Helix packing motif common to the crystal structures of two undecapeptides containing dehydrophenylalanine residues: implications for the de novo design of helical bundle super secondary structural modules

De novo designed peptide based super secondary structures are expected to provide scaffolds for the incorporation of functional sites as in proteins. Self-association of peptide helices of similar screw sense, mediated by weak interactions, has been probed by the crystal structure determination of two closely related peptides: Ac-Gly1-Ala2-ΔPhe3-Leu4-Val5-ΔPhe6-Leu7-Val8-ΔPhe9-Ala10-Gly11-NH2 (I) and Ac-Gly1-Ala2-ΔPhe3-Leu4-Ala5-ΔPhe6-Leu7-Ala8-ΔPhe9-Ala10-Gly11-NH2 (II). The crystal structures determined to atomic resolution and refined to R factors 8.12 and 4.01%, respectively, reveal right-handed 310-helical conformations for both peptides. CD has also revealed the preferential formation of right-handed 310-helical conformations for both molecules. Our aim was to critically analyze the packing of the helices in the solid state with a view to elicit clues for the design of super secondary structural motifs such as two, three, and four helical bundles based on helix-helix interactions. An important finding is that a packing motif could be identified common to both the structures, in which a given peptide helix is surrounded by six other helices reminiscent of transmembrane seven helical bundles. The outer helices are oriented either parallel or antiparallel to the central helix. The helices interact laterally through a combination of N-H ... O, C-H ... O, and C-H ... π hydrogen bonds. Layers of interacting leucine residues are seen in both peptide crystal structures. The packing of the peptide helices in the solid state appears to provide valuable leads for the design of super secondary structural modules such as two, three, or four helix bundles by connecting adjacent antiparallel helices through suitable linkers such as tetraglycine segment

structural insights into n terminal to c terminal interactions and implications for thermostability of a β α 8 triosephosphate isomerase barrel enzyme

Although several factors have been suggested to contribute to thermostability, the stabilization strategies used by proteins are still enigmatic. Studies on a recombinant xylanase from Bacilllus sp. NG-27 (RBSX), which has the ubiquitous (beta/alpha)(8)-triosephosphate isomerase barrel fold, showed that just a single mutation, V1L, although not located in any secondary structural element, markedly enhanced the stability from 70 degrees C to 75 degrees C without loss of catalytic activity. Conversely, the V1A mutation at the same position decreased the stability of the enzyme from 70 degrees C to 68 degrees C. To gain structural insights into how a single extreme N-terminus mutation can markedly influence the thermostability of the enzyme, we determined the crystal structure of RBSX and the two mutants. On the basis of computational analysis of their crystal structures, including residue interaction networks, we established a link between N-terminal to C-terminal contacts and RBSX thermostability. Our study reveals that augmenting N-terminal to C-terminal noncovalent interactions is associated with enhancement of the stability of the enzyme. In addition, we discuss several lines of evidence supporting a connection between N-terminal to C-terminal noncovalent interactions and protein stability in different proteins. We propose that the strategy of mutations at the termini could be exploited with a view to modulate stability without compromising enzymatic activity, or in general, protein function in diverse folds where N and C termini are in close proximity. Database The coordinates of RBSX, V1A and V1L have been deposited in the PDB database under the accession numbers 4QCE, 4QCF, and 4QDM, respectivel

Observation of glycine zipper and unanticipated occurrence of ambidextrous helices in the crystal structure of a chiral undecapeptide

