Field Of Protein Engineering Research Articles

Fluorine in Protein Environments: A QM and MD Study

Noncanonical amino acids with newly designed side-chain functionalities represent powerful tools to improve structural, biological, and pharmacological properties of peptides and proteins. In this context, fluorinated amino acids have increasingly gained importance. Despite the current wide use of fluorination in protein engineering, the basic properties of fluorine in protein environments are still not completely understood. Our aim has been to characterize the physicochemical properties of fluorinated amino acids by using quantum mechanics (QM) and molecular dynamics (MD) approaches. We have analyzed geometry, charges, and hydrogen bonding abilities of several ethane fluorinated derivatives at different QM theory levels and have used them as simplified models for fluorinated amino acid side chains. We have parametrized four fluorinated L-amino acids for the AMBER ff94/99 force field: 4-monofluoroethylglycine (MfeGly), 4,4-difluoroethylglycine (DfeGly), 4,4,4-trifluoroethylglycine (TfeGly), and 4,4-difluoropropylglycine (DfpGly). We have characterized them in terms of molecular volumes, conformational preferences, and hydration properties. The obtained results illustrate that fluorine and hydrogen atoms of fluoromethyl groups could be potential acceptors or donors of weak hydrogen bonds in protein environments. Hydration of the studied fluorinated amino acids was found to be more favorable than for their nonfluorinated analogues, and hydrophobicity was observed to increase with the number of fluorine atoms, which is in accordance with the experimental retention times we obtain for these amino acids. This study broadens our understanding of the properties of fluorine within protein environments, which is important to exploit the full potential of fluorine's unique properties for applications in the field of protein engineering.

Optimizing nucleotide sequence ensembles for combinatorial protein libraries using a genetic algorithm

Protein libraries are essential to the field of protein engineering. Increasingly, probabilistic protein design is being used to synthesize combinatorial protein libraries, which allow the protein engineer to explore a vast space of amino acid sequences, while at the same time placing restrictions on the amino acid distributions. To this end, if site-specific amino acid probabilities are input as the target, then the codon nucleotide distributions that match this target distribution can be used to generate a partially randomized gene library. However, it turns out to be a highly nontrivial computational task to find the codon nucleotide distributions that exactly matches a given target distribution of amino acids. We first showed that for any given target distribution an exact solution may not exist at all. Formulated as a constrained optimization problem, we then developed a genetic algorithm-based approach to find codon nucleotide distributions that match as closely as possible to the target amino acid distribution. As compared with the previous gradient descent method on various objective functions, the new method consistently gave more optimized distributions as measured by the relative entropy between the calculated and the target distributions. To simulate the actual lab solutions, new objective functions were designed to allow for two separate sets of codons in seeking a better match to the target amino acid distribution.

RASMOT-3D PRO: a 3D motif search webserver

Detection of structural motif of residues in protein structures allows identification of structural or functional similarity between proteins. In the field of protein engineering, structural motif identification is essential to select protein scaffolds on which a motif of residues can be transferred to design a new protein with a given function. We describe here the RASMOT-3D PRO webserver (http://biodev.extra.cea.fr/rasmot3d/) that performs a systematic search in 3D structures of protein for a set of residues exhibiting a particular topology. Comparison is based on Cα and Cβ atoms in two steps: inter-atomic distances and RMSD. RASMOT-3D PRO takes in input a PDB file containing the 3D coordinates of the searched motif and provides an interactive list of identified protein structures exhibiting residues of similar topology as the motif searched. Each solution can be graphically examined on the website. The topological search can be conducted in structures described in PDB files uploaded by the user or in those deposited in the PDB. This characteristic as well as the possibility to reject scaffolds sterically incompatible with the target, makes RASMOT-3D PRO a unique webtool in the field of protein engineering.

Supramolecular Control of Split-GFP Reassembly by Conjugation of β-Cyclodextrin and Coumarin Units

