Flexibility Of Peptide Backbone Research Articles

Contribution of the 12-17 hydrophobic region of islet amyloid polypeptide in self-assembly and cytotoxicity.

The islet amyloid polypeptide (IAPP) is a 37-residue aggregation-prone peptide hormone whose deposition as insoluble fibrils in the islets of Langerhans is associated with type II diabetes. Therapeutic interventions targeting IAPP amyloidogenesis, which contributes to pancreatic β-cell degeneration, remain elusive owing to the lack of understanding of the self-assembly mechanisms and of the quaternary proteospecies mediating toxicity. While countless studies have investigated the contributions of the 20–29 amyloidogenic core in self-assembly, IAPP central region, i.e. positions 11 to 19, has been less studied, notwithstanding its potential key role in oligomerization. In this context, the present study aimed at investigating the physicochemical and conformational properties driving IAPP self-assembly and associated cytotoxicity. Computational tools and all-atom molecular dynamics simulation suggested that the hydrophobic 12–17 segment promotes IAPP self-recognition and aggregation. Alanine scanning revealed that the hydrophobic side chains of Leu12, Phe15 and Val17 are critical for amyloid fibril formation. Destabilization of the α-helical folding by Pro substitution enhanced self-assembly when the pyrrolidine ring was successively introduced at positions Ala13, Asn14 and Phe15, in comparison to respective Ala-substituted counterparts. Modulating the peptide backbone flexibility at position Leu16 through successive incorporation of Pro, Gly and α-methylalanine, inhibited amyloid formation and reduced cytotoxicity, while the isobutyl side chain of Leu16 was not critical for self-assembly and IAPP-mediated toxicity. These results highlight the importance of the 12–17 hydrophobic region of IAPP for self-recognition, ultimately supporting the development of therapeutic approaches to prevent oligomerization and/or fibrillization.

Effect of triphenylphosphonium moiety on spatial structure and biointeractions of stereochemical variants of YRFK motif.

Chemical modification of therapeutic peptides is an important approach to improving their physicochemical and pharmacokinetic properties. The triphenylphosphonium (TPP) cation has proved to be a powerful modifier; however, its effects on peptide structure and activity remain uncharacterized. In this study, cytoprotective tetrapeptides based on the YRFK opioid motif with L- or D-Arg residues were linked to (triphenylphosphonio)carboxylic acids with ethylene and pentylene spacers (TPP-3 and TPP-6 groups, respectively). The three-dimensional structure of the oligopeptides was analyzed by NMR spectroscopy, computational methods and circular dichroism (CD). A more compact and bent structure with segregated aromatic groups was revealed for the D-arginine-containing tetrapeptide and its TPP-6 derivative. The TPP moiety caused structure-organizing effect on the tetrapeptides, resulting in transition from random coil to β-sheet structures, and decreased the peptide backbone flexibility up to ten times. The TPP-3-modified oligopeptide with the lowest RMSD value (ca. 0.05Å) was characterized by intrapeptide hydrophobic interactions between the TPP and side groups of Tyr and Phe residues accompanied by strong CD induction. The TPP-6-modified oligopeptides showed enhanced ability to form intermolecular associates and disturb liposomal membranes. The relationship between the spatial structure of the oligopeptides and some of their biologically relevant interactions were additionally revealed and are discussed.

MFPred: Rapid and accurate prediction of protein-peptide recognition multispecificity using self-consistent mean field theory.

Multispecificity–the ability of a single receptor protein molecule to interact with multiple substrates–is a hallmark of molecular recognition at protein-protein and protein-peptide interfaces, including enzyme-substrate complexes. The ability to perform structure-based prediction of multispecificity would aid in the identification of novel enzyme substrates, protein interaction partners, and enable design of novel enzymes targeted towards alternative substrates. The relatively slow speed of current biophysical, structure-based methods limits their use for prediction and, especially, design of multispecificity. Here, we develop a rapid, flexible-backbone self-consistent mean field theory-based technique, MFPred, for multispecificity modeling at protein-peptide interfaces. We benchmark our method by predicting experimentally determined peptide specificity profiles for a range of receptors: protease and kinase enzymes, and protein recognition modules including SH2, SH3, MHC Class I and PDZ domains. We observe robust recapitulation of known specificities for all receptor-peptide complexes, and comparison with other methods shows that MFPred results in equivalent or better prediction accuracy with a ~10-1000-fold decrease in computational expense. We find that modeling bound peptide backbone flexibility is key to the observed accuracy of the method. We used MFPred for predicting with high accuracy the impact of receptor-side mutations on experimentally determined multispecificity of a protease enzyme. Our approach should enable the design of a wide range of altered receptor proteins with programmed multispecificities.

