Conformation of alpha zeins in solid state by Fourier transform IR

The major maize storage proteins (alpha zeins) are deposited as an insoluble mass in the protein bodies of the endosperm. Because they are insoluble in water, most structural studies are performed in alcohol solutions. To solve the question raised by several authors about denaturation of the alpha zein structure by alcohol, we analyze the secondary structure of alpha zeins prepared with and without solubilization in alcohol (corn gluten meal and protein bodies with high concentrations of alpha zeins and traces of beta zeins). The secondary structures of alpha zeins are analyzed in the solid state by Fourier transform IR spectroscopy (FTIR) in KBr pellets and solid-state 13C-NMR spectroscopy. The proportion of secondary structures obtained by FTIR of alpha zeins prepared with and without solubilization in alcohol yield almost identical proportions of alpha helices and beta sheets. The proportion of alpha helices (43%) agrees with that measured by circular dichroism in an alcohol solution. However, the proportion of beta sheets (28%) is higher than the one measured by the same technique. Gluten and protein body samples with high beta zein content showed higher beta sheet and lower alpha helix proportions than that obtained for alpha zein preparations. The solid-state 13C-NMR spectra show the carbonyl peak for the alpha zeins at delta 176 and for the sample rich in beta zeins at delta 172, which demonstrates the presence of a high content of alpha helices and beta sheets, respectively. These results indicate that alcohol solubilization does not affect the conformation of alpha zeins, validating the secondary structure measurements in solution.     

Cooperative effects in hydrogen-bonding of protein secondary structure elements: a systematic analysis of crystal data using Secbase

A systematic analysis of the hydrogen-bonding geometry in helices and beta sheets has been performed. The distances and angles between the backbone carbonyl O and amide N atoms were correlated considering more than 1500 protein chains in crystal structures determined to a resolution better than 1.5 A. They reveal statistically significant trends in the H-bond geometry across the different secondary structural elements. The analysis has been performed using Secbase, a modular extension of Relibase (Receptor Ligand Database) which integrates information about secondary structural elements assigned to individual protein structures with the various search facilities implemented into Relibase. A comparison of the mean hydrogen-bond distances in alpha helices and 3(10) helices of increasing length shows opposing trends. Whereas in alpha helices the mean H-bond distance shrinks with increasing helix length and turn number, the corresponding mean dimension in 3(10) helices expands in a comparable series. Comparing similarly the hydrogen-bond lengths in beta sheets there is no difference to be found between the mean H-bond length in antiparallel and parallel beta sheets along the strand direction. In contrast, an interesting systematic trend appears to be given for the hydrogen bonds perpendicular to the strands bridging across an extended sheet. With increasing number of accumulated strands, which results in a growing number of back-to-back piling hydrogen bonds across the strands, a slight decrease of the mean H-bond distance is apparent in parallel beta sheets whereas such trends are obviously not given in antiparallel beta sheets. This observation suggests that cooperative effects mutually polarizing spatially well-aligned hydrogen bonds are present either in alpha helices and parallel beta sheets whereas such influences seem to be lacking in 3(10) helices and antiparallel beta sheets.     

Benchmarking of TASSER in the ab initio limit

A significant number of protein sequences in a given proteome have no obvious evolutionarily related protein in the database of solved protein structures, the PDB. Under these conditions, ab initio or template-free modeling methods are the sole means of predicting protein structure. To assess its expected performance on proteomes, the TASSER structure prediction algorithm is benchmarked in the ab initio limit on a representative set of 1129 nonhomologous sequences ranging from 40 to 200 residues that cover the PDB at 30% sequence identity and which adopt alpha, alpha + beta, and beta secondary structures. For sequences in the 40-100 (100-200) residue range, as assessed by their root mean square deviation from native, RMSD, the best of the top five ranked models of TASSER has a global fold that is significantly close to the native structure for 25% (16%) of the sequences, and with a correct identification of the structure of the protein core for 59% (36%). In the absence of a native structure, the structural similarity among the top five ranked models is a moderately reliable predictor of folding accuracy. If we classify the sequences according to their secondary structure content, then 64% (36%) of alpha, 43% (24%) of alpha + beta, and 20% (12%) of beta sequences in the 40-100 (100-200) residue range have a significant TM-score (TM-score > or = 0.4). TASSER performs best on helical proteins because there are less secondary structural elements to arrange in a helical protein than in a beta protein of equal length, since the average length of a helix is longer than that of a strand. In addition, helical proteins have shorter loops and dangling tails. If we exclude these flexible fragments, then TASSER has similar accuracy for sequences containing the same number of secondary structural elements, irrespective of whether they are helices and/or strands. Thus, it is the effective configurational entropy of the protein that dictates the average likelihood of correctly arranging the secondary structure elements.     

