Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Protein Engineering”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 865 records · Page 48Linked to original sources
PubMedDate not supplied

Receptins: a novel term for an expanding spectrum of natural and engineered microbial proteins with binding properties for mammalian proteins.

A new term 'receptin', derived from recipere (lat.), is proposed to denote microbial binding proteins that interact with mammalian target proteins. An example of such a 'receptin' is staphyloccocal protein A which binds to the Fc part of many mammalian immunoglobulins. Several other types of 'receptins' are listed. This term may easily be distinguished from the similar term 'receptor', describing a binding site on a cell surface, mostly eukaryotic, where a secondary effect is induced inside the cell upon binding to a ligand. A receptin, however, does not necessarily have to induce a secondary event. Receptins include so called MSCRAMMs, adhesins, and also engineered receptins, affibodies, and engineered ligands. It denotes any protein of microbial origin, cell-bound or soluble, which can bind to a mammalian protein. It fulfills the need for an umbrella terminology for a large group of binding structures. In contrast, the term 'lectin' represents a group of proteins with affinity for carbohydrate structures. The new term 'receptin' includes a number of key microbial proteins involved in host-parasite interactions and in virulence. Some receptins are promising vaccine candidates.

Animals↗
PubMed1995

Understanding and increasing protein stability.

This review surveys the processes leading to loss of protein function and the types of molecular interaction that help stabilize proteins. It considers the effects of organic solvents on stability and the special features of thermophilic proteins. The deliberate manipulation of stability by protein engineering is discussed using the enzyme subtilisin as example. Both random and rational mutations of this protein have led to variants with greatly improved tolerances of high temperatures and organic solvents. One can also use chemical modification to modify protein stability and some of the main approaches are reviewed. The chemical and genetic strategies are complementary and have been combined to stabilize cytochrome c by metal-mediated cross-linking following site-specific mutagenesis. The article concludes by summarizing the beneficial effects of certain additives on protein stability.

Enzyme Stability↗
PubMed2002

A simple method for probing the mechanical unfolding pathway of proteins in detail.

Atomic force microscopy is an exciting new single-molecule technique to add to the toolbox of protein (un)folding methods. However, detailed analysis of the unfolding of proteins on application of force has, to date, relied on protein molecular dynamics simulations or a qualitative interpretation of mutant data. Here we describe how protein engineering Phi value analysis can be adapted to characterize the transition states for mechanical unfolding of proteins. Single-molecule studies also have an advantage over bulk experiments, in that partial Phi values arising from partial structure in the transition state can be clearly distinguished from those averaged over alternate pathways. We show that unfolding rate constants derived in the standard way by using Monte Carlo simulations are not reliable because of the errors involved. However, it is possible to circumvent these problems, providing the unfolding mechanism is not changed by mutation, either by a modification of the Monte Carlo procedure or by comparing mutant and wild-type data directly. The applicability of the method is tested on simulated data sets and experimental data for mutants of titin I27.

Animals↗
PubMed2005

Inductive tissue engineering with protein and DNA-releasing scaffolds.

Cellular differentiation, organization, proliferation and apoptosis are determined by a combination of an intrinsic genetic program, matrix/substrate interactions, and extracellular cues received from the local microenvironment. These molecular cues come in the form of soluble (e.g. cytokines) and insoluble (e.g. ECM proteins) factors, as well as signals from surrounding cells that can promote specific cellular processes leading to tissue formation or regeneration. Recent developments in the field of tissue engineering have employed biomaterials to present these cues, providing powerful tools to investigate the cellular processes involved in tissue development, or to devise therapeutic strategies based on cell replacement or tissue regeneration. These inductive scaffolds utilize natural and/or synthetic biomaterials fabricated into three-dimensional structures. This review summarizes the use of scaffolds in the dual role of structural support for cell growth and vehicle for controlled release of tissue inductive factors, or DNA encoding for these factors. The confluence of molecular and cell biology, materials science and engineering provides the tools to create controllable microenvironments that mimic natural developmental processes and direct tissue formation for experimental and therapeutic applications.

