Investigating Diproline Segments in Proteins: Occurrences, Conformation and Classification

The covalent linkage between the side-chain and the backbone nitrogen atom of proline leads to the formation of the five-membered pyrrolidine ring and hence restriction of the backbone torsional angle phi to values of -60 degrees +/- 30 degrees for the L-proline. Diproline segments constitute a chain fragment with considerably reduced conformational choices. In the current study, the conformational states for the diproline segment ((L)Pro-(L)Pro) found in proteins has been investigated with an emphasis on the cis and trans states for the Pro-Pro peptide bond. The occurrence of diproline segments in turns and other secondary structures has been studied and compared to that of Xaa-Pro-Yaa segments in proteins which gives us a better understanding on the restriction imposed on other residues by the diproline segment and the single proline residue. The study indicates that P(II)-P(II) and P(II)-alpha are the most favorable conformational states for the diproline segment. The analysis on Xaa-Pro-Yaa sequences reveals that the XaaPro peptide bond exists preferably as the trans conformer rather than the cis conformer. The present study may lead to a better understanding of the behavior of proline occurring in diproline segments which can facilitate various designed diproline-based synthetic templates for biological and structural studies. (C) 2011 Wiley Periodicals, Inc. Biopolymers 97: 54-64, 2012.

Hydration dynamics of protein molecules in aqueous solution: Unity among diversity

Dielectric dispersion and NMRD experiments have revealed that a significant fraction of water molecules in the hydration shell of various proteins do not exhibit any slowing down of dynamics. This is usually attributed to the presence of the hydrophobic residues (HBR) on the surface, although HBRs alone cannot account for the large amplitude of the fast component. Solvation dynamics experiments and also computer simulation studies, on the other hand, repeatedly observed the presence of a non-negligible slow component. Here we show, by considering three well-known proteins (lysozyme, myoglobin and adelynate kinase), that the fast component arises partly from the response of those water molecules that are hydrogen bonded with the backbone oxygen (BBO) atoms. These are structurally and energetically less stable than those with the side chain oxygen (SCO) atoms. In addition, the electrostatic interaction energy distribution (EIED) of individual water molecules (hydrogen bonded to SCO) with side chain oxygen atoms shows a surprising two peak character with the lower energy peak almost coincident with the energy distribution of water hydrogen bonded to backbone oxygen atoms (BBO). This two peak contribution appears to be quite general as we find it for lysozyme, myoglobin and adenylate kinase (ADK). The sharp peak of EIED at small energy (at less than 2 k(B)T) for the BBO atoms, together with the first peak of EIED of SCO and the HBRs on the protein surface, explain why a large fraction (similar to 80%) of water in the protein hydration layer remains almost as mobile as bulk water Significant slowness arises only from the hydrogen bonds that populate the second peak of EIED at larger energy (at about 4 k(B)T). Thus, if we consider hydrogen bond interaction alone, only 15-20% of water molecules in the protein hydration layer can exhibit slow dynamics, resulting in an average relaxation time of about 5-10 ps. The latter estimate assumes a time constant of 20-100 ps for the slow component. Interestingly, relaxation of water molecules hydrogen bonded to back bone oxygen exhibit an initial component faster than the bulk, suggesting that hydrogen bonding of these water molecules remains frustrated. This explanation of the heterogeneous and non-exponential dynamics of water in the hydration layer is quantitatively consistent with all the available experimental results, and provides unification among diverse features.

Network properties of protein-decoy structures

Convergence of the vast sequence space of proteins into a highly restricted fold/conformational space suggests a simple yet unique underlying mechanism of protein folding that has been the subject of much debate in the last several decades. One of the major challenges related to the understanding of protein folding or in silico protein structure prediction is the discrimination of non-native structures/decoys from the native structure. Applications of knowledge-based potentials to attain this goal have been extensively reported in the literature. Also, scoring functions based on accessible surface area and amino acid neighbourhood considerations were used in discriminating the decoys from native structures. In this article, we have explored the potential of protein structure network (PSN) parameters to validate the native proteins against a large number of decoy structures generated by diverse methods. We are guided by two principles: (a) the PSNs capture the local properties from a global perspective and (b) inclusion of non-covalent interactions, at all-atom level, including the side-chain atoms, in the network construction accommodates the sequence dependent features. Several network parameters such as the size of the largest cluster, community size, clustering coefficient are evaluated and scored on the basis of the rank of the native structures and the Z-scores. The network analysis of decoy structures highlights the importance of the global properties contributing to the uniqueness of native structures. The analysis also exhibits that the network parameters can be used as metrics to identify the native structures and filter out non-native structures/decoys in a large number of data-sets; thus also has a potential to be used in the protein `structure prediction' problem.