<p>Abstract</p> <p>Background</p> <p>The <it>de novo </it>design of peptides and proteins has recently surfaced as an approach for investigating protein structure and function. This approach vitally tests our knowledge of protein folding and function, while also laying the groundwork for the fabrication of proteins with properties not precedented in nature. The success of these studies relies heavily on the ability to design relatively short peptides that can espouse stable secondary structures. To this end, substitution with α, β-dehydroamino acids, especially α, β-dehydrophenylalanine (ΔPhe) comes in use for spawning well-defined structural motifs. Introduction of ΔPhe induces β-bends in small and 3₁₀-helices in longer peptide sequences.</p> <p>Results</p> <p>The present report is an investigation of the effect of incorporating two glycines in the middle of a ΔPhe containing undecapeptide. A de novo designed undecapeptide, Ac-Gly¹-Ala²-ΔPhe³-Leu⁴-Gly⁵-ΔPhe⁶-Leu⁷-Gly⁸-ΔPhe⁹-Ala¹⁰-Gly¹¹-NH₂, was synthesized and characterized using X-ray diffraction and Circular Dichroism spectroscopic methods. Crystallographic studies suggest that, despite the presence of L-amino acid (L-Ala and L-Leu) residues in the middle of the sequence, the peptide adopts a 3₁₀-helical conformation of ambidextrous screw sense, one of them a left-handed (A) and the other a right-handed (B) 3₁₀-helix with A and B being antiparallel to each other. However, CD studies reveal that the undecapeptide exclusively adopts a right-handed 3₁₀-helical conformation. In the crystal packing, three different interhelical interfaces, Leu-Leu, Gly-Gly and ΔPhe-ΔPhe are observed between the helices A and B. A network of C-H...O hydrogen bonds are observed at ΔPhe-ΔPhe and Gly-Gly interhelical interfaces. An important feature observed is the occurrence of glycine zipper motif at Gly-Gly interface. At this interface, the geometric pattern of interhelical interactions seems to resemble those observed between helices in transmembrane (TM) proteins.</p> <p>Conclusion</p> <p>The present design strategy can thus be exploited in future work on de novo design of helical bundles of higher order and compaction utilizing ΔPhe residues along with GXXG motif.</p

Functionally important segments in proteins dissected using Gene Ontology and geometric clustering of peptide fragments

A geometric clustering algorithm has been developed to dissect protein fragments based on their relevance to function

The Critical Role of Partially Exposed N-Terminal Valine Residue in Stabilizing GH10 Xylanase from Bacillus sp.NG-27 under Poly-Extreme Conditions

BACKGROUND: Understanding the mechanisms that govern protein stability under poly-extreme conditions continues to be a major challenge. Xylanase (BSX) from Bacillus sp. NG-27, which has a TIM-barrel structure, shows optimum activity at high temperature and alkaline pH, and is resistant to denaturation by SDS and degradation by proteinase K. A comparative circular dichroism analysis was performed on native BSX and a recombinant BSX (R-BSX) with just one additional methionine resulting from the start codon. The results of this analysis revealed the role of the partially exposed N-terminus in the unfolding of BSX in response to an increase in temperature. METHODOLOGY: We investigated the poly-extremophilicity of BSX to deduce the structural features responsible for its stability under one set of conditions, in order to gain information about its stability in other extreme conditions. To systematically address the role of the partially exposed N-terminus in BSX stability, a series of mutants was generated in which the first hydrophobic residue, valine (Val1), was either deleted or substituted with various amino acids. Each mutant was subsequently analyzed for its thermal, SDS and proteinase K stability in comparison to native BSX. CONCLUSIONS: A single conversion of Val1 to glycine (Gly) changed R-BSX from being thermo- and alkali- stable and proteinase K and SDS resistant, to being thermolabile and proteinase K-, alkali- and SDS- sensitive. This result provided insight into the structure-function relationships of BSX under poly-extreme conditions. Molecular, biochemical and structural data revealed that the poly-extremophilicity of BSX is governed by a partially exposed N-terminus through hydrophobic interactions. Such hitherto unidentified N-terminal hydrophobic interactions may play a similar role in other proteins, especially those with TIM-barrel structures. The results of the present study are therefore of major significance for protein folding and protein engineering

The Critical Role of N- and C-Terminal Contact in Protein Stability and Folding of a Family 10 Xylanase under Extreme Conditions

Stabilization strategies adopted by proteins under extreme conditions are very complex and involve various kinds of interactions. Recent studies have shown that a large proportion of proteins have their N- and C-terminal elements in close contact and suggested they play a role in protein folding and stability. However, the biological significance of this contact remains elusive.In the present study, we investigate the role of N- and C-terminal residue interaction using a family 10 xylanase (BSX) with a TIM-barrel structure that shows stability under high temperature, alkali pH, and protease and SDS treatment. Based on crystal structure, an aromatic cluster was identified that involves Phe4, Trp6 and Tyr343 holding the N- and C-terminus together; this is a unique and important feature of this protein that might be crucial for folding and stability under poly-extreme conditions. folding and activity. Alanine substitution with Phe4, Trp6 and Tyr343 drastically decreased stability under all parameters studied. Importantly, substitution of Phe4 with Trp increased stability in SDS treatment. Mass spectrometry results of limited proteolysis further demonstrated that the Arg344 residue is highly susceptible to trypsin digestion in sensitive mutants such as ΔF4, W6A and Y343A, suggesting again that disruption of the Phe4-Trp6-Tyr343 (F-W-Y) cluster destabilizes the N- and C-terminal interaction. Our results underscore the importance of N- and C-terminal contact through aromatic interactions in protein folding and stability under extreme conditions, and these results may be useful to improve the stability of other proteins under suboptimal conditions