Animals↗
PubMed1989

A type II DNA-binding protein genetically engineered for fluorescence spectroscopy: the "arm" of transcription factor 1 binds in the DNA grooves.

We examined the fluorescence properties of a mutant TF1 protein (transcription factor 1; a member of the type II class of DNA-binding proteins, DBPII) containing tryptophan in place of phenylalanine (TF1-W61) at position 61 in the "arms" of the protein dimer. The time-resolved fluorescence (excited at 295 nm) of Trp61 decays as a double exponential with lifetimes and amplitudes that are comparable to those found in other tryptophan-containing proteins and peptides, and the time-resolved fluorescence polarization decay indicates that the tryptophan residue possesses considerable internal flexibility, in agreement with crystal studies of the homologous HU protein. The tryptophan emission is quenched when TF1-W61 binds to DNA, and equilibrium studies based on fluorescence show that the nonspecific binding affinity of the TF1-W61 mutant to DNA is similar to that of wild-type TF1. Comparison of the time-resolved fluorescence decay and steady-state fluorescence intensity reveals at least two general classes of Trp61 in the DNA complexes. One class of tryptophans is partially quenched, and the extent of quenching in the complexes with various natural DNAs and synthetic double-stranded polynucleotides correlates with the spectral overlap between tryptophan emission and DNA absorption, indicating that through-space excitation energy transfer contributes to the observed quenching. Comparisons between experimentally determined energy transfer rates and model calculations suggest that the Trp61 is located in one of the DNA grooves at a distance of less than 7.5 A from the DNA helix axis. The second class of Trp61 is "totally" quenched, and we attribute this to tryptophan residues that are in direct contact with the DNA bases.(ABSTRACT TRUNCATED AT 250 WORDS)

Codon↗
PubMed2001

Hydrogen-bond disruption probability in proteins by a modified self-consistent harmonic approach.

Modified self-consistent harmonic approach was employed to calculate the probability for the disruption of each individual hydrogen bonds (H bonds) in x-ray crystal structure of several proteins. The computed probability for 82% of intraprotein and water-protein H bonds studied were found to be roughly consistent with estimated free energies from protein engineering and hydrogen exchange experiments. Hydrogen bonds have been proposed as part of a stereochemical code for protein folding. Proteins fold into unique three-dimensional structures; therefore those bonds involved in the folding code are expected to be stable. We have applied this method to tens of hydrogen bonds in a protein assumed to be involved in the folding code of a protein. 58% of these H bonds were found to have a lower disruption probability (-1.8 kcal/mol). Our results showed that modified self-consistent harmonic approach might be explored as a method supplement to existing methods in analysis of hydrogen bonds in proteins.

Algorithms↗
PubMed2005

N-terminal labeling of proteins using initiator tRNA.

Methodology based on tRNA mediated protein engineering is described for the introduction of fluorophores and other labels at the N-terminus of proteins produced in cell-free translation systems. One method for low-level (trace) N-terminal labeling is based on the use of an Escherichia coli initiator tRNA(fMet) misaminoacylated with methionine modified at the alpha-amino group. In addition to the normal formyl group, the protein translational machinery incorporates the fluorophore BODIPY-FL and the affinity tag biotin at an N-terminal end of the nascent protein. A second method for higher N-terminal labeling uses a chemically aminoacylated amber initiator suppressor tRNA and a DNA template which contains a complementary amber (UAG) codon instead of the normal initiation (AUG) codon. This more versatile approach is demonstrated using a variety of N-terminal markers including fluorescein, biotin, PC-biotin, and a novel dual marker conjugate (Biotin/BODIPY-FL).

Escherichia coli↗
PubMed2005

Chemical biology of protein lipidation: semi-synthesis and structure elucidation of prenylated RabGTPases.

