Packing constraints of hydrophobic side chains in (α/β)8 barrels

An analysis of possible tight packing of hydrophobic groups simultaneously at the both surfaces of β-hyperboloid-8 was conducted. This analysis shows that the disposition of amino acid side chains at the real β-structure's surface is unique. If we sign the mean distance between adjacent β-strands as “a,” and the mean distance along β-strand between C_α atoms, whose side chains are directed to one side of the β-sheet, as “b,” the ratio b/a = √2 very precisely. This ratio ensures the most efficient packing of side hydrophobic groups at the outer surface of β-hyperboloid-8, forming, at the same time, the second by efficiency packing at its inner surface. © 1995 Wiley-Liss, Inc.     

Relationship between structure and amino acid sequence of strongly twisted and coiled β-hairpins in globular proteins

β-Hairpins are widespread in proteins, and it is possible to find them both within β-sheets and separately. In this work, a comparative analysis of amino acid sequences of β-strands within strongly twisted β-hairpins from different structural protein subclasses has been conducted. Strongly twisted and coiled β-hairpin generates in the space a right double helix out of β-strands that are connected by a loop region (connections). The frequencies of amino acid residues on the internal (concave) and external (convex) surfaces of strongly twisted β-hairpins have been determined (220 β-hairpins from nonhomologous proteins were studied). The concave surface of these β-hairpins is mainly generated by hydrophobic residues, while the convex surface by hydrophilic residues; accordingly, the alternation of hydrophobic internal and hydrophilic external residues is observed in their amino acid sequences. Amino acid residues of glycine and alanine (especially in places of the largest twisting of the strands) were anomalously frequently found in internal positions of strongly twisted and coiled β-hairpins. It was established that internal positions never contain the proline residues, while external positions in the twisting region contain them in a relatively large amount. It was demonstrated that at least one amino acid residue in α_L- or ε-conformation is required for generation of relatively short (up to 7 amino acid residues) connection. As a rule, these positions are occupied by glycines. Thus, not only the alternation of hydrophobic and hydrophilic amino acid residues, but also the presence of one or two glycine residues in the connection region and the excess of glycines and alanines in the places of the largest strand twisting on the concave surface, as well as the presence of prolines on the convex surface, are required to generate a strongly twisted and coiled β-hairpin.     

Identification and analysis of extended strands and β-sheets in globular proteins

A method to identify β-sheets in globular proteins from extended strands, using only α-carbon positions, has been developed. The strands that form β-sheets are picked up by means of simple distance criteria. The method has been tested by applying it to three proteins with accurately known secondary structures. It has also been applied to ten other proteins wherein only α-carbon coordinates are available, and the list of β-sheets obtained. The following points are worth noting: (i) The sheets identified by the algorithm are found to agree satisfactorily with the reported ones based on backbone hydrogen bonding, wherever this information is available. (ii) β-Strands that do not form parts of any sheet are a common feature of protein structures. (iii) Such isolated β-strands tend to be short. (iv) The conformation corresponding to the preferred right-handed twist of the sheet is overwhelmingly observed in both the sheet-forming and isolated β-strands.     

Ipomoelin, a jacalin-related lectin with a compact tetrameric association and versatile carbohydrate binding properties regulated by its N terminus

Many proteins are induced in the plant defense response to biotic stress or mechanical wounding. One group is lectins. Ipomoelin (IPO) is one of the wound-inducible proteins of sweet potato (Ipomoea batatas cv. Tainung 57) and is a Jacalin-related lectin (JRL). In this study, we resolved the crystal structures of IPO in its apo form and in complex with carbohydrates such as methyl α-D-mannopyranoside (Me-Man), methyl α-D-glucopyranoside (Me-Glc), and methyl α-D-galactopyranoside (Me-Gal) in different space groups. The packing diagrams indicated that IPO might represent a compact tetrameric association in the JRL family. The protomer of IPO showed a canonical β-prism fold with 12 strands of β-sheets but with 2 additional short β-strands at the N terminus. A truncated IPO (ΔN10IPO) by removing the 2 short β-strands of the N terminus was used to reveal its role in a tetrameric association. Gel filtration chromatography confirmed IPO as a tetrameric form in solution. Isothermal titration calorimetry determined the binding constants (K(A)) of IPO and ΔN10IPO against various carbohydrates. IPO could bind to Me-Man, Me-Glc, and Me-Gal with similar binding constants. In contrast, ΔN10IPO showed high binding ability to Me-Man and Me-Glc but could not bind to Me-Gal. Our structural and functional analysis of IPO revealed that its compact tetrameric association and carbohydrate binding polyspecificity could be regulated by the 2 additional N-terminal β-strands. The versatile carbohydrate binding properties of IPO might play a role in plant defense.     

