th century coincided with the gradual development of quantum mechanics. Holism in physics is the nonseparability of physical systems from their parts, especially quantum phenomena. Classical physics cannot be regarded as holistic, as the behavior of individual parts represents the whole. However, the state of a system in quantum theory resists a certain kind of reductive analysis. For example, two spatially separated quantum systems are described as "entangled," or nonseparable from each other, when a meaningful analysis of one system is indistinguishable from that of the other.[14] There are different conceptions of nonseparability in physics and its exploration is considered to broadly present insight into the ontological problem.

Variants

In one sense, holism for physics is a perspective about the best way to understand the nature of a physical system. In this sense, holism is the methodological claim that systems are accurately understood according to their properties as a whole. A methodological reductionist in physics might seek to explain, for example, the behavior of a liquid by examining its component molecules, atoms, ions or electrons. A methodological holist, on the other hand, believes there is something misguided about this approach; one proponent, a condensed matter physicist, puts it: “the most important advances in this area come about by the emergence of qualitatively new concepts at the intermediate or macroscopic levels—concepts which, one hopes, will be compatible with one's information about the microscopic constituents, but which are in no sense logically dependent on it.” This perspective is considered a conventional attitude among contemporary physicists. In another sense, holism is a metaphysical claim that the nature of a system is not determined by the properties of its component parts. There are three varieties of this sense of physical holism.

  • Ontological holism: some systems are not merely composed of their physical parts
  • Property holism: some systems have properties independent of their physical parts
  • Nomological holism: some systems follow physical laws beyond the laws followed by their physical parts
This Feynman diagram illustrates the path of an electron (e−) and a positron (e+) within a quantum field that could be described in terms of Bohmian mechanics.

The metaphysical claim does not assert that physical systems involve abstract properties beyond the composition of its physical parts, but that there are concrete properties aside from those of its basic physical parts. Theoretical physicist David Bohm (1917-1992) supports this view head-on. Bohm believed that a complete description of the universe would have to go beyond a simple list of all its particles and their positions, there would also have to be a physical quantum field associated with the properties of those particles guiding their trajectories. Bohm's ontological holism concerning the nature of whole physical systems was literal. But Niels Bohr (1885-1962), on the other hand, held ontological holism from an epistemological angle, rather than a literal one. Bohr saw an observational apparatus to be a part of a system under observation, besides the basic physical parts themselves. His theory agrees with Bohm that whole systems were not merely composed of their parts and it identifies properties such as position and momentum as those of whole systems beyond those of its components. But Bohr states that these holistic properties are only meaningful in experimental contexts when physical systems are under observation and that these systems, when not under observation, cannot be said to have meaningful properties, even if these properties took place outside our observation. While Bohr claims these holistic properties exist only insofar as they can be observed, Bohm took his ontological holism one step further by claiming these properties must exist regardless.

Linguistics

Semantic holism suggests that the meaning of individual words depends on the meaning of other words, forming a large web of interconnections. In general, meaning holism states that the properties which determine the meaning of a word are connected such that if the meaning of one word changes, the meaning of every other word in the web changes as well. The set of words that alter in meaning due to a change in the meaning of some other is not necessarily specified in meaning holism, but typically such a change is taken straightforwardly to affect the meaning of every word in the language.

In scientific disciplines, reductionism is the opposing viewpoint to holism. But in the context of linguistics or the philosophy of language, reductionism is typically referred to as atomism. Specifically, atomism states that each word's meaning is independent and so there are no emergent properties within a language. Additionally, there is meaning molecularism which states that a change in one word alters the meaning of only a relatively small set of other words.

The linguistic perspective of meaning holism is traced back to an essay by WV Quine but was subsequently formalized by analytic philosophers Michael Dummett, Jerry Fodor, and Ernest Lepore. While this holistic approach attempts to resolve a classical problem for the philosophy of language concerning how words convey meaning, there is debate over its validity mostly from two angles of criticism: opposition to compositionality and, especially, instability of meaning. The first claims that meaning holism conflicts with the compositionality of language. Meaning in some languages is compositional in that meaning comes from the structure of an expression's parts. Meaning holism suggests that the meaning of words plays an inferential role in the meaning of other words: "pet fish" might imply a meaning of "less than 3 ounces." Since holistic views of meaning assume meaning depends on which words are used and how those words confer meaning onto other words, rather than how they are structured, meaning holism stands in conflict with compositionalism and leaves statements with potentially ambiguous meanings. The second criticism claims that meaning holism makes meaning in language unstable. If some words must be used to infer the meaning of other words, then in order to communicate a message, the sender and the receiver must share an identical set of inferential assumptions or beliefs. If these beliefs were different, meaning may be lost.

Many types of communication would be directly affected by the principles of meaning holism such as informative communication, language learning, and communication about psychological states. Nevertheless, some meaning holists maintain that the instability of meaning holism is an acceptable feature from several different angles. In one example, contextual holists make this point simply by suggesting we often do not actually share identical inferential assumptions but instead rely on context to counter differences of inference and support communication.

Biology

Systems biology aims to understand how genes and proteins function together to form organisms like this animal cell.