Rab/Ypt guanosine triphosphatases (GTPases) represent a family of key membrane traffic regulators in eukaryotic cells. For their function Rab/Ypt proteins require double modification with two covalently bound geranylgeranyl lipid moieties at the C-terminus. Generally, prenylated proteins are very difficult to obtain by recombinant or enzymatic methods. We generated prenylated RabGTPases using a combination of chemical synthesis and protein engineering. This semi-synthesis depends largely on the availability of functionalized prenylated peptides corresponding to the proteins' native structure or modifications. We developed solution phase and solid phase strategies for the generation of peptides corresponding to the prenylated C-terminus of Rab7 GTPase in preparative amounts enabling us to crystallize the mono-prenylated Ypt1:RabGDI complex. The structure of the complex provides a structural basis for the ability of RabGDI to inhibit the release of nucleotide by Rab proteins and a molecular basis for understanding a RabGDI mutant that causes mental retardation in humans.

Models, Molecular↗
PubMed2004

Beyond consensus: statistical free energies reveal hidden interactions in the design of a TPR motif.

Consensus design methods have been used successfully to engineer proteins with a particular fold, and moreover to engineer thermostable exemplars of particular folds. Here, we consider how a statistical free energy approach can expand upon current methods of phylogenetic design. As an example, we have analyzed the tetratricopeptide repeat (TPR) motif, using multiple sequence alignment to identify the significance of each position in the TPR. The results provide information above and beyond that revealed by consensus design alone, especially at poorly conserved positions. A particularly striking finding is that certain residues, which TPR-peptide co-crystal structures show are in direct contact with the ligand, display a marked hypervariability. This suggests a novel means of identifying ligand-binding sites, and also implies that TPRs generally function as ligand-binding domains. Using perturbation analysis (or statistical coupling analysis), we examined site-site interactions within the TPR motif. Correlated occurrences of amino acid residues at poorly conserved positions explain how TPRs achieve their near-neutral surface charge distributions, and why a TPR designed from straight consensus has an unusually high net charge. Networks of interacting sites revealed that TPRs fall into two unrecognized families with distinct sets of interactions related to the identity of position 7 (Leu or Lys/Arg). Statistical free energy analysis provides a more complete description of "What makes a TPR a TPR?" than consensus alone, and it suggests general approaches to extend and improve the phylogenetic design of proteins.

Amino Acid Motifs↗
PubMed2002

Consensus-derived structural determinants of the ankyrin repeat motif.

The ankyrin repeat is one of the most common, modular, protein-protein interaction motifs in nature. To understand the structural determinants of this family of proteins and extract the consensus information that defines the architecture of this motif, we have designed a series of idealized ankyrin repeat proteins containing one, two, three, or four repeats by using statistical analysis of approximately 4,000 ankyrin repeat sequences from the PFAM database. Biophysical and x-ray crystallographic studies of the three and four repeat constructs (3ANK and 4ANK) to 1.26 and 1.5 A resolution, respectively, demonstrate that these proteins are well-folded, monomeric, display high thermostability, and adopt a very regular, tightly packed ankyrin repeat fold. Mapping the degree of amino acid conservation at each position on the 4ANK structure shows that most nonconserved residues are clustered on the surface of the molecule that has been designated as the binding site in naturally occurring ankyrin repeat proteins. Thus, the consensus amino acid sequence contains all information required to define the ankyrin repeat fold. Our results suggest that statistical analysis and the consensus sequence approach can be used as an effective method to design proteins with complex topologies. These generic ankyrin repeat proteins can serve as prototypes for dissecting the rules of molecular recognition mediated by ankyrin repeats and for engineering proteins with novel biological functions.

Amino Acid Sequence↗
PubMed2003

The first semi-synthetic serine protease made by native chemical ligation.