The design of proteins whose structure and function can be manipulated by binding with specific ligands has been of great interest in the field of protein engineering. Some successful examples of small-molecule-dependent proteins have been reported, but their ligand-binding domains have mainly been limited to those derived from natural proteins. The introduction of synthetic components for ligand responsiveness may expand the versatility of small-molecule-dependent proteins. In this study, we designed and constructed a fragmented green fluorescent protein (split GFP) whose reassembly could be modulated by the non-natural supramolecular interaction. In the design of split GFP, beta-cyclodextrin (betaCDx) and coumarin units were introduced into a C-terminal fragment (residues 214-230) of split GFP. The C-terminal peptide with betaCDx and coumarin, DC-M2-betaCDx, which contains both host and guest moieties in the same peptide chain, formed an intramolecular inclusion complex in the absence of exogenous guest molecules. This interaction strongly inhibited reconstitution with the GFP N-terminal fragment (residues 2-214) (GFP 1-10 OPT). However, the addition of suitable guest molecules for betaCDx terminated the intramolecular host-guest interaction in the C-terminal peptide, leading to reassembly of the protein fragments and concomitant fluorescence recovery due to the formation of mature GFP. These results successfully demonstrated direct control of protein structure and function by application of synthetic supramolecular interaction to a fragmented protein. The combined system of fragmented protein and synthetic supramolecular elements is expected to be a useful and flexible strategy for regulation of protein structure and function via binding to synthetic ligands.

Plasmid incompatibility: more compatible than previously thought?

It is generally accepted that plasmids containing the same origin of replication are incompatible. We have re-examined this concept in terms of the plasmid copy number, by introducing plasmids containing the same origin of replication and different antibiotic resistance genes into bacteria. By selecting for resistance to only one antibiotic, we were able to examine the persistence of plasmids carrying resistances to other antibiotics. We find that plasmids are not rapidly lost, but are able to persist in bacteria for multiple overnight growth cycles, with some dependence upon the nature of the antibiotic selected for. By carrying out the experiments with different origins of replication, we have been able to show that higher copy number leads to longer persistence, but even with low copy plasmids, persistence occurs to a significant degree. This observation holds significance for the field of protein engineering, as the presence of two or more plasmids within bacteria weakens, and confuses, the connection between screened phenotype and genotype, with the potential to wrongly assign specific phenotypes to incorrect genotypes.

分子計算のための一点から開始される探索法

This paper discusses DNA-based stochastic optimizations under the constraint that the search starts from a given point in a search space. Generally speaking, a stochastic optimization method explores a search space and finds out the optimum or a sub-optimum after many cycles of trials and errors. This search process could be implemented efficiently by ``molecular computing'', which processes DNA molecules by the techniques of molecular biology to generate and evaluate a vast number of solution candidates at a time. We assume the exploration starting from a single point, and propose a method to embody DNA-based optimization under this constraint, because this method has a promising application in the research field of protein engineering. In this application, a string of nucleotide bases (a base sequence) encodes a protein possessing a specific activity, which could be given as a value of an objective function. Thus, a problem of obtaining a protein with the optimum or a sub-optimum about the desired activity corresponds to a combinatorial problem of obtaining a base sequence giving the optimum or a sub-optimum in the sequence space. Biologists usually modify a base sequence corresponding to a naturally occurring protein into another sequence giving a desired activity. In other words, they explore the space in the proximity of a natural protein as a start point. We first examined if the optimization methods that involve a single start point, such as simulated annealing, Gibbs sampler, and MH algorithms, can be implemented by DNA-based operations. Then, we proposed an application of genetic algorithm, and examined the performance of this application on a model fitness landscape by computer experiments. These experiments gave helpful guidelines in the embodiments of DNA-based stochastic optimization, including a better design of crossover operator.

Synthetic zinc finger peptides: old and novel applications.

In the last decade, the efforts in clarifying the interaction between zinc finger proteins and DNA targets strongly stimulated the creativity of scientists in the field of protein engineering. In particular, the versatility and the modularity of zinc finger (ZF) motives make these domains optimal building blocks for generating artificial zinc finger peptides (ZFPs). ZFPs can act as transcription modulators potentially able to control the expression of any desired gene, when fused to an appropriate effector domain. Artificial ZFPs open the possibility to re-program the expression of specific genes at will and can represent a powerful tool in basic science, biotechnology and gene therapy. In this review we will focus on old, novel and possible future applications of artificial ZFPs.

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.

A structural double-mutant cycle: estimating the strength of a buried salt bridge in barnase

Double-mutant cycles are widely used in the field of protein engineering to measure intermolecular and intramolecular interactions. Ideally, there should be no structural rearrangement of the protein on making the two single mutations and the double mutation within the cycle. However, structural pertubation on mutation does not preclude the use of this method, providing the sum of the changes in the single mutants equals the change in the double mutant. In this way, the energy associated with any structural rearrangement cancels in the double-mutant cycle. Previously, the contribution of a buried salt bridge between Arg69 and Asp93 in barnase to the stability of the folded protein has been determined by double-mutant cycle analysis. In order to determine whether the measured interaction of -14.0 kJ mol(-1) represents the true interaction energy, the crystal structure of each mutant within the double-mutant cycle was solved. Although mutation results in structural shifts, the majority of those in the single mutants are also found in the double mutant; their energetic effects in the double-mutant cycle are therefore cancelled. This study highlights the robust nature of the double-mutant cycle analysis.

