Procedures by which protein structure and function are changed or created in vitro by altering existing or synthesizing new structural genes that direct the synthesis of proteins with sought-after properties. Such procedures may include the design of MOLECULAR MODELS of proteins using COMPUTER GRAPHICS or other molecular modeling techniques; site-specific mutagenesis (MUTAGENESIS, SITE-SPECIFIC) of existing genes; and DIRECTED MOLECULAR EVOLUTION techniques to create new genes.
Directed modification of the gene complement of a living organism by such techniques as altering the DNA, substituting genetic material by means of a virus, transplanting whole nuclei, transplanting cell hybrids, etc.
Methods and techniques used to genetically modify cells' biosynthetic product output and develop conditions for growing the cells as BIOREACTORS.
Directed Molecular Evolution
The techniques used to produce molecules exhibiting properties that conform to the demands of the experimenter. These techniques combine methods of generating structural changes with methods of selection. They are also used to examine proposed mechanisms of evolution under in vitro selection conditions.
The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain).
Molecular Sequence Data
Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.
Amino Acid Sequence
The internal fragments of precursor proteins (INternal proTEINS) that are autocatalytically removed by PROTEIN SPLICING. The flanking fragments (EXTEINS) are ligated forming mature proteins. The nucleic acid sequences coding for inteins are considered to be MOBILE GENETIC ELEMENTS. Inteins are composed of self-splicing domains and an endonuclease domain which plays a role in the spread of the intein's genomic sequence. Mini-inteins are composed of the self-splicing domains only.
A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.
The extent to which an enzyme retains its structural conformation or its activity when subjected to storage, isolation, and purification or various other physical or chemical manipulations, including proteolytic enzymes and heat.
Body of knowledge related to the use of organisms, cells or cell-derived constituents for the purpose of developing products which are technically, scientifically and clinically useful. Alteration of biologic function at the molecular level (i.e., GENETIC ENGINEERING) is a central focus; laboratory methods used include TRANSFECTION and CLONING technologies, sequence and structure analysis algorithms, computer databases, and gene and protein structure function analysis and prediction.
Protein Structure, Secondary
The level of protein structure in which regular hydrogen-bond interactions within contiguous stretches of polypeptide chain give rise to alpha helices, beta strands (which align to form beta sheets) or other types of coils. This is the first folding level of protein conformation.
The excision of in-frame internal protein sequences (INTEINS) of a precursor protein, coupled with ligation of the flanking sequences (EXTEINS). Protein splicing is an autocatalytic reaction and results in the production of two proteins from a single primary translation product: the intein and the mature protein.
Process of generating a genetic MUTATION. It may occur spontaneously or be induced by MUTAGENS.
Linear POLYPEPTIDES that are synthesized on RIBOSOMES and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of AMINO ACIDS determines the shape the polypeptide will take, during PROTEIN FOLDING, and the function of the protein.
Protein Structure, Tertiary
The level of protein structure in which combinations of secondary protein structures (alpha helices, beta sheets, loop regions, and motifs) pack together to form folded shapes called domains. Disulfide bridges between cysteines in two different parts of the polypeptide chain along with other interactions between the chains play a role in the formation and stabilization of tertiary structure. Small proteins usually consist of only one domain but larger proteins may contain a number of domains connected by segments of polypeptide chain which lack regular secondary structure.
Proteins prepared by recombinant DNA technology.
Amino Acid Substitution
The naturally occurring or experimentally induced replacement of one or more AMINO ACIDS in a protein with another. If a functionally equivalent amino acid is substituted, the protein may retain wild-type activity. Substitution may also diminish, enhance, or eliminate protein function. Experimentally induced substitution is often used to study enzyme activities and binding site properties.
Temperate bacteriophage of the genus INOVIRUS which infects enterobacteria, especially E. coli. It is a filamentous phage consisting of single-stranded DNA and is circularly permuted.
Cell Surface Display Techniques
Techniques utilizing cells that express RECOMBINANT FUSION PROTEINS engineered to translocate through the CELL MEMBRANE and remain attached to the outside of the cell.
A rigorously mathematical analysis of energy relationships (heat, work, temperature, and equilibrium). It describes systems whose states are determined by thermal parameters, such as temperature, in addition to mechanical and electromagnetic parameters. (From Hawley's Condensed Chemical Dictionary, 12th ed)
Recombinant Fusion Proteins
Recombinant proteins produced by the GENETIC TRANSLATION of fused genes formed by the combination of NUCLEIC ACID REGULATORY SEQUENCES of one or more genes with the protein coding sequences of one or more genes.
Biologically functional sequences of DNA chemically synthesized in vitro.
Hydroxymethyl and Formyl Transferases
The determination of the concentration of a given component in solution (the analyte) by addition of a liquid reagent of known strength (the titrant) until an equivalence point is reached (when the reactants are present in stoichiometric proportions). Often an indicator is added to make the equivalence point visible (e.g., a change in color).
The molecular designing of drugs for specific purposes (such as DNA-binding, enzyme inhibition, anti-cancer efficacy, etc.) based on knowledge of molecular properties such as activity of functional groups, molecular geometry, and electronic structure, and also on information cataloged on analogous molecules. Drug design is generally computer-assisted molecular modeling and does not include pharmacokinetics, dosage analysis, or drug administration analysis.
Members of the class of compounds composed of AMINO ACIDS joined together by peptide bonds between adjacent amino acids into linear, branched or cyclical structures. OLIGOPEPTIDES are composed of approximately 2-12 amino acids. Polypeptides are composed of approximately 13 or more amino acids. PROTEINS are linear polypeptides that are normally synthesized on RIBOSOMES.
