An optimization approach to predicting protein structural class from amino acid composition.

Proteins are generally classified into four structural classes: all-alpha proteins, all-beta proteins, alpha + beta proteins, and alpha/beta proteins. In this article, a protein is expressed as a vector of 20-dimensional space, in which its 20 components are defined by the composition of its 20 amino acids. Based on this, a new method, the so-called maximum component coefficient method, is proposed for predicting the structural class of a protein according to its amino acid composition. In comparison with the existing methods, the new method yields a higher general accuracy of prediction. Especially for the all-alpha proteins, the rate of correct prediction obtained by the new method is much higher than that by any of the existing methods. For instance, for the 19 all-alpha proteins investigated previously by P.Y. Chou, the rate of correct prediction by means of his method was 84.2%, but the correct rate when predicted with the new method would be 100%! Furthermore, the new method is characterized by an explicable physical picture. This is reflected by the process in which the vector representing a protein to be predicted is decomposed into four component vectors, each of which corresponds to one of the norms of the four protein structural classes.     

Probing the conformation of a human apolipoprotein C-1 by amino acid substitutions and trimethylamine-N-oxide.

To test, at the level of individual amino acids, the conformation of an exchangeable apolipoprotein in aqueous solution and in the presence of an osmolyte trimethylamine-N-oxide (TMAO), six synthetic peptide analogues of human apolipoprotein C-1 (apoC-1, 57 residues) containing point mutations in the predicted alpha-helical regions were analyzed by circular dichroism (CD). The CD spectra and the melting curves of the monomeric wild-type and plasma apoC-1 in neutral low-salt solutions superimpose, indicating 31 +/- 4% alpha-helical structure at 22 degrees C that melts reversibly with T(m,WT) = 50 +/- 2 degrees C and van't Hoff enthalpy deltaH(v,WT)(Tm) = 18 +/- 2 kcal/mol. G15A substitution leads to an increased alpha-helical content of 42 +/- 4% and an increased T(m,G15A) = 57 +/- 2 degrees C, which corresponds to stabilization by delta deltaG(app) = +0.4 +/- 1.5 kcal/mol. G15P mutant has approximately 20% alpha-helical content at 22 degrees C and unfolds with low cooperativity upon heating to 90 degrees C. R23P and T45P mutants are fully unfolded at 0-90 degrees C. In contrast, Q31P mutation leads to no destabilization or unfolding. Consequently, the R23 and T45 locations are essential for the stability of the cooperative alpha-helical unit in apoC-1 monomer, G15 is peripheral to it, and Q31 is located in a nonhelical linker region. Our results suggest that Pro mutagenesis coupled with CD provides a tool for assigning the secondary structure to protein groups, which should be useful for other self-associating proteins that are not amenable to NMR structural analysis in aqueous solution. TMAO induces a reversible cooperative coil-to-helix transition in apoC-1, with the maximal alpha-helical content reaching 74%. Comparison with the maximal alpha-helical content of 73% observed in lipid-bound apoC-1 suggests that the TMAO-stabilized secondary structure resembles the functional lipid-bound apolipoprotein conformation.     

Understanding the roles of amino acid residues in tertiary structure formation of chignolin by using molecular dynamics simulation

Chignolin is a 10-residue peptide (GYDPETGTWG) that forms a stable beta-hairpin structure in water. However, its design template, GPM12 (GYDDATKTFG), does not have a specific structure. To clarify which amino acids give it the ability to form the beta-hairpin structure, we calculated the folding free-energy landscapes of chignolin, GPM12, and their chimeric peptides using multicanonical molecular dynamics (MD) simulation. Cluster analysis of the conformational ensembles revealed that the native structure of chignolin was the lowest in terms of free energy while shallow local minima were widely distributed in the free energy landscape of GPM12, in agreement with experimental observations. Among the chimeric peptides, GPM12(D4P/K7G) stably formed the same beta-hairpin structure as that of chignolin in the MD simulation. This was confirmed by nuclear magnetic resonance (NMR) spectroscopy. A comparison of the free-energy landscapes showed that the conformational distribution of the Asp3-Pro4 sequence was inherently biased in a way that is advantageous both to forming hydrogen bonds with another beta-strand and to initiating loop structure. In addition, Gly7 helps stabilize the loop structure by having a left-handed alpha-helical conformation. Such a conformation is necessary to complete the loop structure, although it is not preferred by other amino acids. Our results suggest that the consistency between the short-range interactions that determine the local geometries and the long-range interactions that determine the global structure is important for stable tertiary structure formation.     