Scientific applications of holism within biology are referred to as systems biology. The opposing analytical approach of systems biology is biological organization which models biological systems and structures only in terms of their component parts. "The reductionist approach has successfully identified most of the components and many of the interactions but, unfortunately, offers no convincing concepts or methods to understand how system properties emerge...the pluralism of causes and effects in biological networks is better addressed by observing, through quantitative measures, multiple components simultaneously and by rigorous data integration with mathematical models." The objective in systems biology is to advance models of the interactions in a system. Holistic approaches to modelling have involved cellular modelling strategies, genomic interaction analysis, and phenotype prediction.

Systems medicine

Systems medicine considers holism to better understand part-whole relationships as with microbial-host coevolution.

Systems medicine is a practical approach to systems biology and accepts its holistic assumptions. Systems medicine takes the systems of the human body as made up of a complete whole and uses this as a starting point in its research and, ultimately, treatment.

Lifestyle applications

The term holism is also sometimes used in the context of various lifestyle practices, such as dieting, education, and healthcare, to refer to ways of life that either supplement or replace conventional practices. In these contexts, holism is not necessarily a rigorous or well-defined methodology for obtaining a particular lifestyle outcome. It is sometimes simply an adjective to describe practices which account for factors that standard forms of these practices may discount, especially in the context of alternative medicine.

Tuesday, August 4, 2026

Protein engineering

From Wikipedia, the free encyclopedia

Protein engineering is the process of developing useful or valuable proteins through the design and production of unnatural polypeptides, often by altering amino acid sequences found in nature. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles. It has been used to improve the function of many enzymes for industrial catalysis. It is also a product and services market, with an estimated value of $168 billion by 2017.

There are two general strategies for protein engineering: rational protein design and directed evolution. These methods are not mutually exclusive; researchers will often apply both. In the future, more detailed knowledge of protein structure and function, and advances in high-throughput screening, may greatly expand the abilities of protein engineering. Eventually, even unnatural amino acids may be included, via newer methods, such as expanded genetic code, that allow encoding novel amino acids in genetic code. The applications in numerous fields, including medicine and industrial bioprocessing, are vast and numerous.

Approaches

Rational design

In rational protein design, a scientist uses detailed knowledge of the structure and function of a protein to make desired changes. In general, this has the advantage of being inexpensive and technically easy, since site-directed mutagenesis methods are well-developed. However, its major drawback is that detailed structural knowledge of a protein is often unavailable, and, even when available, it can be very difficult to predict the effects of various mutations since structural information most often provide a static picture of a protein structure. However, programs such as Folding@home and Foldit have utilized crowdsourcing techniques in order to gain insight into the folding motifs of proteins.

Computational protein design algorithms seek to identify novel amino acid sequences that are low in energy when folded to the pre-specified target structure. While the sequence-conformation space that needs to be searched is large, the most challenging requirement for computational protein design is a fast, yet accurate, energy function that can distinguish optimal sequences from similar suboptimal ones.

Multiple sequence alignment

Without structural information about a protein, sequence analysis is often useful in elucidating information about the protein. These techniques involve alignment of target protein sequences with other related protein sequences. This alignment can show which amino acids are conserved between species and are important for the function of the protein. These analyses can help to identify hot spot amino acids that can serve as the target sites for mutations. Multiple sequence alignment utilizes data bases such as PREFAB, SABMARK, OXBENCH, IRMBASE, and BALIBASE in order to cross reference target protein sequences with known sequences. Multiple sequence alignment techniques are listed below.

This method begins by performing pair wise alignment of sequences using k-tuple or Needleman–Wunsch methods. These methods calculate a matrix that depicts the pair wise similarity among the sequence pairs. Similarity scores are then transformed into distance scores that are used to produce a guide tree using the neighbor joining method. This guide tree is then employed to yield a multiple sequence alignment.

Clustal omega

This method is capable of aligning up to 190,000 sequences by utilizing the k-tuple method. Next sequences are clustered using the mBed and k-means methods. A guide tree is then constructed using the UPGMA method that is used by the HH align package. This guide tree is used to generate multiple sequence alignments.

MAFFT

This method utilizes fast Fourier transform (FFT) that converts amino acid sequences into a sequence composed of volume and polarity values for each amino acid residue. This new sequence is used to find homologous regions.

K-Align

This method utilizes the Wu-Manber approximate string matching algorithm to generate multiple sequence alignments.

Multiple sequence comparison by log expectation (MUSCLE)

This method utilizes Kmer and Kimura distances to generate multiple sequence alignments.

T-Coffee

This method utilizes tree based consistency objective functions for alignment evolution. This method has been shown to be 5–10% more accurate than Clustal W.

Coevolutionary analysis

Coevolutionary analysis is also known as correlated mutation, covariation, or co-substitution. This type of rational design involves reciprocal evolutionary changes at evolutionarily interacting loci. Generally this method begins with the generation of a curated multiple sequence alignments for the target sequence. This alignment is then subjected to manual refinement that involves removal of highly gapped sequences, as well as sequences with low sequence identity. This step increases the quality of the alignment. Next, the manually processed alignment is utilized for further coevolutionary measurements using distinct correlated mutation algorithms. These algorithms result in a coevolution scoring matrix. This matrix is filtered by applying various significance tests to extract significant coevolution values and wipe out background noise. Coevolutionary measurements are further evaluated to assess their performance and stringency. Finally, the results from this coevolutionary analysis are validated experimentally.

Structural prediction

De novo generation of protein benefits from knowledge of existing protein structures. This knowledge of existing protein structure assists with the prediction of new protein structures. Methods for protein structure prediction fall under one of the four following classes: ab initio, fragment based methods, homology modeling, and protein threading.