Limited Proteolysis of Bacillus thuringiensis CryIG and CryIVB δ-Endotoxins Leads to Formation of Active Fragments That Do Not Coincide with the Structural Domains

Bacillus thuringiensis “true” toxins consist of three domains: the N-terminal, α-helical domain followed by two β-structural domains. Their limited proteolysis does not proceed at the domain boundaries, but is directed to the loops within the domains. There are at least two patterns of the limited proteolysis of “true” toxins. The first pattern, observed for CryIA and CryIVD δ-endotoxins, results in the proteolysis of the loops connecting β-strands of the second domain. The second pattern, detected for CryIG and CryIVB proteins, consists in the cleavage of the loop connecting the fifth and the sixth α-helixes of the first domain. The choice between the routes depends on the size, sequence, and dynamics of the loop that define its accessibility to a proteinase. Bioassay of CryIG and CryIVB δ-endotoxin fragments indicates that only two α-helixes, the sixth and the seventh within the first domain, followed by the two β-structural domains are sufficient for the insecticidal activity.     

Simulation Studies on the Stabilities of Aggregates Formed by Fibril-Forming Segments of α-Synuclein

Abstract

We performed molecular dynamics simulations for various oligomers with different β-sheet conformations consisting of α-Synuclein 71–82 residues using an all atom force field and explicit water model. Tetramers of antiparallel β-sheet are shown to be stable, whereas parallel sheets are highly unstable due to the repulsive interactions between bulky and polar side chains as well as the weaker backbone hydrogen bonds. We also investigated the stabilities of double antiparallel β-sheets stacked with asymmetric and symmetric geometries. Our results show that this 12 amino acid residue peptide can form stable β-sheet conformers at 320K and higher temperatures. The backbone hydrogen bonds in β-sheet and the steric packing between hydrophobic side chains between β-sheets are shown to give conformational stabilities.     

Molecular Dynamics Simulations to Investigate the Structural Stability and Aggregation Behavior of the GGVVIA Oligomers Derived from Amyloid β Peptide

Abstract

Several neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Huntington's dis-eases, are associated with amyloid fibrils formed by different polypeptides. Recently, the atomic structure of the amyloid-forming peptide GGVVIA from the C-terminal hydrophobic segment of amyloid-β (Aβ) peptide has been determined and revealed a dry, tightly self-com-plementing structure between two β-sheets, termed as “steric zipper”. In this study, several all-atom molecular dynamics simulations with explicit water were conducted to investigate the structural stability and aggregation behavior of the GGVVIA oligomers with various sizes. The results of our single-layer models suggested that the structural stability of the GGVVIA oligomers increases remarkably with increasing the numbers of β-strands. We fur-ther identified that SH2-ST2 may act as a stable seed in prompting amyloid fibril formations. Our results also demonstrated that hydrophobic interaction is the principle driving force to stabilize and associate the GGVVIA oligomers between β-strands; while the hydrophobic steric zipper formed via the side chains of V3, V4, and I5 plays a critical role in holding the two neighboring β-sheets together. Single glycine substitution at V3, V4, and I5 directly disrupted the hydrophobic steric zipper between these two β-sheets, resulting in the destabili-zation of the oligomers. Our simulation results provided detailed insights into understanding the aggregation behavior of the GGVVIA oligomers in the atomic level. It may also be help-ful for designing new inhibitors able to prevent the fibril formation of Aβ peptide.     

A protein structural class prediction method based on novel features

In this study, a 12-dimensional feature vector is constructed to reflect the general contents and spatial arrangements of the secondary structural elements of a given protein sequence. Among the 12 features, 6 novel features are specially designed to improve the prediction accuracies for α/β and α + β classes based on the distributions of α-helices and β-strands and the characteristics of parallel β-sheets and anti-parallel β-sheets. To evaluate our method, the jackknife cross-validating test is employed on two widely-used datasets, 25PDB and 1189 datasets with sequence similarity lower than 40% and 25%, respectively. The performance of our method outperforms the recently reported methods in most cases, and the 6 newly-designed features have significant positive effect to the prediction accuracies, especially for α/β and α + β classes.     