The arrangement of two or more amino acid or base sequences from an organism or organisms in such a way as to align areas of the sequences sharing common properties. The degree of relatedness or homology between the sequences is predicted computationally or statistically based on weights assigned to the elements aligned between the sequences. This in turn can serve as a potential indicator of the genetic relatedness between the organisms.
Sequence Homology, Amino Acid
Nuclear Magnetic Resonance, Biomolecular
NMR spectroscopy on small- to medium-size biological macromolecules. This is often used for structural investigation of proteins and nucleic acids, and often involves more than one isotope.
The property of objects that determines the direction of heat flow when they are placed in direct thermal contact. The temperature is the energy of microscopic motions (vibrational and translational) of the particles of atoms.
The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.
A basic enzyme that is present in saliva, tears, egg white, and many animal fluids. It functions as an antibacterial agent. The enzyme catalyzes the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrin. EC 188.8.131.52.
A family of SERINE ENDOPEPTIDASES isolated from Bacillus subtilis. EC 3.4.21.-
Short sequences (generally about 10 base pairs) of DNA that are complementary to sequences of messenger RNA and allow reverse transcriptases to start copying the adjacent sequences of mRNA. Primers are used extensively in genetic and molecular biology techniques.
Hydrophobic and Hydrophilic Interactions
A field of biology concerned with the development of techniques for the collection and manipulation of biological data, and the use of such data to make biological discoveries or predictions. This field encompasses all computational methods and theories for solving biological problems including manipulation of models and datasets.
Mixed Function Oxygenases
Widely distributed enzymes that carry out oxidation-reduction reactions in which one atom of the oxygen molecule is incorporated into the organic substrate; the other oxygen atom is reduced and combined with hydrogen ions to form water. They are also known as monooxygenases or hydroxylases. These reactions require two substrates as reductants for each of the two oxygen atoms. There are different classes of monooxygenases depending on the type of hydrogen-providing cosubstrate (COENZYMES) required in the mixed-function oxidation.
Protein Structure, Quaternary
The characteristic 3-dimensional shape and arrangement of multimeric proteins (aggregates of more than one polypeptide chain).
A molecule that binds to another molecule, used especially to refer to a small molecule that binds specifically to a larger molecule, e.g., an antigen binding to an antibody, a hormone or neurotransmitter binding to a receptor, or a substrate or allosteric effector binding to an enzyme. Ligands are also molecules that donate or accept a pair of electrons to form a coordinate covalent bond with the central metal atom of a coordination complex. (From Dorland, 27th ed)
Magnetic Resonance Spectroscopy
DNA molecules capable of autonomous replication within a host cell and into which other DNA sequences can be inserted and thus amplified. Many are derived from PLASMIDS; BACTERIOPHAGES; or VIRUSES. They are used for transporting foreign genes into recipient cells. Genetic vectors possess a functional replicator site and contain GENETIC MARKERS to facilitate their selective recognition.
Sequence Analysis, Protein
A process that includes the determination of AMINO ACID SEQUENCE of a protein (or peptide, oligopeptide or peptide fragment) and the information analysis of the sequence.
A chemical reaction in which an electron is transferred from one molecule to another. The electron-donating molecule is the reducing agent or reductant; the electron-accepting molecule is the oxidizing agent or oxidant. Reducing and oxidizing agents function as conjugate reductant-oxidant pairs or redox pairs (Lehninger, Principles of Biochemistry, 1982, p471).
Tools or devices for generating products using the synthetic or chemical conversion capacity of a biological system. They can be classical fermentors, cell culture perfusion systems, or enzyme bioreactors. For production of proteins or enzymes, recombinant microorganisms such as bacteria, mammalian cells, or insect or plant cells are usually chosen.
Electrophoresis, Polyacrylamide Gel
Materials fabricated by BIOMIMETICS techniques, i.e., based on natural processes found in biological systems.
Organic compounds that generally contain an amino (-NH2) and a carboxyl (-COOH) group. Twenty alpha-amino acids are the subunits which are polymerized to form proteins.
A low-energy attractive force between hydrogen and another element. It plays a major role in determining the properties of water, proteins, and other compounds.
Enzymes that catalyze the hydrolysis of ester bonds within RNA. EC 3.1.-.
A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine).
Escherichia coli Proteins
Proteins obtained from ESCHERICHIA COLI.
Guided Tissue Regeneration
Procedures for enhancing and directing tissue repair and renewal processes, such as BONE REGENERATION; NERVE REGENERATION; etc. They involve surgically implanting growth conducive tracks or conduits (TISSUE SCAFFOLDING) at the damaged site to stimulate and control the location of cell repopulation. The tracks or conduits are made from synthetic and/or natural materials and may include support cells and induction factors for CELL GROWTH PROCESSES; or CELL MIGRATION.
Amino Acid Motifs
Commonly observed structural components of proteins formed by simple combinations of adjacent secondary structures. A commonly observed structure may be composed of a CONSERVED SEQUENCE which can be represented by a CONSENSUS SEQUENCE.
An interdisciplinary field in materials science, ENGINEERING, and BIOLOGY, studying the use of biological principles for synthesis or fabrication of BIOMIMETIC MATERIALS.
Immunoglobulin molecules having a specific amino acid sequence by virtue of which they interact only with the ANTIGEN (or a very similar shape) that induced their synthesis in cells of the lymphoid series (especially PLASMA CELLS).