Ab initio

These methods involve free modeling without using any structural information about the template. Ab initio methods are aimed at prediction of the native structures of proteins corresponding to the global minimum of its free energy. some examples of ab initio methods are AMBER, GROMOS, GROMACS, CHARMM, OPLS, and ENCEPP12. General steps for ab initio methods begin with the geometric representation of the protein of interest. Next, a potential energy function model for the protein is developed. This model can be created using either molecular mechanics potentials or protein structure derived potential functions. Following the development of a potential model, energy search techniques including molecular dynamic simulations, Monte Carlo simulations and genetic algorithms are applied to the protein.

Fragment based

These methods use database information regarding structures to match homologous structures to the created protein sequences. These homologous structures are assembled to give compact structures using scoring and optimization procedures, with the goal of achieving the lowest potential energy score. Webservers for fragment information are I-TASSER, ROSETTA, ROSETTA @ home, FRAGFOLD, CABS fold, PROFESY, CREF, QUARK, UNDERTAKER, HMM, and ANGLOR.

Homology modeling

These methods are based upon the homology of proteins. These methods are also known as comparative modeling. The first step in homology modeling is generally the identification of template sequences of known structure which are homologous to the query sequence. Next the query sequence is aligned to the template sequence. Following the alignment, the structurally conserved regions are modeled using the template structure. This is followed by the modeling of side chains and loops that are distinct from the template. Finally the modeled structure undergoes refinement and assessment of quality. Servers that are available for homology modeling data are listed here: SWISS MODEL, MODELLER, ReformAlign, PyMOD, TIP-STRUCTFAST, COMPASS, 3d-PSSM, SAMT02, SAMT99, HHPRED, FAGUE, 3D-JIGSAW, META-PP, ROSETTA, and I-TASSER.

Protein threading

Protein threading can be used when a reliable homologue for the query sequence cannot be found. This method begins by obtaining a query sequence and a library of template structures. Next, the query sequence is threaded over known template structures. These candidate models are scored using scoring functions. These are scored based upon potential energy models of both query and template sequence. The match with the lowest potential energy model is then selected. Methods and servers for retrieving threading data and performing calculations are listed here: GenTHREADER, pGenTHREADER, pDomTHREADER, ORFEUS, PROSPECT, BioShell-Threading, FFASO3, RaptorX, HHPred, LOOPP server, Sparks-X, SEGMER, THREADER2, ESYPRED3D, LIBRA, TOPITS, RAPTOR, COTH, MUSTER.

For more information on rational design see site-directed mutagenesis.

Multivalent binding

Multivalent binding can be used to increase the binding specificity and affinity through avidity effects. Having multiple binding domains in a single biomolecule or complex increases the likelihood of other interactions to occur via individual binding events. Avidity or effective affinity can be much higher than the sum of the individual affinities providing a cost and time-effective tool for targeted binding.

Multivalent proteins

Multivalent proteins are relatively easy to produce by post-translational modifications or multiplying the protein-coding DNA sequence. The main advantage of multivalent and multispecific proteins is that they can increase the effective affinity for a target of a known protein. In the case of an inhomogeneous target using a combination of proteins resulting in multispecific binding can increase specificity, which has high applicability in protein therapeutics.

The most common example for multivalent binding are the antibodies, and there is extensive research for bispecific antibodies. Applications of bispecific antibodies cover a broad spectrum that includes diagnosis, imaging, prophylaxis, and therapy.

Directed evolution

In directed evolution, random mutagenesis, e.g. by error-prone PCR or sequence saturation mutagenesis, is applied to a protein, and a selection regime is used to select variants having desired traits. Further rounds of mutation and selection are then applied. This method mimics natural evolution and, in general, produces superior results to rational design. An added process, termed DNA shuffling, mixes and matches pieces of successful variants to produce better results. Such processes mimic the recombination that occurs naturally during sexual reproduction. Advantages of directed evolution are that it requires no prior structural knowledge of a protein, nor is it necessary to be able to predict what effect a given mutation will have. Indeed, the results of directed evolution experiments are often surprising in that desired changes are often caused by mutations that were not expected to have some effect. The drawback is that they require high-throughput screening, which is not feasible for all proteins. Large amounts of recombinant DNA must be mutated and the products screened for desired traits. The large number of variants often requires expensive robotic equipment to automate the process. Further, not all desired activities can be screened for easily.

Laboratory methods can be used to imitate aspects of Darwinian evolution for the development of protein properties in applications such as catalysis. Various experimental techniques are available to generate large and diverse protein libraries and to screen or select variants with desired folding and functionality. Folded proteins can arise in random sequence space, which can be utilized in the development of binding proteins and catalysts. An alternative approach involves modifying existing proteins through random mutagenesis followed by selection or screening to optimize or alter their properties. This approach can serve as a basis for more advanced protein engineering efforts. Combining experimental evolution with computational methods may support the development of functional macromolecules not found in nature.

The main challenges of designing high quality mutant libraries have shown significant progress in the recent past. This progress has been in the form of better descriptions of the effects of mutational loads on protein traits. Also computational approaches have shown large advances in the innumerably large sequence space to more manageable screenable sizes, thus creating smart libraries of mutants. Library size has also been reduced to more screenable sizes by the identification of key beneficial residues using algorithms for systematic recombination. Finally a significant step forward toward efficient reengineering of enzymes has been made with the development of more accurate statistical models and algorithms quantifying and predicting coupled mutational effects on protein functions.

