Advances in protein structure prediction and de novo protein design: A review

This review provides an exposition to the important problems of (i) structure prediction in protein folding and (ii) de novo protein design. The recent advances in protein folding are reviewed based on a classification of the approaches in comparative modeling, fold recognition, and first principles methods with and without database information. The advances towards the challenging problem of loop structure prediction and the first principles method, ASTRO-FOLD, along with the developments in the area of force-fields development have been discussed. Finally, the recent progress in the area of de novo protein design is presented with focus on template flexibility, in silico sequence selection, and successful peptide and protein designs.     

Beta-hairpins in proteins revisited: lessons for de novo design

Beta-Hairpins with short connecting loops (1-5 residues) have been identified from a data set of 250 non-homologous, high resolution (< or =2.0 A) protein crystal structures. The conformational preferences of the loop segments have been analyzed with the specific aim of identifying frequently occurring motifs. Type I' and II' beta-turns were found to have a high propensity for occurrence in two residue loops. For three and four residue loops, the major conformational motif in the linking segments is alphaR-alphaR-alphaL (type I beta-turn followed by a residue in a left-handed helical conformation) and alphaR- alphaR-alphaR-alphaL (a pi-turn motif), respectively. The present larger data set confirms the high occurrences of these motifs which have been identified in earlier analyses. In addition to type I' and type II' beta-turns, several examples of type I beta-turn nucleated two residue loop hairpins, in spite of having an opposing sense of twist to that of type I' beta-turn, have also been observed. Examination of these frequently occurring motifs (flanked by extended conformation [beta]) in the data set reveals that the motifs beta-alphaR-alphaR- alphaL-beta and beta-type I'-beta have equal propensity and type II' indeed having highest propensity to nucleate beta-hairpins. The larger number of examples in this study allows the estimation of the specific amino acid preferences for loop positions in two, three and four residue loops. Small polar residues Asn, Asp, Ser, Thr, Gly and Pro in general have a high propensity for the loop positions but they reveal specific positional preferences in these frequently occurring motifs. There are no strong compositional preferences in the strand segments. Amino acid pair correlations across strands also do not show any significant pattern, with the exception of Cys-Cys pairs. Several Cys- Cys pairs have been identified at the non-hydrogen bonded positions of beta-hairpins; as many as six are disulfide bonded pairs. An examination of longer loop length hairpins reveals that the distortions of hairpins nucleated by tight turns (two residues) are much less frequently observed. The results presented in this study provide inputs for the de novo design of consensus loop segments in synthetic hairpins.      

Metal-binding studies for a de novo designed calcium-binding protein

To understand the key determinants in calcium-binding affinity,a calcium-binding site with pentagonal bipyramid geometry wasdesigned into a non-calcium-binding protein, domain 1 of CD2.This metal-binding protein has five mutations with a net chargein the coordination sphere of –5 and is termed DEEEE.Fluorescence resonance energy transfer was used to determinethe metal-binding affinity of DEEEE to the calcium analog terbium.The addition of protein concentration to Tb(III) solution resultsin a large enhancement of Tb(III) fluorescence due to energytransfer between terbium ions and aromatic residues in CD2-D1.In addition, both calcium and lanthanum compete with terbiumfor the same desired metal binding pocket. Our designed proteinexhibits a stronger affinity for Tb(III), with a K_d of 21 µM,than natural calcium-binding proteins with a similar Greek keyscaffold.     

Cooperative deformation of a de novo designed protein

A de novo protein design has been made to understand the uniquepacking of natural proteins that have a ß/-barrelfold. A carefully designed 207 amino acid sequence was synthesizedusing an Escherichia coli expression system and the structuraland thermodynamic characteristics of the purified protein werestudied. At neutral pH the protein is soluble and monomeric,with large amounts of secondary structure and a hydrophobiccore, although the broad resonance peaks of its NMR spectrumsuggest that the designed protein does not have a unique structurewith tightly packed side chains. In an H–D exchange experiment,no amido protons of the designed protein exchanged slowly withdeuterons. At acidic pH, thermal unfolding was observedwitha remarkable change in the excess heat capacity measured directlyby a differential scanning microcalorimeter. The enthalpy andentropy differences at 110°C, extrapolated from analyzedthermodynamic parameters, are 1/3 of the common values for naturalproteins. These measurements indicate that the folding is significantlycooperative as expected, but that the protein is still looselypacked.     