Correlation between biological activity and conformational dynamics properties of tetra- and pentapeptides derived from fetoplacental proteins

In this work, using molecular dynamics simulation, we study conformational and dynamic properties of biologically active penta- and tetrapeptides derived from fetoplacental proteins such as alpha-fetoprotein, pregnancy specific β1-glycoprotein, and carcinoembryonic antigen. Existence of correlation between flexibility of peptide backbone and biological activity of the investigated peptides was shown. It was also demonstrated that flexibility of peptide backbone depends not only on its length, but also on the presence of reactive functional groups in amino acid side chains that participate in intramolecular interactions. Peptides that demonstrate similar biological effects in regulation of proliferation of lymphocytes and expression of differentiation antigens on their surface (LDSYQCT, PYECE, YECE, and YVCE) are characterized by rigidity of their peptide backbone. Increased backbone flexibility in peptides PYQCE, YQCE, SYKCE, YQCT, YQCS, YVCS, YACS, and YACE is correlated with decreased biological activity. Conformational mobility of amino acid residues does not depend on physicochemical properties only, but also on intramolecular interactions. So, evolutionary restrictions should exist to maintain such interactions in the environment of functionally important sites.

Structure, Dynamics and Surface Hydrophobicity of the Cataract-Associated Mutant, Pro23Thr of Human Gamma D-crystallin: Molecular Basis of Cataract Formation

The cataract-associated Pro23Thr (P23T) mutation in human gammaD-crystallin (HGD) is geographically widespread - thus there is considerable interest in determining the molecular basis of opacity. In an earlier study (1), we found that the mutant showed markedly lowered, retrograde solubility compared to wild-type HGD, leading to the conclusion that the aggregation of P23T was mediated by hydrophobic protein-protein interactions. Subsequently, using NMR (2) and a binding assay with the fluorescent dye Bis-ANS (3), we showed that, in fact, hydrophobic patches were generated on the surface of the mutant protein. Those studies (3) also suggested that the binding site for Bis-ANS on P23T may coincide with the self-association site of the mutant and result in its lowered, retrograde solubility. Here we present new NMR-evidence to identify the Bis-ANS binding sites, and using independent NMR dynamics studies, also show that there is a measurable reduction in the flexibility of the peptide backbone near the hydrophobic patches. These two factors, namely the creation of hydrophobic patches and the lowered peptide backbone flexibility, taken together make a compelling argument that the hydrophobic patches may indeed facilitate the nucleation of aggregates of P23T which are held together by net hydrophobic interactions. Such aggregation would explain the retrograde solubility as well as the lens opacity in vivo. 1. Pande et al (2005) Biochemistry, 44, 2491-2500. 2. Pande et al (2009) Biochem. Biophys. Res. Commun., 382, 196-199. 3. Pande et al (2010) Biochemistry,49, 6122-6129. Supported by NIH grants GM085006 to AS and EY010535 to JP.

Modulation of Reconstituted Pig Kidney Na+/K+-ATPase Activity by Cholesterol in Endogenous Lipid Vesicles: Role of Lipid Domains