Generally, directed evolution may be summarized as an iterative two step process which involves generation of protein mutant libraries, and high throughput screening processes to select for variants with improved traits. This technique does not require prior knowledge of the protein structure and function relationship. Directed evolution utilizes random or focused mutagenesis to generate libraries of mutant proteins. Random mutations can be introduced using either error prone PCR, or site saturation mutagenesis. Mutants may also be generated using recombination of multiple homologous genes. Nature has evolved a limited number of beneficial sequences. Directed evolution makes it possible to identify undiscovered protein sequences which have novel functions. This ability is contingent on the proteins ability to tolerant amino acid residue substitutions without compromising folding or stability.

Directed evolution methods can be broadly categorized into two strategies, asexual and sexual methods.

Asexual methods

Asexual methods do not generate any cross links between parental genes. Single genes are used to create mutant libraries using various mutagenic techniques. These asexual methods can produce either random or focused mutagenesis.

Random mutagenesis

Random mutagenic methods produce mutations at random throughout the gene of interest. Random mutagenesis can introduce the following types of mutations: transitions, transversions, insertions, deletions, inversion, missense, and nonsense. Examples of methods for producing random mutagenesis are below.

Error prone PCR

Error prone PCR utilizes the fact that Taq DNA polymerase lacks 3' to 5' exonuclease activity. This results in an error rate of 0.001–0.002% per nucleotide per replication. This method begins with choosing the gene, or the area within a gene, one wishes to mutate. Next, the extent of error required is calculated based upon the type and extent of activity one wishes to generate. This extent of error determines the error prone PCR strategy to be employed. Following PCR, the genes are cloned into a plasmid and introduced to competent cell systems. These cells are then screened for desired traits. Plasmids are then isolated for colonies which show improved traits, and are then used as templates the next round of mutagenesis. Error prone PCR shows biases for certain mutations relative to others. Such as biases for transitions over transversions.

Rates of error in PCR can be increased in the following ways:

  1. Increase concentration of magnesium chloride, which stabilizes non complementary base pairing.
  2. Add manganese chloride to reduce base pair specificity.
  3. Increased and unbalanced addition of dNTPs.
  4. Addition of base analogs like dITP, 8 oxo-dGTP, and dPTP.
  5. Increase concentration of Taq polymerase.
  6. Increase extension time.
  7. Increase cycle time.
  8. Use less accurate Taq polymerase.

Also see polymerase chain reaction for more information.

Rolling circle error-prone PCR

This PCR method is based upon rolling circle amplification, which is modeled from the method that bacteria use to amplify circular DNA. This method results in linear DNA duplexes. These fragments contain tandem repeats of circular DNA called concatamers, which can be transformed into bacterial strains. Mutations are introduced by first cloning the target sequence into an appropriate plasmid. Next, the amplification process begins using random hexamer primers and Φ29 DNA polymerase under error prone rolling circle amplification conditions. Additional conditions to produce error prone rolling circle amplification are 1.5 pM of template DNA, 1.5 mM MnCl2 and a 24 hour reaction time. MnCl2 is added into the reaction mixture to promote random point mutations in the DNA strands. Mutation rates can be increased by increasing the concentration of MnCl2, or by decreasing concentration of the template DNA. Error prone rolling circle amplification is advantageous relative to error prone PCR because of its use of universal random hexamer primers, rather than specific primers. Also the reaction products of this amplification do not need to be treated with ligases or endonucleases. This reaction is isothermal.

Chemical mutagenesis

Chemical mutagenesis involves the use of chemical agents to introduce mutations into genetic sequences. Examples of chemical mutagens follow.

Sodium bisulfate is effective at mutating G/C rich genomic sequences. This is because sodium bisulfate catalyses deamination of unmethylated cytosine to uracil.

Ethyl methane sulfonate alkylates guanidine residues. This alteration causes errors during DNA replication.

Nitrous acid causes transversion by de-amination of adenine and cytosine.

The dual approach to random chemical mutagenesis is an iterative two step process. First it involves the in vivo chemical mutagenesis of the gene of interest via EMS. Next, the treated gene is isolated and cloning into an untreated expression vector in order to prevent mutations in the plasmid backbone. This technique preserves the plasmids genetic properties.

Targeting glycosylases to embedded arrays for mutagenesis (TaGTEAM)

This method has been used to create targeted in vivo mutagenesis in yeast. This method involves the fusion of a 3-methyladenine DNA glycosylase to tetR DNA-binding domain. This has been shown to increase mutation rates by over 800 time in regions of the genome containing tetO sites.

Mutagenesis by random insertion and deletion

This method involves alteration in length of the sequence via simultaneous deletion and insertion of chunks of bases of arbitrary length. This method has been shown to produce proteins with new functionalities via introduction of new restriction sites, specific codons, four base codons for non-natural amino acids.

Transposon based random mutagenesis

Recently many methods for transposon based random mutagenesis have been reported. This methods include, but are not limited to the following: PERMUTE-random circular permutation, random protein truncation, random nucleotide triplet substitution, random domain/tag/multiple amino acid insertion, codon scanning mutagenesis, and multicodon scanning mutagenesis. These aforementioned techniques all require the design of mini-Mu transposons. Thermo scientific manufactures kits for the design of these transposons.