A strategy for the de novo design of helical proteins with stable folds

This paper describes peptide analogs and the design strategythat were used to facilitate the final construction of a denovo-designed protein (ALIN) whose stable tertiary fold hasbeen determined recently by NMR spectroscopy. Previous studieshave suggested that the main problem in the de novo design ofproteins is the attainment of a protein with a defined fold.To effectively overcome this mainchain multiconformation problem,three related steps, with experimental evaluation of the designhypotheses for each step, were pursued in the design process.Firstly, 15-residue sequences with experimentally verified highhelicities were selected for the helical regions. Secondly,hydrophobic and electrostatic interhelical interactions as wellas an interhelical disulfide bridge were designed to favor anantiparallel configuration of the helix axis. Finally, a loopwith sufficient flexibility was inserted to stabilize the helicesin the desired orientation. To assess the design strategy, peptidescorresponding to each design step were synthesized and theirstructures verified experimentally by far-UV CD. As anticipated,ALIN was the most helical, and the SSbridged dimeric peptideswere more helical than their monomeric counterparts. The van'tHoff enthalpy change for ALIN computed from the CD denaturationcurve and assuming a two-state model was 50 kJ/mol, a valueclose to that observed for helical coiled-coils. Overall, thisreport shows that small, simple proteins can be built usingthe current knowledge of protein structures.     

Dietary protein effects on cholelithiasis in hamsters: Interaction with amino acids and bile acids

The objective of this experiment was to study the effects of dietary cottonseed protein and casein on plasma and biliary lipids, plasma amino acids and gallstones in hamsters. Thirty-four male hamsters (60 ± 5 g) were fed either the lithogenic “Dam Diet” (containing 20% casein, 74.3% sucrose and 5.7% vitamin-mineral mix) or a similar diet that contained 20% cottonseed protein for 30 days. Both diets contained protein as a protein isolate. The concentration of alpha-aminobutyric acid was significantly elevated in the casein-fed group. Significant differences in the total plasma cholesterol or lipoprotein cholesterol concentrations were not observed between the two dietary groups. A significant elevation in the absolute concentration of biliary cholesterol was observed in the casein-fed hamsters. Cottonseed protein-fed animals exhibited a significantly elevated concentration of bile acids. The ratio of glycochenodeoxycholic:glycocholic acid was significantly higher in the cotton-seed protein-fed group. This study reports that an elevated concentration of biliary cholesterol with a concomitant decrease in bile acid concentration yields a condition favorable to gallstone formation. It is proposed that cottonseed protein may have a specific effect on the bile acid pool by increasing the ratio of glycochenodeoxycholic acid:glycocholic acid which, in turn, prevents formation of cholesterol gallstones.     