Anabaena apoflavodoxin hydrogen exchange: on the stable exchange core of the alpha/beta(21345) flavodoxin-like family

An important issue in modern protein biophysics is whether structurally homologous proteins share common stability and/or folding features. Flavodoxin is an archetypal alpha/beta protein organized in three layers: a central beta-sheet (strand order 21345) flanked by helices 1 and 5 on one side and helices 2, 3, and 4 on the opposite side. The backbone internal dynamics of the apoflavodoxin from Anabaena is analyzed here by the hydrogen exchange method. The hydrogen exchange rates indicate that 46 amide protons, distributed throughout the structure of apoflavodoxin, exchange relatively slowly at pH 7.0 (k(ex) < 10(-1) min(-1)). According to their distribution in the structure, protein stability is highest on the beta-sheet, helix 4, and on the layer formed by helices 1 and 5. The exchange kinetics of Anabaena apoflavodoxin was compared with those of the apoflavodoxin from Azotobacter, with which it shares a 48% sequence identity, and with Che Y and cutinase, two other alpha/beta (21345) proteins with no significant sequence homology with flavodoxins. Both similarities and differences are observed in the cores of these proteins. It is of interest that a cluster of a few structurally equivalent residues in the central beta-strands and in helix 5 is common to the cores.     

alpha beta Spectrin coiled coil association at the tetramerization site

On the basis of sequence homology studies, it has been suggested that the association of human erythrocytes alpha and beta spectrin at the tetramerization site involves interactions between helices. However, no empirical details are available, presumably due to the experimental difficulties in studying spectrin molecules because of its size and/or its structural flexibility. It has been speculated that erythrocyte tetramerization involves helical bundling rather than coiled coil association. We have used recombinant spectrin peptides to model alpha and beta spectrin to study their association at the tetramerization site. Two alpha peptides, Sp alpha 1-156 and Sp alpha 1-368, and one beta peptide, Sp beta 1898-2083, were used as model peptides to demonstrate the formation of the alpha beta complex. We also found that the replacement of R28 in Sp alpha 1-368 to give Sp alpha 1-368R28C abolished complex formation with the beta peptide. Circular dichroism techniques were used to monitor the secondary structures of the individual peptides and of the complex, and the results showed that both Sp alpha 1-156 and Sp beta 1898-2083 peptides in solution, separately, included helices that were not paired with other helices in the absence of their binding partners. However, in a mixture of Sp alpha 1-156 and Sp beta 1898-2083 and formation of the alpha beta complex, the unpaired helices associated to form coiled coils. Since the sequences of these two peptides that are involved in the coiled coil association are derived from a native protein, the information obtained from this study also provides insight toward a better understanding of naturally occurring coiled coil subunit-subunit association.     

Major structural determinants of transmembrane proteins identified by principal component analysis

We identify amino acid characteristics important in determining the secondary structures of transmembrane proteins, and compare them with characteristics important for cytoplasmic proteins. Using information derived from multiple sequence alignments, we perform a principal component analysis (PCA) to identify the directions in the 20-dimensional amino acid frequency space that comprise the most variance within each protein secondary structure. These vectors represent the important position-specific properties of the amino acids for coils, turns, beta sheets, and alpha helices. As expected, the most important axis for most of the datasets was hydrophobicity. Additional axes, distinct from hydrophobicity, are surprising, especially in the case of transmembrane alpha helices, where the effects of aromaticity and beta-branching are the next two most significant characteristics. The axis representing beta-branching also has equal importance in cytoplasmic and transmembrane helices, a finding that contrasts with some experimental results in membrane-like environments. In a further analysis, we examine trends for some of the PCA axes over averaged transmembrane alpha helices, and find interesting results for aromaticity.     

3D structure and significance of the GPhiXXG helix packing motif in tetramers of the E1beta subunit of pyruvate dehydrogenase from the archeon Pyrobaculum aerophilum.