Random mutagenesis methods altering the target DNA length

These methods involve altering gene length via insertion and deletion mutations. An example is the tandem repeat insertion (TRINS) method. This technique results in the generation of tandem repeats of random fragments of the target gene via rolling circle amplification and concurrent incorporation of these repeats into the target gene.

Mutator strains

Mutator strains are bacterial cell lines which are deficient in one or more DNA repair mechanisms. An example of a mutator strand is the E. coli XL1-RED. This subordinate strain of E. coli is deficient in the MutS, MutD, MutT DNA repair pathways. Use of mutator strains is useful at introducing many types of mutation; however, these strains show progressive sickness of culture because of the accumulation of mutations in the strains own genome.

Focused mutagenesis

Focused mutagenic methods produce mutations at predetermined amino acid residues. These techniques require and understanding of the sequence-function relationship for the protein of interest. Understanding of this relationship allows for the identification of residues which are important in stability, stereoselectivity, and catalytic efficiency. Examples of methods that produce focused mutagenesis are below.

Site saturation mutagenesis

Site saturation mutagenesis is a PCR based method used to target amino acids with significant roles in protein function. The two most common techniques for performing this are whole plasmid single PCR, and overlap extension PCR.

Whole plasmid single PCR is also referred to as site directed mutagenesis (SDM). SDM products are subjected to Dpn endonuclease digestion. This digestion results in cleavage of only the parental strand, because the parental strand contains a GmATC which is methylated at N6 of adenine. SDM does not work well for large plasmids of over ten kilobases. Also, this method is only capable of replacing two nucleotides at a time.

Overlap extension PCR requires the use of two pairs of primers. One primer in each set contains a mutation. A first round of PCR using these primer sets is performed and two double stranded DNA duplexes are formed. A second round of PCR is then performed in which these duplexes are denatured and annealed with the primer sets again to produce heteroduplexes, in which each strand has a mutation. Any gaps in these newly formed heteroduplexes are filled with DNA polymerases and further amplified.

Sequence saturation mutagenesis (SeSaM)

Sequence saturation mutagenesis results in the randomization of the target sequence at every nucleotide position. This method begins with the generation of variable length DNA fragments tailed with universal bases via the use of template transferases at the 3' termini. Next, these fragments are extended to full length using a single stranded template. The universal bases are replaced with a random standard base, causing mutations. There are several modified versions of this method such as SeSAM-Tv-II, SeSAM-Tv+, and SeSAM-III.

Single primer reactions in parallel (SPRINP)

This site saturation mutagenesis method involves two separate PCR reaction. The first of which uses only forward primers, while the second reaction uses only reverse primers. This avoids the formation of primer dimer formation.

Mega primed and ligase free focused mutagenesis

This site saturation mutagenic technique begins with one mutagenic oligonucleotide and one universal flanking primer. These two reactants are used for an initial PCR cycle. Products from this first PCR cycle are used as mega primers for the next PCR.

Ω-PCR

This site saturation mutagenic method is based on overlap extension PCR. It is used to introduce mutations at any site in a circular plasmid.

PFunkel-ominchange-OSCARR

This method utilizes user defined site directed mutagenesis at single or multiple sites simultaneously. OSCARR is an acronym for one pot simple methodology for cassette randomization and recombination. This randomization and recombination results in randomization of desired fragments of a protein. Omnichange is a sequence independent, multisite saturation mutagenesis which can saturate up to five independent codons on a gene.

Trimer-dimer mutagenesis

This method removes redundant codons and stop codons.

Cassette mutagenesis

This is a PCR based method. Cassette mutagenesis begins with the synthesis of a DNA cassette containing the gene of interest, which is flanked on either side by restriction sites. The endonuclease which cleaves these restriction sites also cleaves sites in the target plasmid. The DNA cassette and the target plasmid are both treated with endonucleases to cleave these restriction sites and create sticky ends. Next the products from this cleavage are ligated together, resulting in the insertion of the gene into the target plasmid. An alternative form of cassette mutagenesis called combinatorial cassette mutagenesis is used to identify the functions of individual amino acid residues in the protein of interest. Recursive ensemble mutagenesis then utilizes information from previous combinatorial cassette mutagenesis. Codon cassette mutagenesis allows you to insert or replace a single codon at a particular site in double stranded DNA.

Sexual methods

Sexual methods of directed evolution involve in vitro recombination which mimic natural in vivo recombination. Generally these techniques require high sequence homology between parental sequences. These techniques are often used to recombine two different parental genes, and these methods do create cross overs between these genes.

In vitro homologous recombination

Homologous recombination can be categorized as either in vivo or in vitro. In vitro homologous recombination mimics natural in vivo recombination. These in vitro recombination methods require high sequence homology between parental sequences. These techniques exploit the natural diversity in parental genes by recombining them to yield chimeric genes. The resulting chimera show a blend of parental characteristics.

DNA shuffling

This in vitro technique was one of the first techniques in the era of recombination. It begins with the digestion of homologous parental genes into small fragments by DNase1. These small fragments are then purified from undigested parental genes. Purified fragments are then reassembled using primer-less PCR. This PCR involves homologous fragments from different parental genes priming for each other, resulting in chimeric DNA. The chimeric DNA of parental size is then amplified using end terminal primers in regular PCR.