Noncanonical amino acids in the interrogation of cellular protein synthesis

Proteins in living cells can be made receptive to bioorthogonal chemistries through metabolic labeling with appropriately designed noncanonical amino acids (ncAAs). In the simplest approach to metabolic labeling, an amino acid analog replaces one of the natural amino acids specified by the protein's gene (or genes) of interest. Through manipulation of experimental conditions, the extent of the replacement can be adjusted. This approach, often termed residue-specific incorporation, allows the ncAA to be incorporated in controlled proportions into positions normally occupied by the natural amino acid residue. For a protein to be labeled in this way with an ncAA, it must fulfill just two requirements: (i) the corresponding natural amino acid must be encoded within the sequence of the protein at the genetic level, and (ii) the protein must be expressed while the ncAA is in the cell. Because this approach permits labeling of proteins throughout the cell, it has enabled us to develop strategies to track cellular protein synthesis by tagging proteins with reactive ncAAs. In procedures similar to isotopic labeling, translationally active ncAAs are incorporated into proteins during a "pulse" in which newly synthesized proteins are tagged. The set of tagged proteins can be distinguished from those made before the pulse by bioorthogonally ligating the ncAA side chain to probes that permit detection, isolation, and visualization of the labeled proteins. Noncanonical amino acids with side chains containing azide, alkyne, or alkene groups have been especially useful in experiments of this kind. They have been incorporated into proteins in the form of methionine analogs that are substrates for the natural translational machinery. The selectivity of the method can be enhanced through the use of mutant aminoacyl tRNA synthetases (aaRSs) that permit incorporation of ncAAs not used by the endogenous biomachinery. Through expression of mutant aaRSs, proteins can be tagged with other useful ncAAs, including analogs that contain ketones or aryl halides. High-throughput screening strategies can identify aaRS variants that activate a wide range of ncAAs. Controlled expression of mutant synthetases has been combined with ncAA tagging to permit cell-selective metabolic labeling of proteins. Expression of a mutant synthetase in a portion of cells within a complex cellular mixture restricts labeling to that subset of cells. Proteins synthesized in cells not expressing the synthetase are neither labeled nor detected. In multicellular environments, this approach permits the identification of the cellular origins of labeled proteins. In this Account, we summarize the tools and strategies that have been developed for interrogating cellular protein synthesis through residue-specific tagging with ncAAs. We describe the chemical and genetic components of ncAA-tagging strategies and discuss how these methods are being used in chemical biology.     

Comparative nutritional value for amino acids,oligopeptides and soybean protein

In recent years our view on the absorption of protein digestion products has undergone a radical change. We now believe that intralumen hydrolysis of intake proteins to free amino acids is only partial. It has thus been hypothesized that partial protein hydrolysates or oligopeptide mixtures are nutritionally superior to corresponding amino acid mixtures. With this consideration for a background, we developed a sophisticated technique of protease-catalyzed modification of soy protein to produce enzymatically modified proteins (EMP) having different levels of covalently attached methionine. We also carried out feeding tests with protein-malnourished rats, with the result that for their recovery from a state of malnutrition, an EMP containing peptide-bound methionine at 3% was significantly more effective than the corresponding amino acid mixture and even the soy protein itself. Discussions stress the importance of applying such a technique to production of an oligopeptide rather than a free amino acid nitrogen source as a foodstuff for therapeutic use.     

Extraction of amino acids from protein hydrolysates by electrodialysis

Protein hydrolysates were obtained by acid hydrolysis from animal or human residues, such as poultry feathers, ox blood and human hair. After neutralization and discolouration with active charcoal, the hydrolysates were treated by successive electrodialysis (ED) in order to extract amino acids into several fractions. The current density and pH were optimized for each ED operation performed with preindustrial pilot scale equipment. The first step was the demineralization of amino acid mixtures using an ED stack with two compartments. The salt removal was achieved with extraction degrees higher than 90% and current efficiencies of about 80%. In the most favourable case, the amino acid losses did not exceed 10%. The second step was the extraction of the charged amino acids using an ED stack with four compartments. Three fractions were obtained, corresponding to the acidic, basic and neutral amino acids. The extraction degrees varied from 80% to 100%. In the third step, the fractionation of basic amino acids on the one hand, and neutral amino acids on the other hand, was carried out with enrichment degrees varying from 50% to 80%. © 1998 SCI     

Predicting surface exposure of amino acids from protein sequence

The amino acid residues on a protein surface play a key rolein interaction with other molecules, determine many physicalproperties, and constrain the structure of the folded protein.A database of monomeric protein crystal structures was usedto teach computer-simulated neural networks rules for predictingsurface exposure from local sequence. These trained networksare able to correctly predict surface exposure for 72% of residuesin a testing set using a binary model (buried/exposed) and for54% of residues using a ternary model (buried/intermediate/exposed).In the ternary model, only 11% of the exposed residues are predictedas buried and only 5% of the buried residues are predicted asexposed. Also, since the networks are able to predict exposurewith a quantitative confidence estimate, it is possible to assignexposure for over half of the residues in a binary model with>80% accuracy. Even more accurate predictions are obtainedby making a consensus prediction of exposure for a homologousfamily. The effect of the local environment of an amino acidon its accessibility, though smaller than expected, is significantand accounts for the higher success rate of prediction thanobtained with previously used criteria. In the absence of athree-dimensional structure, the ability to predict surfaceaccessibility of amino acids directly from the sequence is avaluable tool in choosing sites of chemical modification orspecific mutations and in studies of molecular interaction.     