Structural characterization of folded pentapeptides containing centrally positioned beta(R)Val, gamma(R)Val and gamma(S)Val residues

A cylindrical pore of similar to 7.5 angstrom diameter containing a one-dimensional water wire, within the confines of a hydrophobic channel lined with the valine side chain, has been observed in crystals of the peptide Boc-D-Pro-Aib-Val-Aib-Val-OMe (1) (Raghavender et al., 2009, 2010). The synthesis and structural characterization in crystals of three backbone homologated analogues Boc-D-Pro-Aib-beta(3)(R) Val-Aib-Val-OMe (2), Boc-D-Pro-Aib-gamma(4)(R)Val-Aib-Val-OMe (3), Boc-D-Pro-Aib-gamma(4)(S)Val-Aib-Val-OMe (4) are described. Crystal structures of peptides 2, 3 and 4 reveal close-packed arrangements in which no pore was formed. In peptides 2 and 3 the N-terminus D-Pro-Aib segment adopted conformations closely related to Type II' beta-turns, while residues 2-4 form one turn of an alpha beta right-handed C-11 helix in 2 and an alpha gamma C-12 helix in 3. In peptide 4, a continuous left-handed helical structure was observed with the D-Pro-Aib segment forming a Type III' beta-turn, followed by one turn of a left-handed alpha gamma C-12 helix. (C) 2012 Elsevier Ltd. All rights reserved.

A touch of history and a peep into the future of the lipid-quinone known as coenzyme Q and ubiquinone

Coenzyme Q (ubiquinone), a fully substituted benzoquinone with polyprenyl side chain, participates in many cellular redox activities. Paradoxically it was discovered only in 1957, albeit being ubiquitous. It required a person, F. L. Crane, a place, Enzyme Institute, Madison, USA, and a time when D. E. Green was directing vigorous research on mitochondria. Located at the transition of 2-electron flavoproteins and 1-electron cytochrome carriers, it facilitates electron transfer through the elegant Q-cycle in mitochondria to reduce O-2 to H2O, and to H2O2, now a significant signal-transducing agent, as a minor activity in shunt pathway (animals) and alternative oxidase (plants). The ability to form Q-radical by losing an electron and a proton was ingeniously used by Mitchell to explain the formation of the proton gradient, considered the core of energy transduction, and also in acidification in vacuoles. Known to be a mobile membrane constituent (microsomes, plasma membrane and Golgi apparatus), allowing it to reach multiple sites, coenzyme Q is expected to have other activities. Coenzyme Q protects circulating lipoproteins being a better lipid antioxidant than even vitamin E. Binding to proteins such as QPS, QPN, QPC and uncoupling protein in mitochondria, QA and QB in the reaction centre in R. sphaeroides, and disulfide bond-forming protein in E. coli (possibly also in Golgi), coenzyme Q acquires selective functions. A characteristic of orally dosed coenzyme Q is its exclusive absorption into the liver, but not the other tissues. This enrichment of Q is accompanied by significant decrease of blood pressure and of serum cholesterol. Inhibition of formation of mevalonate, the common precursor in the branched isoprene pathway, by the minor product, coenzyme Q, decreases the major product, cholesterol. Relaxation of contracted arterial smooth muscle by a side-chain truncated product of coenzyme Q explains its effect of decreasing blood pressure. Extensive clinical studies carried out on oral supplements of coenzyine Q, initially by K. Folkers and Y. Yamamura and followed many others, revealed a large number of beneficial effects, significantly in cardiovascular diseases. Such a variety of effects by this lipid quinone cannot depend on redox activity alone. The fat-soluble vitamins (A, D, E and K) that bear structural relationship with coenzyme Q are known to be active in their polar forms. A vignette of modified forms of coenzyme Q taking active role in its multiple effects is emerging.