Random priming in vitro recombination (RPR)

This in vitro homologous recombination method begins with the synthesis of many short gene fragments exhibiting point mutations using random sequence primers. These fragments are reassembled to full length parental genes using primer-less PCR. These reassembled sequences are then amplified using PCR and subjected to further selection processes. This method is advantageous relative to DNA shuffling because there is no use of DNase1, thus there is no bias for recombination next to a pyrimidine nucleotide. This method is also advantageous due to its use of synthetic random primers which are uniform in length, and lack biases. Finally this method is independent of the length of DNA template sequence, and requires a small amount of parental DNA.

Truncated metagenomic gene-specific PCR

This method generates chimeric genes directly from metagenomic samples. It begins with isolation of the desired gene by functional screening from metagenomic DNA sample. Next, specific primers are designed and used to amplify the homologous genes from different environmental samples. Finally, chimeric libraries are generated to retrieve the desired functional clones by shuffling these amplified homologous genes.

Staggered extension process (StEP)

This in vitro method is based on template switching to generate chimeric genes. This PCR based method begins with an initial denaturation of the template, followed by annealing of primers and a short extension time. All subsequent cycle generate annealing between the short fragments generated in previous cycles and different parts of the template. These short fragments and the templates anneal together based on sequence complementarity. This process of fragments annealing template DNA is known as template switching. These annealed fragments will then serve as primers for further extension. This method is carried out until the parental length chimeric gene sequence is obtained. Execution of this method only requires flanking primers to begin. There is also no need for Dnase1 enzyme.

Random chimeragenesis on transient templates (RACHITT)

This method has been shown to generate chimeric gene libraries with an average of 14 crossovers per chimeric gene. It begins by aligning fragments from a parental top strand onto the bottom strand of a uracil containing template from a homologous gene. 5' and 3' overhang flaps are cleaved and gaps are filled by the exonuclease and endonuclease activities of Pfu and taq DNA polymerases. The uracil containing template is then removed from the heteroduplex by treatment with a uracil DNA glcosylase, followed by further amplification using PCR. This method is advantageous because it generates chimeras with relatively high crossover frequency. However it is somewhat limited due to the complexity and the need for generation of single stranded DNA and uracil containing single stranded template DNA.

Synthetic shuffling

Shuffling of synthetic degenerate oligonucleotides adds flexibility to shuffling methods, since oligonucleotides containing optimal codons and beneficial mutations can be included.

In vivo Homologous Recombination

Cloning performed in yeast involves PCR dependent reassembly of fragmented expression vectors. These reassembled vectors are then introduced to, and cloned in yeast. Using yeast to clone the vector avoids toxicity and counter-selection that would be introduced by ligation and propagation in E. coli.

Mutagenic organized recombination process by homologous in vivo grouping (MORPHING)

This method introduces mutations into specific regions of genes while leaving other parts intact by utilizing the high frequency of homologous recombination in yeast.

Phage-assisted continuous evolution (PACE)

This method utilizes a bacteriophage with a modified life cycle to transfer evolving genes from host to host. The phage's life cycle is designed in such a way that the transfer is correlated with the activity of interest from the enzyme. This method is advantageous because it requires minimal human intervention for the continuous evolution of the gene.

In vitro non-homologous recombination methods

These methods are based upon the fact that proteins can exhibit similar structural identity while lacking sequence homology.

Exon shuffling

Exon shuffling is the combination of exons from different proteins by recombination events occurring at introns. Orthologous exon shuffling involves combining exons from orthologous genes from different species. Orthologous domain shuffling involves shuffling of entire protein domains from orthologous genes from different species. Paralogous exon shuffling involves shuffling of exon from different genes from the same species. Paralogous domain shuffling involves shuffling of entire protein domains from paralogous proteins from the same species. Functional homolog shuffling involves shuffling of non-homologous domains which are functional related. All of these processes being with amplification of the desired exons from different genes using chimeric synthetic oligonucleotides. This amplification products are then reassembled into full length genes using primer-less PCR. During these PCR cycles the fragments act as templates and primers. This results in chimeric full length genes, which are then subjected to screening.

Incremental truncation for the creation of hybrid enzymes (ITCHY)

Fragments of parental genes are created using controlled digestion by exonuclease III. These fragments are blunted using endonuclease, and are ligated to produce hybrid genes. THIOITCHY is a modified ITCHY technique which utilized nucleotide triphosphate analogs such as α-phosphothioate dNTPs. Incorporation of these nucleotides blocks digestion by exonuclease III. This inhibition of digestion by exonuclease III is called spiking. Spiking can be accomplished by first truncating genes with exonuclease to create fragments with short single stranded overhangs. These fragments then serve as templates for amplification by DNA polymerase in the presence of small amounts of phosphothioate dNTPs. These resulting fragments are then ligated together to form full length genes. Alternatively the intact parental genes can be amplified by PCR in the presence of normal dNTPs and phosphothioate dNTPs. These full length amplification products are then subjected to digestion by an exonuclease. Digestion will continue until the exonuclease encounters an α-pdNTP, resulting in fragments of different length. These fragments are then ligated together to generate chimeric genes.

SCRATCHY

This method generates libraries of hybrid genes inhibiting multiple crossovers by combining DNA shuffling and ITCHY. This method begins with the construction of two independent ITCHY libraries. The first with gene A on the N-terminus. And the other having gene B on the N-terminus. These hybrid gene fragments are separated using either restriction enzyme digestion or PCR with terminus primers via agarose gel electrophoresis. These isolated fragments are then mixed together and further digested using DNase1. Digested fragments are then reassembled by primerless PCR with template switching.