Diverse experimental and theoretical evidence suggests that plasma membranes contain cholesterol-induced segregated domains that could play a key role in the modulation of membrane functions, including intrinsic enzyme activity. To gain insight into the role of cholesterol, we reconstituted pig kidney Na+/K+-ATPase into unilamellar vesicles of endogenous lipids mimicking the natural membrane and addressed the question of how modification of the cholesterol content could affect the ATPase activity via changes in the membrane lipid phase and in the protein structure and dynamics. We used steady-state and time-resolved fluorescence spectroscopy with the lipid phase probes DPH and Laurdan and the protein probe fluorescein and also used infrared spectroscopy using attenuated total reflectance. Upon modification of membrane cholesterol content, the ATPase activity did not change monotonically but instead exhibited abrupt changes resulting in two peaks at or close to critical cholesterol mole fractions (25 and 33.3 mol %) predicted by the superlattice or regular distribution model. Fluorescence parameters associated with the membrane probes also showed abrupt changes with peaks, coincident with the cholesterol concentrations associated with the peaks in the enzyme activity, while parameters associated with the protein probes also showed slight but abrupt changes resulting in dips at the same cholesterol concentrations. Notably, the IR amide I band maximum also showed spectral shifts, characterized by a frequency variation pattern with peaks at the same cholesterol concentrations. Overall, these results indicate that the lipid phase had slightly lower hydration, at or near the two critical cholesterol concentrations predicted by the superlattice theory. However, in the protein domains monitored there was a slight but significant hydration increase along with increased peptide backbone flexibility at these cholesterol concentrations. We propose that in the vicinity of the critical mole fractions, where superlattice formation can occur, minute changes in cholesterol concentration produce abrupt changes in the membrane organization, increasing interdomain surfaces. These changes, in turn, induce small changes in the protein's structure and dynamics, therefore acting to fine-tune the enzyme.

Conformational Analysis of the C-Terminal Gly-Leu-Met-NH 2 Tripeptide of Substance P Bound to the NK-1 Receptor

We examined the effect of simultaneously incorporating proline or proline-amino acid chimeras in positions 9, 10, and/or 11 of substance P, on the affinity for the two NK-1 binding sites and on second-messenger activation. Because these 3-substituted prolines constrain not only the (phi,psi) values of the peptide backbone, but also the chi space of the amino acid side chain, we were able to gather data on the structural requirements for high-affinity binding to the NK-1 receptor. We were able to confirm that this C-terminal component is crucial and that it should adopt an extended conformation close to a polyproline II structure when bound to the receptor. The partial additivity of these constraints, more specifically, for the NK-1M site, suggests that the peptide backbone flexibility around the hinge-point residue Gly9 is essential to subtly position crucial side chains.

Nanosecond Dynamics of Acetylcholinesterase Near the Active Center Gorge

To delineate the role of peptide backbone flexibility and rapid molecular motion in acetylcholinesterase catalysis and inhibitor association, we investigated the decay of fluorescence anisotropy at three sites of fluorescein conjugation to cysteine-substitution mutants of the enzyme. One cysteine was placed in a loop at the peripheral site near the rim of the active center gorge (H287C); a second was in a helical region outside of the active center gorge (T249C); a third was at the tip of a small, flexible omega loop well separated from the gorge (A262C). Mutation and fluorophore conjugation did not appreciably alter catalytic or inhibitor binding parameters of the enzyme. The results show that each site examined was associated with a high degree of segmental motion; however, the A262C and H287C sites were significantly more flexible than the T249C site. Association of the active center inhibitor, tacrine, and the peripheral site peptide inhibitor, fasciculin, had no effect on the anisotropy decay of fluorophores at positions 249 and 262. Fasciculin, but not tacrine, on the other hand, dramatically altered the decay profile of the fluorophore at the 287 position, in a manner consistent with fasciculin reducing the segmental motion of the peptide chain in this local region. The results suggest that the motions of residues near the active center gorge and across from the Cys(69)-Cys(96) omega loop are uncoupled and that ligand binding at the active center or the peripheral site does not influence acetylcholinesterase conformational dynamics globally, but induces primarily domain localized decreases in flexibility proximal to the bound ligand.

Increased detection of structural templates using alignments of designed sequences

Protein structure prediction by comparative modeling benefits greatly from the use of multiple sequence alignment information to improve the accuracy of structural template identification and the alignment of target sequences to structural templates. Unfortunately, this benefit is limited to those protein sequences for which at least several natural sequence homologues exist. We show here that the use of large diverse alignments of computationally designed protein sequences confers many of the same benefits as natural sequences in identifying structural templates for comparative modeling targets. A large-scale massively parallelized application of an all-atom protein design algorithm, including a simple model of peptide backbone flexibility, has allowed us to generate 500 diverse, non-native, high-quality sequences for each of 264 protein structures in our test set. PSI-BLAST searches using the sequence profiles generated from the designed sequences ("reverse" BLAST searches) give near-perfect accuracy in identifying true structural homologues of the parent structure, with 54% coverage. In 41 of 49 genomes scanned using reverse BLAST searches, at least one novel structural template (not found by the standard method of PSI-BLAST against PDB) is identified. Further improvements in coverage, through optimizing the scoring function used to design sequences and continued application to new protein structures beyond the test set, will allow this method to mature into a useful strategy for identifying distantly related structural templates.