Designed three-stranded beta-sheet in an alpha/beta hybrid peptide

The incorporation of beta-amino acid residues into the antiparallel beta-strand segments of a multi-stranded beta-sheet peptide is demonstrated for a 19-residue peptide, Boc-LV(beta)FV(D)PGL(beta)FVVL(D)PGLVL(beta)FVV-OMe (BBH19). Two centrally positioned (D)Pro-Gly segments facilitate formation of a stable three-stranded beta-sheet, in which beta-phenylalanine ((beta)Phe) residues occur at facing positions 3, 8 and 17. Structure determination in methanol solution is accomplished by using NMR-derived restraints obtained from NOEs, temperature dependence of amide NH chemical shifts, rates of H/D exchange of amide protons and vicinal coupling constants. The data are consistent with a conformationally well-defined three-stranded beta-sheet structure in solution. Cross-strand interactions between (beta)Phe3/(beta)Phe17 and (beta)Phe3/Val15 residues define orientations of these side-chains. The observation of close contact distances between the side-chains on the N- and C-terminal strands of the three-stranded beta-sheet provides strong support for the designed structure. Evidence is presented for multiple side-chain conformations from an analysis of NOE data. An unusual observation of the disappearance of the Gly NH resonances upon prolonged storage in methanol is rationalised on the basis of a slow aggregation step, resulting in stacking of three-stranded beta-sheet structures, which in turn influences the conformational interconversion between type I' and type II' beta-turns at the two (D)Pro-Gly segments. Experimental evidence for these processes is presented. The decapeptide fragment Boc-LV(beta)FV(D)PGL(beta)FVV-OMe (BBH10), which has been previously characterized as a type I' beta-turn nucleated hairpin, is shown to favour a type II' beta-turn conformation in solution, supporting the occurrence of conformational interconversion at the turn segments in these hairpin and sheet structures.

Chemical unfolding of chicken villin headpiece in aqueous dimethyl sulfoxide solution: cosolvent concentration dependence, pathway, and microscopic mechanism

Unfolding of a protein often proceeds through partial unfolded intermediate states (PUIS). PUIS have been detected in several experimental and simulation studies. However, complete analyses of transitions between different PUIS and the unfolding trajectory are sparse. To understand such dynamical processes, we study chemical unfolding of a small protein, chicken villin head piece (HP-36), in aqueous dimethyl sulfoxide (DMSO) solution. We carry out molecular dynamics simulations at various solution compositions under ambient conditions. In each concentration, the initial step of unfolding involves separation of two adjacent native contacts, between phenyl alanine residues (11-18 and 7-18). This first step induces, under appropriate conditions, subsequent separation among other hydrophobic contacts, signifying a high degree of cooperativity in the unfolding process. The observed sequence of structural changes in HP-36 on increasing DMSO concentration and the observed sequence of PUIS, are in approximate agreement with earlier simulation results (in pure water) and experimental observations on unfolding of HP-36. Peculiar to water-DMSO mixture, an intervening structural transformation (around 15% of DMSO) in the binary mixture solvent retards the progression of unfolding as composition is increased. This is reflected in a remarkable nonmonotonic composition dependence of RMSD, radius of gyration and the fraction of native contacts. At 30% mole fraction of DMSO, we find the extended randomly coiled structure of the unfolded protein. The molecular mechanism of DMSO induced unfolding process is attributed to the initial preferential solvation of the hydrophobic side chain atoms through the methyl groups of DMSO, followed by the hydrogen bonding of the oxygen atom of DMSO to the exposed backbone NH groups of HP-36.

Structure, molecular dynamics, and stress in a linear polymer

We present a study correlating uniaxial stress in a polymer with its underlying structure when it is strained. The uniaxial stress is significantly influenced by the mean-square bond length and mean bond angle. In contrast, the size and shape of the polymer, typically represented by the end-to-end length, mass ratio, and radius of gyration, contribute negligibly. Among externally set control variables, density and polymer chain length play a critical role in influencing the anisotropic uniaxial stress. Short chain polymers more or less behave like rigid molecules. Temperature and rate of loading, in the range considered, have a very mild effect on the uniaxial stress.

Mammalian neuronal sodium channel Blocker mu-conotoxin BuIIIB has a structured N-terminus that influences potency

Among the mu-conotoxins that block vertebrate voltage-gated sodium channels (VGSCs), some have been shown to be potent analgesics following systemic administration in mice. We have determined the solution structure of a new representative of this family, mu-BuIIIB, and established its disulfide connectivities by direct mass spectrometric collision induced dissociation fragmentation of the peptide with disulfides intact The major oxidative folding product adopts a 1-4/2-5/3-6 pattern with the following disulfide bridges: Cys5-Cys17, Cys6-Cys23, and Cys13-Cys24. The solution structure reveals that the unique N-terminal extension in mu-BuIIIB, which is also present in mu-BuIIIA and mu-BuIIIC but absent in other mu-conotoxins, forms part of a short a-helix encompassing Glu3 to Asn8. This helix is packed against the rest of the toxin and stabilized by the Cys5-Cys17 and Cys6-Cys23 disulfide bonds. As such, the side chain of Val1 is located close to the aromatic rings of Trp16 and His20, which are located on the canonical helix that displays several residues found to be essential for VGSC blockade in related mu-conotoxins. Mutations of residues 2 and 3 in the N-terminal extension enhanced the potency of mu-BuIIIB for Na(v)1.3. One analogue, D-Ala2]BuIIIB, showed a 40-fold increase, making it the most potent peptide blocker of this channel characterized to date and thus a useful new tool with which to characterize this channel. On the basis of previous results for related mu-conotoxins, the dramatic effects of mutations at the N-terminus were unanticipated and suggest that further gains in potency might be achieved by additional modifications of this region.