Fishing new proteins in the twilight zone of genomes: the test case of outer membrane proteins in Escherichia coli K12, Escherichia coli O157:H7, and other Gram-negative bacteria

We address the problem of clustering the whole protein content of genomes into three different categories-globular, all-alpha, and all-beta membrane proteins-with the aim of fishing new membrane proteins in the pool of nonannotated proteins (twilight zone). The focus is then mainly on outer membrane proteins. This is performed by using an integrated suite of programs (Hunter) specifically developed for predicting the occurrence of signal peptides in proteins of Gram-negative bacteria and the topography of all-alpha and all-beta membrane proteins. Hunter is tested on the well and partially annotated proteins (2160 and 760, respectively) of Escherichia coli K 12 scoring as high as 95.6% in the correct assignment of each chain to the category. Of the remaining 1253 nonannotated sequences, 1099 are predicted globular, 136 are all-alpha, and 18 are all-beta membrane proteins. In Escherichia coli 0157:H7 we filtered 1901 nonannotated proteins. Our analysis classifies 1564 globular chains, 327 inner membrane proteins, and 10 outer membrane proteins. With Hunter, new membrane proteins are added to the list of putative membrane proteins of Gram-negative bacteria. The content of outer membrane proteins per genome (nine are analyzed) ranges from 1.5% to 2.4%, and it is one order of magnitude lower than that of inner membrane proteins. The finding is particularly relevant when it is considered that this is the first large-scale analysis based on validated tools that can predict the content of outer membrane proteins in a genome and can allow cross-comparison of the same protein type between different species.     

Molecular dynamics as a tool to detect protein foldability. A mutant of domain B1 of protein G with non-native secondary structure propensities

The usefulness of molecular dynamics to assess the structural integrity of mutants containing several mutations has been investigated. Our goal was to determine whether molecular dynamics would be able to discriminate mutants of a protein having a close-to-wild-type fold, from those that are not folded under the same conditions. We used as a model the B1 domain of protein G in which we replaced the unique central alpha-helix by the sequence of the second beta-hairpin, which has a strong intrinsic propensity to form this secondary structure in solution. In the resulting protein, one-third of the secondary structure has been replaced by a non-native one. Models of the mutants were built based on the three-dimensional structure of the wild-type GB1 domain. During 2 ns of molecular dynamics simulations on these models, mutants containing up to 10 mutations in the helix retained the native fold, while another mutant with an additional mutation unfolded. This result is in agreement with our circular dichroism and NMR experiments, which indicated that the former mutants fold into a structure similar to the wild-type, as opposed to the latter mutant which is partly unfolded. Additionally, a mutant containing six mutations scattered through the surface of the domain, and which is unfolded, was also detected by the simulation. This study suggests that molecular dynamics calculations could be performed on molecular models of mutants of a protein to evaluate their foldability, prior to a mutagenesis experiment.     

Three-dimensional model and quaternary structure of the human eye lens protein gamma S-crystallin based on beta- and gamma-crystallin X-ray coordinates and ultracentrifugation.

A 3-dimensional model of the human eye lens protein gamma S-crystallin has been constructed using comparative modeling approaches encoded in the program COMPOSER on the basis of the 3-dimensional structure of gamma-crystallin and beta-crystallin. The model is biased toward the monomeric gamma B-crystallin, which is more similar in sequence. Bovine gamma S-crystallin was shown to be monomeric by analytical ultracentrifugation without any tendency to form assemblies up to concentrations in the millimolar range. The connecting peptide between domains was therefore built assuming an intramolecular association as in the monomeric gamma-crystallins. Because the linker has 1 extra residue compared with gamma B and beta B2, the conformation of the connecting peptide was constructed by using a fragment from a protein database. gamma S-crystallin differs from gamma B-crystallin mainly in the interface region between domains. The charged residues are generally paired, although in a different way from both beta- and gamma-crystallins, and may contribute to the different roles of these proteins in the lens.     

A method for determining the positions of polar hydrogens added to a protein structure that maximizes protein hydrogen bonding.

An automated method for the optimal placement of polar hydrogens in a protein structure is described. This method treats the polar, side chain hydrogens of lysine, serine, threonine, and tyrosine and the amino terminus of a protein. The program, called NETWORK, divides the potential hydrogen-bonding pairs of a protein into groups of interacting donors and acceptors. A search is conducted on each of the local groups to find an arrangement which forms the most hydrogen bonds. If two or more arrangements have the same number of hydrogen bonds, the arrangement with the shortest set of hydrogen bonds is selected. The polar hydrogens of the histidyl side chain are specifically treated, and the ionization state of this residue is allowed to change, if this change results in additional hydrogen bonds for the local group. The program will accept Protein Data Bank as well as Biosym-format coordinate files. Input and output routines can be easily modified to accept other coordinate file formats. The predictions from this method are compared to known hydrogen positions for bovine pancreatic trypsin inhibitor, insulin, RNase-A, and trypsin for which the neutron diffraction structures have been determined. The usefulness of this program is further demonstrated by a comparison of molecular dynamics simulations for the enzyme cytochrome P-450cam with and without using NETWORK.     