Site-directed mutagenesis of Escherichia coli translation initiation factor IF1. Identification of the amino acids involved in its ribosomal binding and recycling

Starting from a synthetic modular gene (infA^*) encoding Escherichiacoli translation initiation factor IF1, we have constructedmutants in which amino acids are deleted from the carboxyl terminusor in which His29 or His34 are replaced by Tyr or Asp residues.The mutant proteins were overproduced, purified and tested invitro for their properties in several partial reactions of thetranslation initiation pathway and for their capacity to stimulateMS2 RNA-dependent protein synthesis. The results allow for theconclusion that: (i) Arg69 is part of the 30S ribosomal subunitbinding site of IF1 and its deletion results in the substantialloss of all IF1 functions; (ii) neither one of its two histidinesis essential for the binding of IF1 to the 30S ribosomal subunit,for the stimulation of fMet-tRNA binding to 30S or 70S ribosomalparticles or for MS2 RNA-dependent protein synthesis; but (iii)His29 is involved in the 50S subunit-induced ejection of IF1from the 30S ribosomal subunit.     

De novo design of helical bundles as models for understanding protein folding and function

De novo protein design has proven to be a powerful tool for understanding protein folding, structure, and function. In this Account, we highlight aspects of our research on the design of dimeric, four-helix bundles. Dimeric, four-helix bundles are found throughout nature, and the history of their design in our laboratory illustrates our hierarchic approach to protein design. This approach has been successfully applied to create a completely native-like protein. Structural and mutational analysis allowed us to explore the determinants of native protein structure. These determinants were then applied to the design of a dinuclear metal-binding protein that can now serve as a model for this important class of proteins.     

Use of propensities of amino acids to the local structural environments to understand effect of substitution mutations on protein stability

Advances in site-directed mutagenesis and other genetic engineeringtechniques have made it possible to create novel proteins of interest. Achallenging aspect of these studies is to understand the effect ofsubstitution mutations on folding and stability of natural proteins. Wepresent an analysis of protein structure data, available from theliterature, for which substitution mutations have been made and changes instability characteristics are reported. Amino acid structural environmentparameters have been computed for a set of 304 non- homologous bestresolved protein structures. The structural environment parameters wereused to calculate each of the 20 amino acid propensities to a givenstructural environment. The observed increase or decrease in stability uponmutation was found to be correlated with the average residue structuralenvironment propensity of wild-type residue versus mutant residue. Theanalysis presented here helps identification of less optimally placedresidues in a given protein structure, and suggests possible substitutionmutations to a residue with higher propensity to the corresponding localstructural environment. We propose that such substitution mutations,suggested based on amino acid propensities to local structuralenvironments, should bestow higher stability to the protein structure.     

The de novo protein with grafted biological function: transferring of interferon blast-transforming activity to albebetin

The de novo protein albebetin has been designed recently toform a predetermined tertiary fold that has not yet been observedin natural proteins. An eight amino acid fragment (131–138)of human interferon ₂ carrying the blasttransforming activityof the protein was attached to the N-terminus of albebetin nextto its initiatory methionine residue. The gene of chimeric proteinwas expressed in a wheat germ cell-free translation system andsynthesized protein was tested for its compactness and stability.Its ability for receptor binding was also studied. We have shownthat albebetin with attached octapeptide is practically as compactas natural proteins of corresponding molecular weight and possesseshigh stability toward the urea-induced unfolding. It binds murinethymocyte receptor at a high affinity and activates the thymocyteblast transformation efficiently at a concentration of 10^-11M.     