Structural Characterization of Polyglutamine Fibrils by Solid-State NMR Spectroscopy

Protein aggregation via polyglutamine stretches occurs in a number of severe neurodegenerative diseases such as Huntington's disease. We have investigated fibrillar aggregates of polyglutamine peptides below, at, and above the toxicity limit of around 37 glutamine residues using solid-state NMR and electron microscopy. Experimental data are consistent with a dry fibril core of at least 70-80 Å in width for all constructs. Solid-state NMR dipolar correlation experiments reveal a largely β-strand character of all samples and point to tight interdigitation of hydrogen-bonded glutamine side chains from different sheets. Two approximately equally frequent populations of glutamine residues with distinct sets of chemical shifts are found, consistent with local backbone dihedral angles compensating for β-strand twist or with two distinct sets of side-chain conformations. Peptides comprising 15 glutamine residues are present as single extended β-strands. Data obtained for longer constructs are most compatible with a superpleated arrangement with individual molecules contributing β-strands to more than one sheet and an antiparallel assembly of strands within β-sheets.     

Ribosomal subunit orientations determined in the monomeric ribosome by single and by double-labeling immune electron microscopy

The energies of two and three-chain antiparallel and parallel β-sheets have been minimized. The chains were considered to be equivalent. In each case, chains consisting of four and of eight l-alanine residues, respectively, with CH₃CO- and -NHCH₃ end groups were examined. Computations were carried out both for chains constrained to have a regular structure (i.e. the same φ and ψ dihedral angles for each residue) and for chains in which the regularity constraint was relaxed. All computed minimum-energy β-sheets were found to have a right-handed twist, as observed in proteins. As in the case of right-handed α-helices, it is the intrastrand non-bonded interaction energy that plays the key role in forcing β-sheets of l-amino acid residues to adopt a right-handed twist. The non-bonded energy contribution favoring the right-handed twist is the result of many small pairwise interatomic interactions involving the C^βH₃ groups. Polyglycine β-sheets, lacking the C^βH₃ side-chains, are not twisted. The twist of the poly-l-alanine sheet diminishes as the number of residues per chain increases, in agreement with observations. The twist of the four-residue chain increases somewhat (because of interstrand non-bonded interactions, also involving the C^βH₃ groups) in going from a single chain to a two-chain antiparallel structure, but then decreases slightly in going from a two-chain to a three-chain structure. β-Sheets in observed protein structures sometimes have a larger twist than those in the structures computed here. This may be due to irregularities in amino acid sequence and in hydrogenbonding patterns in the observed sheets, or to long-range interactions in proteins. The minimized energies of parallel β-sheets are considerably higher than those of the corresponding antiparallel β-sheets, indicating that parallel β-sheets are intrinsically less stable. This finding about the two kinds of β-sheets agrees with suggestions based on analyses of β-sheets observed in proteins. The energy difference between antiparallel and parallel β-sheets is due to closer packing of the chains and a more favorable alignment of the peptide dipoles in the antiparallel structures. The hydrogen-bond geometry in the computed antiparallel structures is very close to that proposed by Arnott et al. (1967) for the β-form of poly-l-alanine.     

Role of cholesterol in functional association between K(+)-Cl(-) cotransporter-3a and Na+,K(+)-ATPase

The conformation of amyloid-beta peptide (Aβ) determines if toxic aggregates are formed. The peptide structure by its turn depends on the environment and molecule-molecule interactions. We characterized the secondary structure of Aβ-(1-40) in surfactant solutions and interacting with monolayers. The peptide adopts β-sheet structure in solutions of ionic surfactants at sub-micelle concentrations and α-helix in the presence of ionic micelles. Uncharged micelles induce β-sheets. Aβ-(1-40) alters the critical micelle concentration value of the non-ionic surfactant, underlining hydrophobic interactions. At ionic monolayers the peptide forms β-sheets when its concentration at the surface is high enough. These results suggest that only electrostatic interactions of charged micelles that surround completely the peptide are able to induce non-aggregated α-helix structure.     