Modeling the membrane environment has implications for membrane protein structure and function: Influenza A M2 protein

The M2 protein, a proton channel, from Influenza A has been structurally characterized by X‐ray diffraction and by solution and solid‐state NMR spectroscopy in a variety of membrane mimetic environments. These structures show substantial backbone differences even though they all present a left‐handed tetrameric helical bundle for the transmembrane domain. Variations in the helix tilt influence drug binding and the chemistry of the histidine tetrad responsible for acid activation, proton selectivity and transport. Some of the major structural differences do not arise from the lack of precision, but instead can be traced to the influences of the membrane mimetic environments. The structure in lipid bilayers displays unique chemistry for the histidine tetrad, which binds two protons cooperatively to form a pair of imidazole‐imidazolium dimers. The resulting interhistidine hydrogen bonds contribute to a three orders of magnitude enhancement in tetramer stability. Integration with computation has provided detailed understanding of the functional mechanism for proton selectivity, conductance and gating of this important drug target.     

Conformationally constrained single-chain peptide mimics of relaxin B-chain secondary structure.

Relaxin is a member of the insulin superfamily and has many biological actions including angiogenesis and collagen degradation. It is a 6 kDa peptide hormone consisting of two peptide chains (A and B) tethered by two disulphide bonds. Past structure-function relationship studies have shown the key receptor binding site of relaxin to be principally situated within the B-chain alpha-helix. Molecular dynamic simulations were performed to aid the design of conformationally constrained relaxin B-chain analogues that possess alpha-helical structure and relaxin-like activity. Restraints included disulphide bonds, both single and double, and lactam bonds. Each peptide was prepared by solid phase synthesis and, following purification, subjected to detailed conformational analysis by circular dichroism spectroscopy. Of 15 prepared relaxin B-chain mimetics, one was able to mimic the secondary structure of the native ligand as indicated by biomolecular recognition/interaction analysis using surface enhanced laser desorption ionization mass spectroscopy together with a relaxin antibody. However, none of the mimetics possess characteristic relaxin-like biological activity which strongly indicates that the pharmacophore comprises additional structural elements other than the relaxin B-chain alpha-helix. These findings will assist in the design and preparation of novel relaxin agonists and antagonists.     

A new approach for structure analysis of two-dimensional membrane protein crystals using X-ray powder diffraction data

The application of powder diffraction methods to problems in structural biology is generally regarded as intractable because of the large number of unresolved, overlapping X‐ray reflections. Here, we use information about unit cell lattice parameters, space group transformations, and chemical composition as a priori information in a bootstrap process that resolves the ambiguities associated with overlapping reflections. The measured ratios of reflections that can be resolved experimentally are used to refine the position, the shape, and the orientation of low‐resolution molecular structures within the unit cell, in leading to the resolution of the overlapping reflections. The molecular model is then made progressively more sophisticated as additional diffraction information is included in the analysis. We apply our method to the recovery of the structure of the bacteriorhodopsin molecule (bR) to a resolution of 7 Å using experimental data obtained from two‐dimensional purple membrane crystals. The approach can be used to determine the structure factors directly or to provide reliable low‐resolution phase information that can be refined further by the conventional methods of protein crystallography.     

A new computational model for protein folding based on atomic solvation.

A new model for calculating the solvation energy of proteins is developed and tested for its ability to identify the native conformation as the global energy minimum among a group of thousands of computationally generated compact non-native conformations for a series of globular proteins. In the model (called the WZS model), solvation preferences for a set of 17 chemically derived molecular fragments of the 20 amino acids are learned by a training algorithm based on maximizing the solvation energy difference between native and non-native conformations for a training set of proteins. The performance of the WZS model confirms the success of this learning approach; the WZS model misrecognizes (as more stable than native) only 7 of 8,200 non-native structures. Possible applications of this model to the prediction of protein structure from sequence are discussed.     

Thermal folding and mechanical unfolding pathways of protein secondary structures

Mechanical stretching of secondary structures is studied through molecular dynamics simulations of a Go-like model. Force versus displacement curves are studied as a function of the stiffness and velocity of the pulling device. The succession of stretching events, as measured by the order in which contacts are ruptured, is compared to the sequencing of events during thermal folding and unfolding. Opposite cross-correlations are found for an alpha-helix and a beta-hairpin structure. In a tandem of two alpha-helices, the two constituent helices unravel nearly simultaneously. A simple condition for simultaneous versus sequential unraveling of repeat units is presented.     