Radiation chemical studies of protein reactions: Effect of amino acids on optical rotation

Monosodium 1-glutamate, disodium inosine-5′-monophosphate, disodium guanosine-5′-monophosphate, calcium d-pantothanate, 1-arginine, and 1-aspartic acid were found to protect the changes in the internal relationships of the atoms in the protein molecule from radiation damage. The behavior of the optical rotation was determined. The empirical equation for the protective effect is given by [α] _f = b ? a log X, where [α] f is the final specific rotation of the solution, X is the concentration of the amino acids, and a and b are adjustable constants.     

Computer-based de novo design, synthesis, and evaluation of boronic acid-based artificial receptors for selective recognition of dopamine

Dopamine is an important neurotransmitter that plays important roles in various physiological and pathological processes, such as Parkinson's disease. Chemosensors for dopamine have a number of potential applications. On the basis both of the strong and reversible complexation between the boronic acid moiety and a diol functional group and computational chemistry studies, we have designed a series of four compounds for selective three-point recognition of dopamine, which include boronic acid-diol complexation, aromatic-hydrophobic interactions, and ionic interactions between a carboxylate and a protonated amino group. These compounds were synthesized in seven or eight linear steps and showed dopamine selectivity of up to tenfold over epinephrine. NMR spectroscopy experiments were conducted to probe the structures of the receptor-dopamine complexes. These receptors are the first to show such significant selectivity for dopamine over epinephrine in aqueous solution under near physiological conditions.     

Identification by site-directed mutagenesis of amino acids in the subsite of bovine pancreatic ribonuclease A

In addition to hydrolysing RNA, bovine pancreatic ribonucleasesplits esters of pyrimidine nucleoside 3'-phosphates, includingdinucleotides. For a series of 3':5'-linked dinucleotides ofgeneral structure CpN, where N is a 5' linked nucleoside, k_calfor the release of N varies enormously with the precise structureof N. Structural studies have been interpreted to indicate thatthe group N interacts with a subsite, B2, on the enzyme thatcomprises Gln69, Asn71 and Glulll. We report studies by site-directedmutagenesis that indicate that Gln69 is not involved in productiveinteractions with any of the dinucleotide substrates and thatAsn71 is an important component of subsite B2 for all dinucleotidesubstrates tested. Glulll appears to be functionally involvedin catalysis for dinucleotide substrates solely when N is guanosine.     

Nanomechanics for MEMS: a structural design perspective

Nanomechanics and structural design of MEMS are intimately tied together. As mechanical properties of hard materials are found to be strongly sample-size dependent, new criteria are in demand for size-dependent structural analysis and design of MEMS components. The paper offers a critical survey of some of the most interesting and challenging advances in nanomechanics of metals from a MEMS design standpoint. The emphasis is not just on sample size effects in intrinsic properties (in plasticity, elasticity and fracture) but also on extrinsic effects arising in material testing of super hard nano-sized samples and crucially affecting MEMS performance if discarded.     

二 恶英生成抑制剂及其阻滞机理研究现状与展望

固体废物焚烧过程产生的二英类物质具剧毒性且可在环境中造成持久性污染,国内外学者一直致力于探究高效的二英类物质源头减量化技术以保护公众健康。本文综述了近年来二英类物质从头合成的生成阻滞研究现状,对比分析了实验研究中常采用的含硫类抑制剂、含氮类抑制剂、含OH基抑制剂及复合抑制剂对二英类物质的抑制效果及其阻滞机理,进而剖析二英生成抑制剂的研发思路进展。在当前以抑制氯源和催化剂活性为主的抑制途径基础上,依据二英类物质的从头合成机理,探讨了通过阻滞碳源或氧气与反应物接触进而阻滞从头合成反应的抑制新途径,并展望了未来新型阻滞剂研发的方向,以期为二英类物质的源头减量和污染控制提供理论支持。抑制剂工业化应用和推广仍处于尝试阶段,在实际规模的焚烧厂应用时抑制效果不稳定,未来仍需进一步开展中试和现场试验以验证抑制作用实效及其影响因素。