Autotransporter passenger domain secretion requires a hydrophobic cavity at the extracellular entrance of the β-domain pore

Whooping cough (pertussis) is a highly contagious acute respiratory illness of humans caused by the Gram-negative bacterial pathogen Bordetella pertussis. The AT (autotransporter) BrkA (Bordetella serum-resistance killing protein A) is an important B. pertussis virulence factor that confers serum resistance and mediates adherence. In the present study, we have solved the crystal structure of the BrkA β-domain at 3 ? (1 ?=0.1 nm) resolution. Special features are a hairpin-like structure formed by the external loop L4, which is observed fortuitously sitting inside the pore of the crystallographic adjacent β-domain, and a previously undiscovered hydrophobic cavity formed by patches on loop L4 and β-strands S5 and S6. This adopts a ubiquitous structure characteristic of all AT β-domains. Mutagenesis studies have demonstrated that the hairpin-like structure and hydrophobic cavity are crucial for BrkA passenger domain (virulence effector) translocation. This structure helps in understanding the molecular mechanism of AT assembly and secretion and provides a potential target for anti-pertussis drug design.     

A novel structural tree for wrap-proteins,a subclass of (α+β)-proteins

In this paper, a novel structural subclass of (α+β)-proteins is presented. A characteristic feature of these proteins and domains is that they consist of strongly twisted and coiled β-sheets wrapped around one or two α-helices, so they are referred to here as wrap-proteins. It is shown that overall folds of the wrap-proteins can be obtained by stepwise addition of α-helices and/or β-strands to the strongly twisted and coiled β-hairpin taken as the starting structure in modeling. As a result of modeling, a structural tree for the wrap-proteins was constructed that includes 201 folds of which 49 occur in known nonhomologous proteins.     

Crystal structure of the human collagen XV trimerization domain: A potent trimerizing unit common to multiplexin collagens

Correct folding of the collagen triple helix requires a self-association step which selects and binds α-chains into trimers. Here we report the crystal structure of the trimerization domain of human type XV collagen. The trimerization domain of type XV collagen contains three monomers each composed of four β-sheets and an α-helix. The hydrophobic core of the trimer is devoid of solvent molecules and is shaped by β-sheet planes from each monomer. The trimerization domain is extremely stable and forms at picomolar concentrations. It is found that the trimerization domain of type XV collagen is structurally similar to that of type XVIII, despite only 32% sequence identity. High structural conservation indicates that the multiplexin trimerization domain represents a three dimensional fold that allows for sequence variability while retaining structural integrity necessary for tight and efficient trimerization.     

Analysis and prediction of the packing of α-helices against a β-sheet in the tertiary structure of globular proteins

The packing of α-helices and β-sheets in six $\text{α}\text{β}$ proteins (e.g. flavodoxin) has been analysed. The results provide the basis for a computer algorithm to predict the tertiary structure of an $\text{α}\text{β}$ protein from its amino acid sequence and actual assignment of secondary structure.The packing of an individual α-helix against a β-sheet generally involves two adjacent ± 4 rows of non-polar residues on the α-helix at the positions i, i + 4, i + 8, i + 1, i + 5, i + 9. The pattern of interacting β-sheet residues results from the twisted nature of the sheet surface and the attendant rotation of the side-chains. At a more detailed level, four of the α-helical residues (i + 1, i + 4, i + 5 and i + 8) form a diamond that surrounds one particular β-sheet residue, generally isoleucine, leucine or valine. In general, the α-helix sits 10 Å above the sheet and lies parallel to the strand direction.The prediction follows a combinational approach. First, a list of possible β-sheet structures (10⁶ to 10¹⁴) is constructed by the generation of all β-sheet topologies and β-strand alignments. This list is reduced by constraints on topology and the location of non-polar residues to mediate the sheet/helix packing, and then rank-ordered on the extent of hydrogen bonding. This algorithm was uniformly applied to 16 $\text{α}\text{β}$ domains in 13 proteins. For every structure, one member of the reduced list was close to the crystal structure; the root-mean-square deviation between equivalenced C^α atoms averaged 5.6 Å for 100 residues. For the $\text{α}\text{β}$ proteins with pure parallel β-sheets, the total number of structures comparable to or better than the native in terms of hydrogen bonds was between 1 and 148. For proteins with mixed β-sheets, the worst case is glyceraldehyde-3-phosphate dehydrogenase, where as many as 3800 structures would have to be sampled. The evolutionary significance of these results as well as the potential use of a combinatorial approach to the protein folding problem are discussed.     