As part of a structural genomics project, we have determined the 2.0 A structure of the E1beta subunit of pyruvate dehydrogenase from Pyrobaculum aerophilum (PA), a thermophilic archaeon. The overall fold of E1beta from PA is closely similar to the previously determined E1beta structures from humans (HU) and P. putida (PP). However, unlike the HU and PP structures, the PA structure was determined in the absence of its partner subunit, E1alpha. Significant structural rearrangements occur in E1beta when its E1alpha partner is absent, including rearrangement of several secondary structure elements such as helix C. Helix C is buried by E1alpha in the HU and PP structures, but makes crystal contacts in the PA structure that lead to an apparent beta(4) tetramer. Static light scattering and sedimentation velocity data are consistent with the formation of PA E1beta tetramers in solution. The interaction of helix C with its symmetry-related counterpart stabilizes the tetrameric interface, where two glycine residues on the same face of one helix create a packing surface for the other helix. This GPhiXXG helix-helix interaction motif has previously been found in interacting transmembrane helices, and is found here at the E1alpha-E1beta interface for both the HU and PP alpha(2)beta(2) tetramers. As a case study in structural genomics, this work illustrates that comparative analysis of protein structures can identify the structural significance of a sequence motif.     

Structures of membrane proteins

Summary The possible conformations of integral membrane proteins are restricted by the nature of their environment. In order to satisfy the requirement of maximum hydrogen bonding, those protions of the polypeptide chain which are in contact with lipid hydrocarbon must be organized into regions of regular secondary structure. As possible models of the intramembranous regions of integral membrane proteins, three types of regular structues are discussed. Two, the alpha helix and the beta-pleated sheet, are regularly occurring structural features of soluble proteins. The third is a newly proposed class of conformations called beta helices. These helices have unique features which make them particularly well-suited to the lipid bilayer environment. The central segment of the membrane-spanning protein glycophorin can be arranged into a beta helix with a hydrophobic exterior and a polar interior containing charged amino-acid side chains. Such structures could function as transmembrane ion channels. A model of the activation process based on a hypothetical equilibrium between alpha and beta helical forms of a transmembrane protein is presented. The model can accurately reproduce the kinetics and voltage dependence of the channels in nerve.     

Novel protein folds and their nonsequential structural analogs

Newly determined protein structures are classified to belong to a new fold, if the structures are sufficiently dissimilar from all other so far known protein structures. To analyze structural similarities of proteins, structure alignment tools are used. We demonstrate that the usage of nonsequential structure alignment tools, which neglect the polypeptide chain connectivity, can yield structure alignments with significant similarities between proteins of known three-dimensional structure and newly determined protein structures that possess a new fold. The recently introduced protein structure alignment tool, GANGSTA, is specialized to perform nonsequential alignments with proper assignment of the secondary structure types by focusing on helices and strands only. In the new version, GANGSTA+, the underlying algorithms were completely redesigned, yielding enhanced quality of structure alignments, offering alignment against a larger database of protein structures, and being more efficient. We applied DaliLite, TM-align, and GANGSTA+ on three protein crystal structures considered to be novel folds. Applying GANGSTA+ to these novel folds, we find proteins in the ASTRAL40 database, which possess significant structural similarities, albeit the alignments are nonsequential and in some cases involve secondary structure elements aligned in reverse orientation. A web server is available at http://agknapp.chemie.fu-berlin.de/gplus for pairwise alignment, visualization, and database comparison.     

Relative stability of protein structures determined by X-ray crystallography or NMR spectroscopy: a molecular dynamics simulation study

The relative stability of protein structures determined by either X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy has been investigated by using molecular dynamics simulation techniques. Published structures of 34 proteins containing between 50 and 100 residues have been evaluated. The proteins selected represent a mixture of secondary structure types including all alpha, all beta, and alpha/beta. The proteins selected do not contain cysteine-cysteine bridges. In addition, any crystallographic waters, metal ions, cofactors, or bound ligands were removed before the systems were simulated. The stability of the structures was evaluated by simulating, under identical conditions, each of the proteins for at least 5 ns in explicit solvent. It is found that not only do NMR-derived structures have, on average, higher internal strain than structures determined by X-ray crystallography but that a significant proportion of the structures are unstable and rapidly diverge in simulations.     

Dissection of the de novo designed peptide alpha t alpha: stability and properties of the intact molecule and its constituent helices

Alpha t alpha is a de novo designed 38-residue peptide [Fezoui et al. (1995) Protein Sci. 4, 286-295] that adopts a helical hairpin conformation in solution [Fezoui et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3675-3679; Fezoui et al. (1997) Protein Sci. 6, 1869-1877]. Since alpha t alpha was developed as a model system for protein folding at the stage where secondary structures interact and become mutually stabilizing, it is of interest to investigate the increase in stability that occurs with helix association. alpha t alpha was dissected into its component helices and the relative stabilities of the individual helices and the parent molecule were assessed. The Delta G0 of unfolding of alpha t alpha measured by guanidine hydrochloride denaturation was determined to be 3.4 kcal/mol. The equilibrium constant for folding of alpha t alpha was estimated from the Delta G0 as 338 and from hydrogen exchange measurements as 259. The stability of the helices in intact alpha t alpha over the individual helices increased by a factor of at least 37 based on amide proton exchange measurements. Sedimentation equilibrium studies showed very little association of the peptides to form either homo- or heterodimers, suggesting that helix association is stabilized by the high effective concentration of the helices caused by the presence of the connecting turn. The effects of salt and pH on the helicity of the component peptides are largely reflected in the intact molecule, implying that short-range interactions still make important contributions to the conformation of the intact molecule even though significant stabilization is caused by helix association.     