Effects of the N2144S mutation on backbone dynamics of a TB-cbEGF domain pair from human fibrillin-1

The calcium-binding epidermal growth factor-like (cbEGF) module and the transforming growth factor β-binding protein-like (TB) module are the two major structural motifs found in fibrillin-1, the extracellular matrix (ECM) protein defective in the Marfan syndrome (MFS). An MFS-causing mutation, N2144S, which removes a calcium ligand in cbEGF32, does not detectably affect fibrillin-1 biosynthesis, rate of secretion, processing, or deposition of reducible fibrillin-1 into the ECM. Since the residue at position 2144 is normally engaged in calcium ligation, it is unable to mediate intermolecular interactions. We have shown previously that this mutation does not affect the folding properties of the TB or cbEGF domains in vitro, but does decrease calcium-binding in cbEGF and TB-cbEGF domain constructs. Here, we use NMR spectroscopy to probe the effects of the N2144S mutation on backbone dynamic properties of TB6-cbEGF32. Analysis of the backbone 15N relaxation data of wild-type TB6-cbEGF32 has revealed a flexible inter-domain linkage. Parallel dynamics analysis of the N2144S mutant has shown increased flexibility in the region joining the two domains as well as in the calcium-binding site at the N terminus of cbEGF32. This research demonstrates that a small change in peptide backbone flexibility, which does not enhance proteolytic susceptibility of the domain pair, is associated with an MFS phenotype. Flexibility of the TB-cbEGF linkage is likely to contribute to the biomechanical properties of fibrillin-rich connective tissue microfibrils, and may play a role in the microfibril assembly process.

Val(659)-->Glu mutation within the transmembrane domain of ErbB-2: effects measured by (2)H NMR in fluid phospholipid bilayers.

Certain point mutations within the hydrophobic transmembrane domains of class I receptor tyrosine kinases have been associated with oncogenic transformation in vitro and in vivo [Gullick, J., and Srinivasan, R. (1998) Breast Cancer Res. Treat. 52, 43-53]. An important example is the replacement of a single (hydrophobic) valine by (charged) glutamate in the rat protein, Neu, and in the homologous human protein, ErbB-2. It has been suggested that the oncogenic nature of this Val-->Glu substitution may derive from alteration of the transmembrane domain's ability to take part in direct side-to-side associations. In the present work, we examined the basis of this phenomenon by studying transmembrane portions of ErbB-2 in fluid bilayer membranes. An expression system was designed to produce such peptides from the wild-type ErbB-2, and from an identical region of the transforming mutant in which Val(659) is replaced by Glu. All peptides were 50-mers, containing the appropriate transmembrane domain plus contiguous stretches of amino acids from the cytoplasmic and extracellular domains. Deuterium heteronuclear probes were incorporated into alanine side chains (thus, each alanine -CH(3) side chain became -CD(3)). Given the presence of natural alanine residues at positions 648 and 657 within ErbB-2, this approach afforded heteronuclear probes within the motif Ser(656)AlaValValGlu(660), thought to be important for homodimer formation, and nine residues upstream of this site. Further peptides were produced, by site-directed mutagenesis, to confirm spectral assignments and to provide an additional probe location at position 670 (11 residues downstream of the motif region). On SDS-polyacrylamide gels, the transmembrane peptides migrated as predominant monomers in equilibrium with smaller populations of homodimers/-oligomers. CD spectra of both wild-type and transforming mutant peptides were consistent with the transmembrane portions being basically alpha-helical. (2)H NMR spectra of each transmembrane peptide were obtained in fluid phospholipid bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) from 35 to 65 degrees C. Results were consistent with the concept that the glutamic acid residue characterizing the mutant is uncharged at neutral pH. Narrowed spectral components from species rotating rapidly and symmetrically within the membrane appeared to represent monomeric peptide. Mutation of Val(659) to Glu within the hydrophobic domain induced changes in side chain angulation of at least 6-8 degrees at Ala(657) (i.e., within the five amino acid motif thought to be involved in homodimer formation), and downstream of this site to residue 670. There was little evidence of effect at the upstream site (Ala(648)) at the membrane surface. This result argues that the transforming mutation is associated with significant intramolecular rearrangement of the monomeric transmembrane helix-extending over some four helix turns-which could influence its lateral associations. In addition, temperature effects on spectral quadrupole splittings suggested that there is greater peptide backbone flexibility for the wild-type transmembrane region.