Exploration of sequence space for protein engineering.

The process of protein engineering is currently evolving towards a heuristic understanding of the sequence-function relationship. Improved DNA sequencing capacity, efficient protein function characterization and improved quality of data points in conjunction with well-established statistical tools from other industries are changing the protein engineering field. Algorithms capturing the heuristic sequence-function relationships will have a drastic impact on the field of protein engineering. In this review, several alternative approaches to quantitatively assess sequence space are discussed and the relatively few examples of wet-lab validation of statistical sequence-function characterization/correlation are described.

Molecular Mechanisms for Antibody-Mediated Modulation of Peptide-Displaying Enzyme Sensors

The generation of molecular sensors based on peptide-displaying enzymes for the detection of antibodies or antigens represents an innovative field of protein engineering. The knowledge of the underlying molecular mechanisms of enzymatic modulation in such sensors would be of great importance for the rational construction and improvement of responsiveness of new peptide–enzyme molecules. Here we analyze the enzymatic characteristics of three different kinds of sensors based in engineered β-galactosidase, alkaline phosphatase and β-lactamase, to explore a common activation basis. We describe two different categories of enzyme sensors. In one of them, including only some modified β-lactamases, the enzymatic activity is inhibited upon ligand binding and it seems to be caused by the steric coverage of the active site by the bound antibody. In a second group, embracing members of the three studied enzymes, the ability to be modulated upon effector binding depends on the ratio between the kcat of the engineered enzyme and the kcat of the intact enzyme. This proves a common mechanism for enzymatic modulation of enzyme biosensors that is probably caused by conformational effects induced by the bound antibody on the enzyme.

A Method of Protein Molecular Surface Comparison Based on Geometric Hashing

The results of recent researches in protein engineering field have clarified that the function of a protein depends on not only its structure [2] but its molecular surface [1]. Therefore, extracting relationships between the surface and the function is an important subject for medical chemistry, chemical industry, agricultural engineering, and so on. On the other hand, the proteins that have the same function, but different surfaces have been discovered. This fact suggests that a protein surface can be divided into an active site, which determines the protein function, and a non-active site. Therefore, we propose a method to compare 3D molecular surface data of proteins, which have information of the shape and the material features. Molecular surface data consists of a set of thousands of vertexes, which includes material feature data such as electrostatic potential and hydrophobic property. As the number of vertexes is very large, in this method, in order to reduce calculation time to compare molecular surface data, geometric hashing method [3], which finds the relationship between two pictures effectively by using a hash table, is applied.

New strategies of protein engineering—directed evolution of enzymein vitro

New and highly effective strategies for directed enzyme evolutionin vitro have been developed in the protein engineering field. They allow engineering all kinds of enzymesin vitro so that new ones with novel functions and features can be obtained by the methods of errorprone PCR, DNA shuffling (exon shuffling), hybrid enzyme, random-primingin vitro recombination (RPR), stagger extension process (StEP), random-directed mutagenesisin vitro, etc., even with little knowlodge of spatial structure and catalytic mechanism of enzymes in advance. The process that would take millions of years in nature can in principle be accomplished in the test within several years.

Capillary zone electrophoresis with optimized temperature control for studying thermal denaturation of proteins at various pH.

Capillary electrophoresis (CE) was used to analyze the thermal denaturation of bovine beta-lactoglobulin at different pH. This model protein exhibits complex pH- and temperature association/dissociation dependence balances in its quaternary structure. The study was possible after modification and improvement of a capillary electrophoresis apparatus. The improvement allowed both efficient control (temperature fluctuations <0.05 degrees C) and accurate measurement of the temperature (+/- 0.1 degrees C) within the capillary cartridge. CE allowed the thermodynamic parameters of beta-lactoglobulin thermal denaturation to be estimated. The transition temperature, Tm, was determined at acidic, neutral and alkaline pH. Van't Hoff analysis was performed through direct measurement of native and unfolded protein populations in the slow-time regime. This allowed estimation of thermodynamic parameters (deltaH, deltaS, deltaCp). Finally, the stability curve, i.e., the temperature dependence of the free energy change (deltaG) of protein unfolding was drawn. The accuracy of the parameters values compares with parameters obtained by calorimetric measurements. The available parameters and the requirement of minute amount of protein sample are of potential interest in the field of protein engineering and biological pharmaceuticals. Accordingly, CE can be proposed as a convenient tool to study protein stability and denaturation processes.