Antimicrobial peptide RP-1 structure and interactions with anionic versus zwitterionic micelles

Topologically, platelet factor-4 kinocidins consist of distinct N-terminal extended, C-terminal helical, and interposing gamma-core structural domains. The C-terminal alpha-helices autonomously confer direct microbicidal activity, and the synthetic antimicrobial peptide RP-1 is modeled upon these domains. In this study, the structure of RP-1 was assessed using several complementary techniques. The high-resolution structure of RP-1 was determined by NMR in anionic sodium dodecyl sulfate (SDS) and zwitterionic dodecylphosphocholine (DPC) micelles, which approximate prokaryotic and eukaryotic membranes, respectively. NMR data indicate the peptide assumes an amphipathic alpha-helical backbone conformation in both micelle environments. However, small differences were observed in the side-chain orientations of lysine, tyrosine, and phenylalanine residues in SDS versus DPC environments. NMR experiments with a paramagnetic probe indicated differences in positioning of the peptide within the two micelle types. Molecular dynamics (MD) simulations of the peptide in both micelle types were also performed to add insight into the peptide/micelle interactions and to assess the validity of this technique to predict the structure of peptides in complex with micelles. MD independently predicted RP-1 to interact only peripherally with the DPC micelle, leaving its spherical shape intact. In contrast, RP-1 entered deeply into and significantly distorted the SDS micelle. Overall, the experimental and MD results support a preferential specificity of RP-1 for anionic membranes over zwitterionic membranes. This specificity likely derives from differences in RP-1 interaction with distinct lipid systems, including subtle differences in side chain orientations, rather than gross changes in RP-1 structure in the two lipid environments.     

New Fourier transform infrared based computational method for peptide secondary structure determination. I. Description of method

Fourier transform infrared (FTIR) experiments in dimethylsulfoxide, a solvent incapable of H donation, demonstrate that H --> D isotopic replacement on the amide side of peptide bonds involves modifications of both the position and intensity of the amide I band. The effect of the isotopic substitution is particularly significant in the 1710-1670 and 1670-1650 cm(-1) regions, which are generally associated with beta-turns and alpha-helices. This behavior, attributed to the existence of intramolecular H-bonds in the polypeptide chain, is directly correlated to the presence of different secondary structures. Utilizing the effects induced by isotopic substitution, a method for the quantitative determination of the percentage of intramolecular H-bonds and the correlated secondary structures is proposed. The method consists of three principal steps: resolution of the fine structure of the amide I band with the determination of the number and position of the different components; reconstruction of the experimentally measured amide I band as a combination of Gaussian and Lorentzian functions, centered on the wave numbers set by band-narrowing methods, through a curve-fitting program; and quantitative determination of the population of the H-bonded carbonyls and the correlated secondary structures by comparison of the integrated intensities pertaining to the components with homologous wave numbers before and after isotopic exchange. The method is tested on a synthetic fragment of proocytocin that was previously analyzed by NMR techniques using the same solvent systems.     

Role of amino acids in translational mechanisms governing milk protein synthesis in murine and ruminant mammary epithelial cells

The role of amino acids (AA) on translational regulation in mammary epithelial cells cultured under lactogenic conditions was studied. The rates of total protein synthesis and beta-lactoglobulin (BLG) synthesis in mouse CID-9 cells were 2.1- or 3.1-fold higher, respectively, than in their bovine L-1 counterparts. Total AA deprivation or selective deprivation of Leu had a negative protein-specific effect on BLG synthesis that was more pronounced in bovine cells than in murine cells. Dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and S6 kinase (S6K1) on Thr(389) but not on Ser(411) was also more prominent in bovine cells. Noteably, deprivation of Leu had a less marked effect on BLG synthesis and 4E-BP1 or S6K1 phosphorylation than deprivation of all AA. In AA-deprived CID-9 cells, Leu specifically restored BLG synthesis from pre-existing mRNA whereas AA also restored total protein synthesis. This restoration was associated with a more pronounced effect on 4E-BP1 and S6K1 phosphorylation in bovine versus murine cells. Rapamycin specifically reduced Leu- and AA-stimulated BLG translation initiation in a dose-dependent manner. A further reduction was observed for Leu-treated cells in the presence of LY294002, a PI3K (phosphatidylinositol 3-kinase) inhibitor, which also reduced total protein synthesis. These findings suggest that direct signaling from AA to the translational machinery is involved in determining the rates of milk protein synthesis in mammary epithelial cells.     