Do parallel β-strands have dipole moments? An ab initio molecular-orbital-direct reaction field study

Recently, it was suggested that parallel β-sheets have a significant dipole moment, in contrast to antiparallel sheets. Ab initio molecular-orbital (MO) calculations on parallel and antiparallel β-strands of tetra(Gly) show that they have very similar charge distributions. Interaction energies between two and three strands of tetra(Gly), obtained using the direct reaction field Hamiltonian, show that a particular choice of point charges is probably not crucial for calculating interactions within β-sheets, but that it might be for calculating interactions between these sheets and other parts of a protein, in particular, α-helices. The point-charge representation of our MO-SCF results will probably reduce the hazard of introducing artefacts in electrostatic calculations of protein conformational energies, provided the short-range interactions are treated in a more realistic way, i.e., such that intra- and interchain induction effects are included.     

Protein Structures and Neutral Theory of Evolution

Abstract

The neutral theory of evolution is extended to the origin of protein molecules. Arguments are presented which suggest that the amino acid sequences of many globular proteins mainly represent “memorized” random sequences while biological evolution reduces to the “editing” these random sequences. Physical requirements for a functional globular protein are formulated and it is shown that many of these requirements do not involve strategical selection of amino acid sequences during biological evolution but are inherent also for typical random sequences. In particular, it is shown that random sequences of polar and unpolar amino acid residues can form α-helices and β-strands with lengths and arrangement along the chain similar to those in real globular proteins. These α- and β-regions in random sequences can form three-dimensional folding patterns also similar to those in real proteins. The arguments are presented suggesting that even the tight packing of side groups inside protein core do not require very strong biological selection of amino acid sequences either. Thus many structural features of real proteins can exist also in random sequences and the biological selection is needed mainly for the creation of active sites of proteins and for their stability under physiological conditions.     

Secondary-structure analysis of alcohol-denatured proteins by vacuum-ultraviolet circular dichroism spectroscopy

To elucidate the structural characteristics of alcohol-denatured proteins, we measured the vacuum-ultraviolet circular dichroism (VUVCD) spectra of six proteins-myoglobin, human serum albumin, α-lactalbumin, thioredoxin, β-lactoglobulin, and α-chymotrypsinogen A-down to 170 nm in trifluoroethanol solutions (TFE: 0-50%) and down to 175 nm in methanol solutions (MeOH: 0-70%) at pH 2.0 and 25°C, using a synchrotron-radiation VUVCD spectrophotometer. The contents of α-helices, β-strands, turns, poly-L-proline type II helices (PPIIs), and unordered structures of these proteins were estimated using the SELCON3 program, including the numbers of α-helix and β-strand segments. Furthermore, the positions of α-helices and β-strands on amino acid sequences were predicted by combining these secondary-structure data with a neural-network method. All alcohol-denatured proteins showed higher α-helix contents (up to ~ 90%) compared with the native states, and they consisted of several long helical segments. The helix-forming ability was higher in TFE than in MeOH, whereas small amounts of β-strands without sheets were formed in the MeOH solution. The produced α-helices were transformed dominantly from the β-strands and unordered structures, and slightly from the turns. The content and mean length of α-helix segments decreased as the number of disulfide bonds in the proteins increased, suggesting that disulfide bonds suppress helix formation by alcohols. These results demonstrate that alcohol-denatured proteins constitute an ensemble of many long α-helices, a few β-strands and PPIIs, turns, and unordered structures, depending on the types of proteins and alcohols involved.     

The Erythrocyte/Brain Glucose Transporter (GLUT1) May Adopt a Two–Channel Transmembrane α/β Structure.

There are two models of topology for the membrane domains of the erythrocyte/brain facilitative glucose transporter, GLUT1. The first is composed of 12 membrane–spanning α–helices, the second of 16 membrane-spanning β-strands. We have used Jähnig′s and Eisenberg′s methods to identify possible transmembrane segments (10 spanning α-helices and 4 β-strands). The topology proposed is more consistent with available experimental data from FTIR, CD and mapping experiment than the previous models . We suggest that GLUT1 might form two channels, one of which is responsible for glucose transport. This agrees with the theoretical and experimental arguments. Finally, an analysis of the mutation periodicity and of the mean hydrophobicity for the GLUT family is provided in order to evaluate the packing of the protein in the membrane.