Recombined extension on truncated templates (RETT)

This method generates libraries of hybrid genes by template switching of uni-directionally growing polynucleotides in the presence of single stranded DNA fragments as templates for chimeras. This method begins with the preparation of single stranded DNA fragments by reverse transcription from target mRNA. Gene specific primers are then annealed to the single stranded DNA. These genes are then extended during a PCR cycle. This cycle is followed by template switching and annealing of the short fragments obtained from the earlier primer extension to other single stranded DNA fragments. This process is repeated until full length single stranded DNA is obtained.

Sequence homology-independent protein recombination (SHIPREC)

This method generates recombination between genes with little to no sequence homology. These chimeras are fused via a linker sequence containing several restriction sites. This construct is then digested using DNase1. Fragments are made are made blunt ended using S1 nuclease. These blunt end fragments are put together into a circular sequence by ligation. This circular construct is then linearized using restriction enzymes for which the restriction sites are present in the linker region. This results in a library of chimeric genes in which contribution of genes to 5' and 3' end will be reversed as compared to the starting construct.

Sequence independent site directed chimeragenesis (SISDC)

This method results in a library of genes with multiple crossovers from several parental genes. This method does not require sequence identity among the parental genes. This does require one or two conserved amino acids at every crossover position. It begins with alignment of parental sequences and identification of consensus regions which serve as crossover sites. This is followed by the incorporation of specific tags containing restriction sites followed by the removal of the tags by digestion with Bac1, resulting in genes with cohesive ends. These gene fragments are mixed and ligated in an appropriate order to form chimeric libraries.

Degenerate homo-duplex recombination (DHR)

This method begins with alignment of homologous genes, followed by identification of regions of polymorphism. Next the top strand of the gene is divided into small degenerate oligonucleotides. The bottom strand is also digested into oligonucleotides to serve as scaffolds. These fragments are combined in solution are top strand oligonucleotides are assembled onto bottom strand oligonucleotides. Gaps between these fragments are filled with polymerase and ligated.

Random multi-recombinant PCR (RM-PCR)

This method involves the shuffling of plural DNA fragments without homology, in a single PCR. This results in the reconstruction of complete proteins by assembly of modules encoding different structural units.

User friendly DNA recombination (USERec)

This method begins with the amplification of gene fragments which need to be recombined, using uracil dNTPs. This amplification solution also contains primers, PfuTurbo, and Cx Hotstart DNA polymerase. Amplified products are next incubated with USER enzyme. This enzyme catalyzes the removal of uracil residues from DNA creating single base pair gaps. The USER enzyme treated fragments are mixed and ligated using T4 DNA ligase and subjected to Dpn1 digestion to remove the template DNA. These resulting dingle stranded fragments are subjected to amplification using PCR, and are transformed into E. coli.

Golden Gate shuffling (GGS) recombination

This method allows you to recombine at least 9 different fragments in an acceptor vector by using type 2 restriction enzyme which cuts outside of the restriction sites. It begins with sub cloning of fragments in separate vectors to create Bsa1 flanking sequences on both sides. These vectors are then cleaved using type II restriction enzyme Bsa1, which generates four nucleotide single strand overhangs. Fragments with complementary overhangs are hybridized and ligated using T4 DNA ligase. Finally these constructs are then transformed into E. coli cells, which are screened for expression levels.

Phosphoro thioate-based DNA recombination method (PRTec)

This method can be used to recombine structural elements or entire protein domains. This method is based on phosphorothioate chemistry which allows the specific cleavage of phosphorothiodiester bonds. The first step in the process begins with amplification of fragments that need to be recombined along with the vector backbone. This amplification is accomplished using primers with phosphorothiolated nucleotides at 5' ends. Amplified PCR products are cleaved in an ethanol-iodine solution at high temperatures. Next these fragments are hybridized at room temperature and transformed into E. coli which repair any nicks.

Integron

This system is based upon a natural site specific recombination system in E. coli. This system is called the integron system, and produces natural gene shuffling. This method was used to construct and optimize a functional tryptophan biosynthetic operon in trp-deficient E. coli by delivering individual recombination cassettes or trpA-E genes along with regulatory elements with the integron system.

Y-Ligation based shuffling (YLBS)

This method generates single stranded DNA strands, which encompass a single block sequence either at the 5' or 3' end, complementary sequences in a stem loop region, and a D branch region serving as a primer binding site for PCR. Equivalent amounts of both 5' and 3' half strands are mixed and formed a hybrid due to the complementarity in the stem region. Hybrids with free phosphorylated 5' end in 3' half strands are then ligated with free 3' ends in 5' half strands using T4 DNA ligase in the presence of 0.1 mM ATP. Ligated products are then amplified by two types of PCR to generate pre 5' half and pre 3' half PCR products. These PCR product are converted to single strands via avidin-biotin binding to the 5' end of the primes containing stem sequences that were biotin labeled. Next, biotinylated 5' half strands and non-biotinylated 3' half strands are used as 5' and 3' half strands for the next Y-ligation cycle.

Semi-rational design

Semi-rational design uses information about a proteins sequence, structure and function, in tandem with predictive algorithms. Together these are used to identify target amino acid residues which are most likely to influence protein function. Mutations of these key amino acid residues create libraries of mutant proteins that are more likely to have enhanced properties.