Selective incorporation of non-natural amino acid residues into proteins is a powerful approach to delineate structure-function relationships. Although many methodologies are available for chemistry-based protein engineering, more facile methods are needed to make this approach suitable for routine laboratory practice. Here, we describe a new strategy and provide a proof of concept for engineering semi-synthetic proteins. We chose a serine protease Streptomyces griseus trypsin (SGT) for this study to show that it is possible to efficiently couple a synthetic peptide containing a catalytically critical residue to a recombinant fragment containing the other active site residues. The 223-residue hybrid SGT molecule was prepared by fusing a chemically synthesized N-terminal peptide to a large C-terminal fragment of recombinant origin using native chemical ligation. This C-terminal polypeptide was produced from full-length SGT by cyanogen bromide cleavage at a genetically engineered Met57 position. This semi-synthetic hybrid trypsin is fully active, showing kinetics identical to the wild-type enzyme. Thus, we believe that it is an ideal model enzyme for studying the catalytic mechanisms of serine proteases by providing a straightforward approach to incorporate non-natural amino acids in the N-terminal region of the protein. In particular, this strategy will allow for replacement of the catalytic His57 residue and the buried N-terminus, which is thought to help align the active site, with synthetic analogs. Our approach relies on readily available recombinant proteins and small synthetic peptides, thus having general applications in chemical engineering of large proteins where the N-terminal region is the focal interest.

Amino Acid Sequence↗
PubMed2001

Intein-mediated ligation and cyclization of expressed proteins.

Protein splicing is a posttranslational processing event that releases an internal protein sequence from a protein precursor. During the splicing process the internal protein sequence, termed an intein, embedded in the protein precursor self-catalyzes its excision and the ligation of the flanking protein regions, termed exteins. The dissection of the splicing pathway, which involves the precise cleavage and formation of peptide bonds, and the identification of key catalytic residues at the splice junctions have led to the modulation of the protein splicing process as a protein engineering tool. Novel strategies have been developed to use intein-catalyzed reactions for the production and manipulation of proteins and peptides. These new approaches have broken down the size limitation barrier of chemical synthetic methods and are less technically demanding. The purpose of this article is to describe how to use self-splicing inteins in protein semisynthesis and backbone cyclization. The first two sections of the article provide a brief review of the distinct chemical steps that underlie protein splicing and intein enabled technology.

Bacterial Proteins↗
PubMed1991

Deletions, fusions and domain rearrangements.

The techniques of protein engineering are proving to be powerful analytical tools for the study of the structure and function of complex multidomain proteins. In particular, the overexpression of individual functional modules is providing proteins for three-dimensional structural analyses. Progress is also being made in the design and construction of novel multidomain proteins with potential therapeutic applications.

Animals↗
PubMed2004

Chemical and biochemical strategies for the randomization of protein encoding DNA sequences: library construction methods for directed evolution.

Directed molecular evolution and combinatorial methodologies are playing an increasingly important role in the field of protein engineering. The general approach of generating a library of partially randomized genes, expressing the gene library to generate the proteins the library encodes and then screening the proteins for improved or modified characteristics has successfully been applied in the areas of protein-ligand binding, improving protein stability and modifying enzyme selectivity. A wide range of techniques are now available for generating gene libraries with different characteristics. This review will discuss these different methodologies, their accessibility and applicability to non-expert laboratories and the characteristics of the libraries they produce. The aim is to provide an up to date resource to allow groups interested in using directed evolution to identify the most appropriate methods for their purposes and to guide those moving on from initial experiments to more ambitious targets in the selection of library construction techniques. References are provided to original methodology papers and other recent examples from the primary literature that provide details of experimental methods.

Base Sequence↗
PubMed1996

Structure of the transition state for folding of the 129 aa protein CheY resembles that of a smaller protein, CI-2.