A short commentary on indents and edges of β-sheets

Abstractβ-sheets in proteins are formed by extended polypeptide chains, called β-strands. While there is a general consensus on two types of β-strands, viz. ‘edge strands’ (or ‘edges’) and ‘inner strands’ (or ‘central strands’), the possibility of distinguishing between different regions of inner strands remains less explored. In this paper, we address the portions of inner strands of β-sheets that stick out on either or both sides. We call these portions the ‘indent strands’ or ‘indents’ because they give the typical indented appearance to β-sheets. Similar to the edge strands, the indent strands also have β-bridge partner residues on one side while the other side is still open for backbone hydrogen bonds. Despite this similarity, the indent strands differ from the edge strands in terms of various properties such as β-bulges and amino acid composition due to their localization within β-sheets and therefore within folded proteins to certain extent. The localization of indents and edges within folded proteins seems to govern the strategies deployed to deter unhindered β-sheet propagation through β-strand stacking interactions. Our findings suggest that, edges and indents differ in their strategies to avoid further β-strand stacking. Short length itself is a good strategy to avoid stacking and a majority of indents are two residue or shorter in length. Edge strands on the other hand are overall longer. While long edges are known to use various negative design strategies like β-bulges, prolines, strategically placed charges, inward-pointing charged side chains and loop coverage to avoid further β-strand stacking, long indents seem to favor mechanisms such as enrichment in flexible residues with high solvation potential and depletion in hydrophobic residues in response to their less solvent exposed nature. Such subtle differences between indents and edges could be leveraged for designing novel β-sheet architectures.</jats:p

Phylogenetic Studies and Inhibitor Design Targeting Protein Interacting Interface of Nucleoid-Associated Protein HU

AbstractThe formations of nucleoprotein structures by promiscuous DNA binding proteins like HU are assisted with their protein protein interaction capability with other proteins. InE. coliGal repressosome assembly, GalR piggybacks HU to the critical position on the DNA (hbs site) through a specific GalR–HU interaction using an interface at the bottom of alpha helical region, which we termed as HUpb interface. Similarly, MtbHU also interact with Topoisomerase I with the same interface to enhance its relaxation activity. In an earlier study, we determined the crystal structure of MtbHU, inhibited it using stilbene derivatives which inhibited the cell growth. It motivated us to understand the evolutionary and structural characteristics of the HUpb interface, which has not been investigated previously for HU or for any other NAPs. Our analyses found residue positions corresponding to MtbHU Thr11 to Gln20 form the interface while Ala23 serves the pocket lining residue. Due to ancestral mutations in the duplication event before the HU and IHF split, physicochemical properties of the interface vary among clades. Thus, this interface could engage different proteins in different HU oligomeric states inProteobacteria. Protein-protein interfaces are usually a challenging target due to its flatter surface. In case of MtbHUpb interface, we observed that due to the presence of a partially hydrophobic pocket, small molecule scaffolds could fit into it, while the ligand can be further designed to interact with D17, which is the crucial residue for Topoisomerase I interaction. We used a two-step virtual screening protocol with known drug like molecules as starting set to an aim to re-purpose drugs. Our docking results showed compounds like Maltotetraose, Valrubicin, Iodixanol, Enalkiren, indinavir, Carfilzomib, oxytetracycline, quinalizarin, Raltitrexed, Epigallocatechin and their analogues exhibit high scoring binding at MtbHUpb interface. Our present report gives a model example of an evolutionary study of an interface of nucleoid associated protein, which is used to computationally design inhibitors. This strategy could be in general useful for designing inhibitors for various types of protein-protein interfaces using evolutionary guided design.</jats:p