Family of G protein alpha chains: amphipathic analysis and predicted structure of functional domains

The G proteins transduce hormonal and other signals into regulation of enzymes such as adenylyl cyclase and retinal cGMP phosphodiesterase. Each G protein contains an alpha subunit that binds and hydrolyzes guanine nucleotides and interacts with beta gamma subunits and specific receptor and effector proteins. Amphipathic and secondary structure analysis of the primary sequences of five different alpha chains (bovine alpha s, alpha t1 and alpha t2, mouse alpha i, and rat alpha o) predicted the secondary structure of a composite alpha chain (alpha avg). The alpha chains contain four short regions of sequence homologous to regions in the GDP binding domain of bacterial elongation factor Tu (EF-Tu). Similarities between the predicted secondary structures of these regions in alpha avg and the known secondary structure of EF-Tu allowed us to construct a three-dimensional model of the GDP binding domain of alpha avg. Identification of the GDP binding domain of alpha avg defined three additional domains in the composite polypeptide. The first includes the amino terminal 41 residues of alpha avg, with a predicted amphipathic alpha helical structure; this domain may control binding of the alpha chains to the beta gamma complex. The second domain, containing predicted beta strands and alpha helices, several of which are strongly amphipathic, probably contains sequences responsible for interaction of alpha chains with effector enzymes. The predicted structure of the third domain, containing the carboxy terminal 100 amino acids, is predominantly beta sheet with an amphipathic alpha helix at the carboxy terminus. We propose that this domain is responsible for receptor binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of stabilization centers in 4 helix bundle proteins

Stabilization centers (SCs) were shown to play an important role in preventing decay of three-dimensional protein structures. These residue clusters, stabilized by cooperative long-range interactions, were proposed to serve as anchoring points for arranging secondary structure elements. In all-alpha proteins, SC elements appear less frequently than in all-beta, alpha/beta, and alpha+beta proteins suggesting that tertiary structure formation of all-alpha proteins is governed by different principles than in other protein classes. Here we analyzed the relation between the formation of stabilization centers and the inter-axial angles (Omega) of alpha-helices in 4 helix bundle proteins. In the distance range, where dipoles have dominant effect on the helix pair arrangement, those helix pairs, where residues from both helices participate in SC elements, appear as parallel more frequently than those helices where no SC elements are present. For SC containing helix pairs, the energetic difference between the parallel and anti-parallel states decreases considerably from 1.1 kcal/mol to 0.4 kcal/mol. Although the observed effect is weak for more distant helices, a competition between the SC element formation and the optimal dipole-dipole interaction of alpha-helices is proposed as a mechanism for tertiary structure formation in 4 helix bundle proteins. The SC-forming potential of different arrangements as well as the pitfalls of the SC definition are also discussed.     

Secondary structure switching in Cro protein evolution

We report the solution structure of the Cro protein from bacteriophage P22. Comparisons of its sequence and structure to those of lambda Cro strongly suggest an alpha-to-beta secondary structure switching event during Cro evolution. The folds of P22 Cro and lambda Cro share a three alpha helix fragment comprising the N-terminal half of the domain. However, P22 Cro's C terminus folds as two helices, while lambda Cro's folds as a beta hairpin. The all-alpha fold found for P22 Cro appears to be ancestral, since it also occurs in cI proteins, which are anciently duplicated paralogues of Cro. PSI-BLAST and transitive homology analyses strongly suggest that the sequences of P22 Cro and lambda Cro are globally homologous despite encoding different folds. The alpha+beta fold of lambda Cro therefore likely evolved from its all-alpha ancestor by homologous secondary structure switching, rather than by nonhomologous replacement of both sequence and structure.     

Predicted secondary structures of four penicillin beta-lactamases and a comparison with two lysozymes.