BACKGROUND: Protein engineering analysis has been used as a tool to determine the structure of the transition state of two different proteins: CI-2 and barnase. CI-2 belongs to the group of small, globular proteins with no disulphide bonds that fold via a two-state mechanism. Barnase is a larger protein (110 aa) and displays a folding intermediate. The structure of the transition state of both proteins is quite different. Whereas in CI-2 no region is fully native and it looks like an expanded form of the folded state, in barnase several regions are folded and the rate-limiting step seems to be the consolidation of the hydrophobic core. On the basis of these results, a unified scheme for the transition state of protein folding has been presented. We decided to characterize the folding pathway, or pathways, present in the alpha/beta parallel family of proteins using one of the smallest members, CheY (129 aa), as a model case. RESULTS: The folding pathway of CheY contains, as does that of barnase, a kinetic intermediate. The picture obtained for CheY from the equilibrium and kinetic analyses of several mutations scattered throughout the whole protein is different from that found for barnase. On the basis of the experimental results and the structure of CheY, the protein can be divided into two subdomains (from beta-strand 1 to beta-strand 3 and from beta-strand 3 to the C terminus). Whereas the structure of the first subdomain in the transition state resembles that found for the CI-2 protein, the second subdomain is compact but unstructured. The packing of the first alpha-helix against beta-strands 1 and 2 seems to be the nucleus around which the rest of the protein folds. CONCLUSIONS: Comparison of the transition state of barnase with those of CheY and CI-2 indicates that different proteins have different transition states, probably depending on the energetics and the position of the rate-limiting step in the folding pathway. CheY appears to fold through a nucleation/condensation mechanism as has been found for CI-2. The rate-determining step in some multimodular proteins could be the formation of a stable domain, with the less stable domains folding after the major rate-determining step.

Amino Acid Sequence↗
PubMed2001

The 1.6 A resolution crystal structure of a mutant plastocyanin bearing a 21-25 engineered disulfide bridge.

Plastocyanin is an electron-transfer protein which has been largely used for biophysical studies as well as for protein-engineering experiments. A surface disulfide bridge has been engineered in poplar plastocyanin to allow protein chemisorption on gold substrates. The mutated plastocyanin crystal structure has been studied at 1.6 A resolution (R factor = 0.145, R(free) = 0.205) to characterize the effects of the engineered disulfide on the overall protein structure and on the Cu-coordination sphere in view of biophysical applications. The new orthorhombic crystal form isolated for the mutated plastocyanin displays two protein molecules per asymmetric unit.

Crystallization↗
PubMed1992

Rapid evolution of peptide and protein binding properties in vitro.

A significant bottleneck in protein engineering arises from the problem of identifying particular molecules with new functions from a potentially enormous range of peptide or protein variants. Two areas of emerging technology, phage display and multiple peptide synthesis, provide new means of screening huge libraries in vitro for novel binding properties. This review is also published in Current Opinion in Structural Biology 1992, 2:597-604.

Bacteriophages↗
PubMed1998

Construction of a family of Cys2His2 zinc binding sites in the hydrophobic core of thioredoxin by structure-based design.

A semi-automated, rational design strategy has been used to introduce a family of seven single, mononuclear Cys2His2 zinc sites at various locations in the hydrophobic core of Escherichia colithioredoxin, a protein that is normally devoid of metal centers. The electronic absorption spectra of the CoII complexes show that five of these designed proteins bind metal with the intended tetrahedral geometry. The designed sites differ in their metal-binding constants and effects on protein stability. Since these designs are constructed within the same host protein framework, comparison of their behavior allows a qualitative evaluation of dominant factors that contribute to metal-binding and metal-mediated protein stabilization. Metal-binding constants are dominated by steric interactions between the buried, designed coordination sphere and the surrounding protein matrix. Metal-mediated stability is the consequence of differential binding to the native and unfolded states. Increased interactions with the unfolded state decrease the stabilizing effect of metal binding. The affinity for the unfolded state is dependent on the placement of the primary coordination sphere residues within the linear protein sequence. These results indicate that a protein fold can have a remarkably broad potential for accommodating metal-mediated cross-links and suggest strategies for engineering protein stability by constructing metal sites that maximize metal binding to the native state and minimize binding to the unfolded state.

Amino Acid Sequence↗