An automated approach to network features of protein structure ensembles

Network theory applied to protein structures provides insights into numerous problems of biological relevance. The explosion in structural data available from PDB and simulations establishes a need to introduce a standalone-efficient program that assembles network concepts/parameters under one hood in an automated manner. Herein, we discuss the development/application of an exhaustive, user-friendly, standalone program package named PSN-Ensemble, which can handle structural ensembles generated through molecular dynamics (MD) simulation/NMR studies or from multiple X-ray structures. The novelty in network construction lies in the explicit consideration of side-chain interactions among amino acids. The program evaluates network parameters dealing with topological organization and long-range allosteric communication. The introduction of a flexible weighing scheme in terms of residue pairwise cross-correlation/interaction energy in PSN-Ensemble brings in dynamical/chemical knowledge into the network representation. Also, the results are mapped on a graphical display of the structure, allowing an easy access of network analysis to a general biological community. The potential of PSN-Ensemble toward examining structural ensemble is exemplified using MD trajectories of an ubiquitin-conjugating enzyme (UbcH5b). Furthermore, insights derived from network parameters evaluated using PSN-Ensemble for single-static structures of active/inactive states of 2-adrenergic receptor and the ternary tRNA complexes of tyrosyl tRNA synthetases (from organisms across kingdoms) are discussed. PSN-Ensemble is freely available from http://vishgraph.mbu.iisc.ernet.in/PSN-Ensemble/psn_index.html.

A Histidine Aspartate Ionic Lock Gates the Iron Passage in Miniferritins from Mycobacterium smegmatis

Background: DNA-binding protein from starved cells (Dps) are nano-compartments that can oxidize and store iron rendering protection from free radicals. Results: A histidine-aspartate ionic cluster in mycobaterial Dps2 modulates the rate of iron entry and exit in these proteins. Conclusion: Substitutions that disrupt the cluster interface alter the iron uptake/release properties with localized structural changes. Significance: Identifying important gating residues can help in designing nano-delivery vehicles. Dps (DNA-binding protein from starved cells) are dodecameric assemblies belonging to the ferritin family that can bind DNA, carry out ferroxidation, and store iron in their shells. The ferritin-like trimeric pore harbors the channel for the entry and exit of iron. By representing the structure of Dps as a network we have identified a charge-driven interface formed by a histidine aspartate cluster at the pore interface unique to Mycobacterium smegmatis Dps protein, MsDps2. Site-directed mutagenesis was employed to generate mutants to disrupt the charged interactions. Kinetics of iron uptake/release of the wild type and mutants were compared. Crystal structures were solved at a resolution of 1.8-2.2 for the various mutants to compare structural alterations vis a vis the wild type protein. The substitutions at the pore interface resulted in alterations in the side chain conformations leading to an overall weakening of the interface network, especially in cases of substitutions that alter the charge at the pore interface. Contrary to earlier findings where conserved aspartate residues were found crucial for iron release, we propose here that in the case of MsDps2, it is the interplay of negative-positive potentials at the pore that enables proper functioning of the protein. In similar studies in ferritins, negative and positive patches near the iron exit pore were found to be important in iron uptake/release kinetics. The unique ionic cluster in MsDps2 makes it a suitable candidate to act as nano-delivery vehicle, as these gated pores can be manipulated to exhibit conformations allowing for slow or fast rates of iron release.

Allosteric Transition Induced by Mg2+ Ion in a Transactivator Monitored by SERS

We demonstrate the utility of the surface-enhanced Raman spectroscopy (SERS) to monitor conformational transitions in protein upon ligand binding. The changes in protein's secondary and tertiary structures were monitored using amide and aliphatic/aromatic side chain vibrations. Changes in these bands are suggestive of the stabilization of the secondary and tertiary structure of transcription activator protein C in the presence of Mg2+ ion, whereas the spectral fingerprint remained unaltered in the case of a mutant protein, defective in Mg2+ binding. The importance of the acidic residues in Mg2+ binding, which triggers an overall allosteric transition in the protein, is visualized in the molecular model. The present study thus opens up avenues toward the application of SERS as a potential tool for gaining structural insights into the changes occurring during conformational transitions in proteins.