Adding 'splice' to protein engineering.

The field of protein engineering has always attracted a multidisciplinary cast of characters ranging from biologists to organic chemists. Consequently, a variety of different protein engineering techniques have been described which encompass the purely synthetic and the purely biosynthetic. Of these, heterologous expression of recombinantly cloned genes is by far the most commonly employed route to engineered proteins — in part because the approach is readily accessible to most researchers. Although site-directed mutagenesis is facilitated by the ‘replicative fidelity and efficiency of living organisms’ [1], this of course also constrains the approach to the naturally occurring amino acids. This limitation is largely overcome by elegant chemistry-driven approaches, such as unnatural amino acid mutagenesis [2] and protein total or semi-synthesis via the chemical [3–9] or enzymatic [10–13] ligation of unprotected peptides. These approaches are, however, technically demanding, a feature which has prevented their widespread use by the biological community. Recently, a novel protein semi-synthesis approach was reported [14–16] that harnesses the phenomenon of protein splicing (for review see [17]), and in the process renders proteins harboring unnatural building blocks accessible to most researchers. This short review describes the genesis of this methodology, termed ‘expressed protein ligation’ [14,15], its applications thus far and its future potential in protein engineering.

Redesigning secondary structure to invert coenzyme specificity in isopropylmalate dehydrogenase.

Rational engineering of enzymes involves introducing key amino acids guided by a knowledge of protein structure to effect a desirable change in function. To date, all successful attempts to change specificity have been limited to substituting individual amino acids within a protein fold. However, the infant field of protein engineering will only reach maturity when changes in function can be generated by rationally engineering secondary structures. Guided by x-ray crystal structures and molecular modeling, site-directed mutagenesis has been used to systematically invert the coenzyme specificity of Thermus thermophilus isopropylmalate dehydrogenase from a 100-fold preference for NAD to a 1000-fold preference for NADP. The engineered mutant, which is twice as active as wild type, contains four amino acid substitutions and an alpha-helix and loop that replaces the original beta-turn. These results demonstrate that rational engineering of secondary structures to produce enzymes with novel properties is feasible.

Free energy perturbation techniques applied to subtilisin BPN' stability.

The inability to predict the effect of specific mutations on protein stability has been an area of concern to researchers in the field of protein engineering. Small stabilization free energies (5 to 15 kcal/mol) distinguish the native and the denatured states of a protein, making the rational design of protein stability a difficult challenge. Free Energy Perturbation Technique (FEPT) appears to be a method that will be important for protein engineering to meet this challenge. Not only is it a method to evaluate potential sites for mutation prior to synthesis, it identifies important atomic contributions that are responsible for the free energy changes of interest. Accuracy and speed are the principal limitations of the technique, but the powerful combination of structure, energy, dynamics and solvent make the investment of time and effort very attractive. Our examples illustrate both the power and limitations of FEPT. Using the program CHARMm, FEPT has been applied to the well known stabilizing mutations--asparagine to Serine--at residue 218 in subtilisin BPN'. In analyzing the atomic contributions that result in the increase in stability, two mutations at residue 203 were chosen to test the predictive power of FEPT. Sometimes extraordinary measures must be undertaken to sample sufficient conformational space to achieve accurate FEPT results. However we believe the method will be invaluable in the development of rules for designing a more stable subtilisin BPN'.

Protein Folding: New Twists

The solution of the “protein folding problem” is critical to the burgeoning field of protein engineering and design. In this review, we discuss some developments that prompt us to seek an explicit stereo–chemical code for protein assembly. Our analysis leads to the conclusion that, to a good approximation, protein molecules are structural hierarchies of just four types of primitive folding units—including a novel unit called the Ω–loop. This conclusion, in turn, suggests a model of protein assembly called hierarchic condensation in which these nascent structural primitives associate in step–wise fashion, leading to larger modules and further association. Hierarchic organization is a familiar and simplifying pattern in the macroscopic world, so the existence of hierarchic architecture in proteins is a tantalizing finding. Despite their apparent structural complexity, protein molecules may yet turn out to be governed by stereo–chemical rules that are remarkably simple.