We have predicted the secondary structures of four beta-lactamases (Bacillus cereus, Bacillus licheniformis, Staphylococcus aureus, and Escherichia coli R-TEM) by the statistical method of Chou & Fasman as well as by the information theory method of Garnier et al. The secondary structures of all four beta-lactamases are of the alpha/beta type (Levitt & Chothia's nomenclature), with helices at N- and C-termini. There are about eight short regions each of alpha-helical (30--50%) and beta-strand (10--20%) structure separated by about 20 reverse turns. The conformation of the Gram-positive and Gram-negative beta-lactamases are generally similar although a few differences are predicted between the S.aureus and E.coli structures. Surprisingly, the two bacilli structures differ significantly in three short regions. In all four enzymes the region near the catalytically-implicated tyrosine has similar secondary structure. The secondary structure of hen egg white lysozyme, a penicillin-binding enzyme, as well as T4 phage lysozyme, has similarities to the N-terminal half of the penicillin-destroying beta-lactamases.     

Structure of the Escherichia coli TolB protein determined by MAD methods at 1.95 A resolution.

BACKGROUND: The periplasmic protein TolB from Escherichia coli is part of the Tol-PAL (peptidoglycan-associated lipoprotein) multiprotein complex used by group A colicins to penetrate and kill cells. TolB homologues are found in many gram-negative bacteria and the Tol-PAL system is thought to play a role in bacterial envelope integrity. TolB is required for lethal infection by Salmonella typhimurium in mice. RESULTS: The crystal structure of the selenomethionine-substituted TolB protein from E. coli was solved using multiwavelength anomalous dispersion methods and refined to 1. 95 A. TolB has a two-domain structure. The N-terminal domain consists of two alpha helices, a five-stranded beta-sheet floor and a long loop at the back of this floor. The C-terminal domain is a six-bladed beta propeller. The small, possibly mobile, contact area (430 A(2)) between the two domains involves residues from the two helices and the first and sixth blades of the beta propeller. All available genomic sequences were used to identify new TolB homologues in gram-negative bacteria. The TolB structure was then interpreted using the observed conservation pattern. CONCLUSIONS: The TolB beta-propeller C-terminal domain exhibits sequence similarities to numerous members of the prolyl oligopeptidase family and, to a lesser extent, to class B metallo-beta-lactamases. The alpha/beta N-terminal domain shares a structural similarity with the C-terminal domain of transfer RNA ligases. We suggest that the TolB protein might be part of a multiprotein complex involved in the recycling of peptidoglycan or in its covalent linking with lipoproteins.     

Analysis of secondary structure in proteins by chemical cross-linking coupled to MS

Chemical cross-linking is an attractive technique for the study of the structure of protein complexes due to its low sample consumption and short analysis time. Furthermore, distance constraints obtained from the identification of cross-linked peptides by MS can be used to construct and validate protein models. If a sufficient number of distance constraints are obtained, then determining the secondary structure of a protein can allow inference of the protein's fold. In this work, we show how the distance constraints obtained from cross-linking experiments can identify secondary structures within the protein sequence. Molecular modeling of alpha helices and beta sheets reveals that each secondary structure presents different cross-linking possibilities due to the topological distances between reactive residues. Cross-linking experiments performed with amine reactive cross-linkers with model alpha helix containing proteins corroborated the molecular modeling predictions. The cross-linking patterns established here can be extended to other cross-linkers with known lengths for the determination of secondary structures in proteins.     

A method of calculating the discrete secondary structures of globular proteins

The model of formation of alpha-helices and beta-structures determined by joint action of the three elements: N-terminal, internal and C-terminal fragments are presented. Algorithm for calculation of their localization in a given amino acid sequence was constructed on the base of this model. The preference of the fragments of the amino acid sequence to a definite type of the secondary structure was estimated on the base of corresponding average values of linear discriminant functions dsk (s = alpha, beta, k = N, in, C). The latter were constructed in the previous paper on the base of the revealed significant characteristics. These integral characteristics are used for calculating the localisation of discrete secondary structures. The total prediction for 3 states (alpha, beta, c) given 71% correctly predicted residues (for 4 states alpha, beta, c, t) 62% for the training set, consisting of 72 proteins. For the control set (15 proteins) the accuracy of prediction is about 65%. The essential advantages of this method are: 1) the possibility to localize the discrete secondary structures; 2) the high accuracy of prediction of long secondary structures (for alpha-helices approximately 90%, for beta-structures approximately 80%), which is important for the determination of the protein folding. The influence of mutation on the secondary structure of proteins was investigated. The anormally high stability of the secondary structures of immunoglobulins to mutations was revealed. This probably results from the selection during evolution of such variants of amino acid sequences, which are able to provide the functional variability of antigenic determinants, but keep invariant the tertially structure of protein.