Role of beta-lactam carboxyl group on binding of penicillins and cephalosporins to class C beta-lactamases

Molecular models for the Henry Michaelis complexes of Enterobacter cloacae, a class C beta-lactamase, with penicillin G and cephalotin have been constructed by using molecular mechanic calculations, based on the AMBER force field, to examine the molecular differentiation mechanisms between cephalosporins and penicillins in beta-lactamases. Ser318Ala and Thr316Ala mutations in both complexes and Asn346Ala and Thr316Ala/Asn346Ala double mutation in penicillin G complex have also been studied. Results confirm that Thr316, Ser318, and Asn346 play a crucial role in the substrate recognition, via their interactions with one of the oxygens of the antibiotic carboxyl group. Both mutation Ser318Ala and Thr316Ala strongly affect the correct binding of cephalotin to P99, the first mainly by precluding the discriminating salt bridge between carboxyl and serine OH groups, and the second one by the Ser318, Lys315, and Tyr150 spatial rearrangements. On the other hand, Ser318Ala mutation has little effect on penicillin G binding, but the Thr316Ala/Asn346Ala double mutation causes the departure of the antibiotic from the oxyanion hole. Molecular dynamic simulations allow us to interpret the experimental results of some class C and A beta-lactamases.     

Biphasic recognition chiral extraction: A novel method for separation of mandelic acid enantiomers

This article reports a new chiral separation method-biphasic recognition chiral extraction for the separation of mandelic acid enantiomers. Distribution behavior of mandelic acid enantiomers was studied in the extraction system with O,O'-di-benzoyl-(2S,3S)-4-toluoyl-tartaric acid (D-(+)-DTTA) in organic phase and beta-CD derivatives in aqueous phase, and the influence of the types and concentrations of extractants and pH on extraction efficiency was investigated. Hydroxypropyl-beta-cyclodextrin (HP-beta-CD), hydroxyethyl-beta-cyclodextrin (HE-beta-CD), and methyl-beta-cyclodextrin (Me-beta-CD) have stronger recognition abilities for S-mandelic acid than those for R-mandelic acid, among which HP-beta-CD has the strongest ability. D-(+)-DTTA preferentially recognizes R-mandelic acid. pH and the concentrations of extractants have great effects on chiral separation ability. A high enantioseparation efficiency with a maximum enantioselectivity of 1.527 is obtained at pH of 2.7 and the ratio of 2:1 of [D-(+)-DTTA] to [HP-beta-CD]. The obtained results indicate that the biphasic recognition chiral extraction is of stronger chiral separation ability than the monophasic recognition chiral extraction. It may be very helpful to optimize the extraction systems and realize the large-scale production of pure enantiomers.     

Topography of a 2.0 A structure of alpha1-antitrypsin reveals targets for rational drug design to prevent conformational disease

Members of the serpin family of serine proteinase inhibitors play important roles in the inflammatory, coagulation, fibrinolytic, and complement cascades. An inherent part of their function is the ability to undergo a structural rearrangement, the stressed (S) to relaxed (R) transition, in which an extra strand is inserted into the central A beta-sheet. In order for this transition to take place, the A sheet has to be unusually flexible. Malfunctions in this flexibility can lead to aberrant protein linkage, serpin inactivation, and diseases as diverse as cirrhosis, thrombosis, angioedema, emphysema, and dementia. The development of agents that control this conformational rearrangement requires a high resolution structure of an active serpin. We present here the topology of the archetypal serpin alpha1-antitrypsin to 2 A resolution. This structure allows us to define five cavities that are potential targets for rational drug design to develop agents that will prevent conformational transitions and ameliorate the associated disease.     

Disruption of the beta-sheet structure of a protected pentapeptide, related to the beta-amyloid sequence 17-21, induced by a single, helicogenic C(alpha)-tetrasubstituted alpha-amino acid.

Fifteen years ago it was shown that an alpha-aminoisobutyric acid (Aib) residue is significantly more effective than an L-Pro or a D-amino acid residue in inducing beta-sheet disruption in short model peptides. As this secondary structure element is known to play a crucial role in the neuropathology of Alzheimer's disease, it was decided to check the effect of Aib (and other selected, helix inducer, C(alpha)-tetrasubstituted alpha-amino acids) on the beta-sheet conformation adopted by a protected pentapeptide related to the sequence 17-21 of the beta-amyloid peptide. By use of FT-IR absorption and 1H NMR techniques it was found that the strong self-association characterizing the pentapeptide molecules in weakly polar organic solvents is completely abolished by replacing a single residue with Aib or one of its congeners.