Ramachandran analysis of conserved glycyl residues in homologous proteins of known structure

High conservation of glycyl residues in homologous proteins is fairly frequent. It is commonly understood that glycine tends to be highly conserved either because of its unique Ramachandran angles or to avoid steric clash that would arise with a larger side chain. Using a database of aligned 3D structures of homologous proteins we identified conserved Gly in 288 alignment positions from 85 families. Ninety-six of these alignment positions correspond to conserved Gly residue with (phi, ) values allowed for non-glycyl residues. Reasons for this observation were investigated by in-silico mutation of these glycyl residues to Ala. We found in 94% of the cases a short contact exists between the C atom of the introduced Ala with the atoms which are often distant in the primary structure. This suggests the lack of space even for a short side chain thereby explaining high conservation of glycyl residues even when they adopt (phi, ) values allowed for Ala. In 189 alignment positions, the conserved glycyl residues adopt (phi, ) values which are disallowed for Ala. In-silico mutation of these Gly residues to Ala almost always results in steric hindrance involving C atom of Ala as one would expect by comparing Ramachandran maps for Ala and Gly. Rare occurrence of the disallowed glycyl conformations even in ultrahigh resolution protein structures are accompanied by short contacts in the crystal structures and such disallowed conformations are not conserved in the homologues. These observations raise the doubt on the accuracy of such glycyl conformations in proteins.

INDUCTION OF FOLDED STRUCTURES IN DESIGNED PEPTIDES USING CONFORMATIONALLY CONSTRAINED RESIDUES

Folding into compact globular structures, with well-defined modules of secondary structure, appears to be a characteristic of long polypeptide chains, with a specific patterning of coded amino acid residues along the length of sequence. Cooperative hydrogen bond driven secondary structure formation and solvent forces, which contribute favorably to the entropy of folding, by promoting compaction of the polymeric chain, have long been discussed as major determinants of the folding process. First principles design approaches, which use non-coded amino acids, employ an alternative structure directing strategy, by using amino acid residues which exhibit a strong conformational bias for specific regions of the Ramachandran map. This overview of ongoing studies in the authors' laboratory, attempts to explore the use of conformationally restricted amino acid residues in the design of peptides with well-defined secondary structures. Short peptides composed of 20 genetically coded amino acids usually exist in solution as an ensemble of equilibrating conformations. Apolar peptide sequences, which are readily soluble in organic solvents like chloroform and methanol, facilitate formation of structures which are predominately driven by intramolecular hydrogen bond formation. The choice of sequences containing residues with a limited range of conformational choices strongly favors formation of local turn structures, stabilized by short range intramolecular hydrogen bonds. Two residue beta-turns can nucleate either helical or hairpin folding, depending on the precise conformation of the turn segment Restriction of the conformational space available to amino acid residues is easily achieved by introduction of an additional alkyl group at the C alpha carbon atom or by side chain backbone cyclization, as in proline. Studies of synthetic sequences incorporating two prototype residues alpha-aminoisobutyric acid (Aib) and D-proline (DPro) illustrate the utility of the strategy in construction of helices and hairpins. Extensions to the design of conformationally switchable sequences and structurally defined hybrid peptides containing backbone homologated residues are also surveyed.

Sparsely populated residue conformations in protein structures: Revisiting ``experimental'' Ramachandran maps

The Ramachandran map clearly delineates the regions of accessible conformational (phi-) space for amino acid residues in proteins. Experimental distributions of phi, values in high-resolution protein structures, reveal sparsely populated zones within fully allowed regions and distinct clusters in apparently disallowed regions. Conformational space has been divided into 14 distinct bins. Residues adopting these relatively rare conformations are presented and amino acid propensities for these regions are estimated. Inspection of specific examples in a completely arid, fully allowed region in the top left quadrant establishes that side-chain and backbone interactions may provide the energetic compensation necessary for populating this region of phi- space. Asn, Asp, and His residues showed the highest propensities in this region. The two distinct clusters in the bottom right quadrant which are formally disallowed on strict steric considerations correspond to the gamma turn (C7 axial) conformation (Bin 12) and the i + 1 position of Type II turns (Bin 13). Of the 516 non-Gly residues in Bin 13, 384 occupied the i + 1 position of Type II turns. Further examination of these turn segments revealed a high propensity to occur at the N-terminus of helices and as a tight turn in hairpins. The strand-helix motif with the Type II turn as a connecting element was also found in as many as 57 examples. Proteins 2014; 82:1101-1112. (c) 2013 Wiley Periodicals, Inc.