Advances in semi-rational enzyme engineering and de novo enzyme design offer researchers efficient frameworks for manipulating biocatalysts. The integration of sequence- and structure-based approaches in enzyme redesign has proven to significantly reduce variant iterations, eliminating the necessity for high-throughput analysis. However, current computational de novo and redesign methods are inferior to evolved variants in catalytic performance. To close this gap, design algorithm refinements — such as integrating protein dynamics into future simulations — are expected to further improve accuracy in structure predictions and enhance catalytic ability.

Biochemical and biophysical studies, along with fine-tuning of predictive frameworks will be useful to experimentally evaluate the functional significance of individual design features. Better understanding of these functional contributions will then give feedback for the improvement of future designs.

Directed evolution will likely not be replaced as the method of choice for protein engineering, although computational protein design has fundamentally changed the way protein engineering can manipulate bio-macromolecules. Smaller, more focused and functionally-rich libraries may be generated by using in methods which incorporate predictive frameworks for hypothesis-driven protein engineering. New design strategies and technical advances have begun a departure from traditional protocols, such as directed evolution, which represents the most effective strategy for identifying top-performing candidates in focused libraries. Whole-gene library synthesis is replacing shuffling and mutagenesis protocols for library preparation. Also highly specific low throughput screening assays are increasingly applied in place of monumental screening and selection efforts of millions of candidates. Together, these developments are poised to take protein engineering beyond directed evolution and towards practical, more efficient strategies for tailoring biocatalysts.

Screening and selection techniques

Once a protein has undergone directed evolution, ration design or semi-ration design, the libraries of mutant proteins must be screened to determine which mutants show enhanced properties. Phage display methods are one option for screening proteins. This method involves the fusion of genes encoding the variant polypeptides with phage coat protein genes. Protein variants expressed on phage surfaces are selected by binding with immobilized targets in vitro. Phages with selected protein variants are then amplified in bacteria, followed by the identification of positive clones by enzyme linked immunosorbent assay. These selected phages are then subjected to DNA sequencing.

Cell surface display systems can also be utilized to screen mutant polypeptide libraries. The library mutant genes are incorporated into expression vectors which are then transformed into appropriate host cells. These host cells are subjected to further high throughput screening methods to identify the cells with desired phenotypes.

Cell free display systems have been developed to exploit in vitro protein translation or cell free translation. These methods include mRNA display, ribosome display, covalent and non covalent DNA display, and in vitro compartmentalization.

Enzyme engineering

Enzyme engineering is the application of modifying an enzyme's structure (and, thus, its function) or modifying the catalytic activity of isolated enzymes to produce new metabolites, to allow new (catalyzed) pathways for reactions to occur, or to convert from certain compounds into others (biotransformation). These products are useful as chemicals, pharmaceuticals, fuel, food, or agricultural additives.

An enzyme reactor consists of a vessel containing a reactional medium that is used to perform a desired conversion by enzymatic means. Enzymes used in this process are free in the solution.

Examples of engineered proteins

Computing methods have been used to design a protein with a novel fold, such as Top7, and sensors for unnatural molecules. The engineering of fusion proteins has yielded rilonacept, a pharmaceutical that has secured Food and Drug Administration (FDA) approval for treating cryopyrin-associated periodic syndrome.

Another computing method, IPRO, successfully engineered the switching of cofactor specificity of Candida boidinii xylose reductase. Iterative Protein Redesign and Optimization (IPRO) redesigns proteins to increase or give specificity to native or novel substrates and cofactors. This is done by repeatedly randomly perturbing the structure of the proteins around specified design positions, identifying the lowest energy combination of rotamers, and determining whether the new design has a lower binding energy than prior ones. The iterative nature of this process allows IPRO to make additive mutations to a protein sequence that collectively improve the specificity toward desired substrates and/or cofactors.

Computation-aided design has also been used to engineer complex properties of a highly ordered nano-protein assembly. A protein cage, E. coli bacterioferritin (EcBfr), which naturally shows structural instability and an incomplete self-assembly behavior by populating two oligomerization states, is the model protein in this study. Through computational analysis and comparison to its homologs, it has been found that this protein has a smaller-than-average dimeric interface on its two-fold symmetry axis due mainly to the existence of an interfacial water pocket centered on two water-bridged asparagine residues. To investigate the possibility of engineering EcBfr for modified structural stability, a semi-empirical computational method is used to virtually explore the energy differences of the 480 possible mutants at the dimeric interface relative to the wild type EcBfr. This computational study also converges on the water-bridged asparagines. Replacing these two asparagines with hydrophobic amino acids results in proteins that fold into alpha-helical monomers and assemble into cages as evidenced by circular dichroism and transmission electron microscopy. Both thermal and chemical denaturation confirm that, all redesigned proteins, in agreement with the calculations, possess increased stability. One of the three mutations shifts the population in favor of the higher order oligomerization state in solution as shown by both size exclusion chromatography and native gel electrophoresis.

A in silico method, PoreDesigner, was developed to redesign bacterial channel protein (OmpF) to reduce its 1 nm pore size to any desired sub-nm dimension. Transport experiments on the narrowest designed pores revealed complete salt rejection when assembled in biomimetic block-polymer matrices.

Drake equation

From Wikipedia, the free encyclopedia https://en.wikipedia.org/wiki/Drake_equation   Frank Drake ...