Phase transfer catalysis in solid phase peptide synthesis

Relatively small cyclic peptides that contain functionalized side chains provide interesting model compounds for studying side chain-side chain interactions, peptide backbone flexibility (especially if X-Pro bonds are included), and as potential enzyme mimetics. In order to develop more efficient synthetic routes to compounds such as cyclo(Xxx-Pro-Gly-Yyy-Pro-Gly), using the Merrifield method, we have investigated several orthogonal solid phase synthesis strategies and contrasted the use of two solid phase peptide-resin cleavage techniques for preparing partially protected linear sequences. Phase transfer catalysis using tetrabutyl ammonium hydrogen sulfate in THF with saturated aqueous K2CO3 provides peptide acid salts in which most of the common protecting groups (Arg(NO2), Tyr(Bzl), Z-Lys, Lys(Boc), and Glu(tBu)) are not affected. Using 500 MHz proton NMR, peptides having a cyclo (L-L-Gly-L-L-Gly) sequence generally display two conformers in DMSO-d6 with the major isomer being the bis-cis conformer, while the minor form contains two beta turns. For peptides with a cyclo(D-L-Gly-L-L-Gly) sequence, the major conformer contains one cis and one trans X-Pro bond and one Type II beta turn, as previously predicted for related structure by Kopple and others.

Structural requirements for thioester bond formation in human complement component C3. Reassessment of the role of thioester bond integrity on the conformation of C3.

A unique thioester bond, formed between the side chains of neighboring C and Q residues, is present in complement components C3 and C4 and the protease inhibitor alpha 2-macroglobulin. This structure is essential for mediating covalent attachment to target acceptors and also for maintaining these proteins in their native conformation. An examination of the residues in the immediate vicinity of the C and Q reveals a very high degree of sequence similarity among the three proteins which crosses species barriers. The following is the sequence flanking the thioester residues in C3, the highly conserved amino acids being underlined and the the thioester-forming residues being indicated by italics: 1005V-T-P-S-G-C-G-E-Q-N-M-I-G-M-T-P-T1021. Through a site-directed mutagenesis and cDNA expression approach, we have examined the importance of the conserved amino acids in the formation, stability, and function of the thioester bond in C3. The behavior of the mutants fell into three categories. The potential loss in peptide backbone flexibility by the replacement of G1009 by A or S was permissive to thioester formation and function as was replacement of M1015 by the still fairly bulky residue F. In contrast, replacement of M1015 by A resulted in an alpha-chain which was highly unstable toward proteolytic degradation. The third category, which included mutant molecules P1007G, P1020G, E1012Q, and Q1013N, displayed an unusual phenotype in which both the autolytic fragmentation and the hemolytic activity characteristics of thioester-intact molecules were absent. However, like their wildtype counterpart, these molecules retained the ability to be cleaved by C3 convertase (C4b2a), a conformation-dependent property that is normally lost in the conversion of native C3 to thioester-hydrolyzed C3(H2O). Since an identical functional profile was obtained when the thioester was deliberately prevented from forming in the mutant C1010A, we conclude that if a stable thioester fails to form during biosynthesis, at least parts of the mature protein can adopt a more native-like conformation than is the case when the thioester is first formed and then hydrolyzed in the mature protein. In view of these new findings, the interpretation of the previously observed correlation between the loss of thioester integrity and the adoption of a C3b-like conformation must be reassessed.

Primary Structural Determinants Essential for Potent Inhibition of cAMP-dependent Protein Kinase by Inhibitory Peptides Corresponding to the Active Portion of the Heat-Stable Inhibitor Protein

PKI-(5-24)-amide is a 20-residue peptide with the sequence, Thr5-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-A la-Ile-His- Asp24-NH2, that corresponds to the active portion of the heat-stable inhibitor protein of cAMP-dependent protein kinase (Cheng, H.-C., Kemp, B. E., Pearson, R. B., Smith, A. J., Misconi, L., Van Patten, S. M., and Walsh, D. A. (1986) J. Biol. Chem. 261, 989-992). Amino acid residues in PKI-(5-24)-amide responsible for the potent inhibition (Ki = 2.3 nM) of the catalytic subunit of protein kinase were further investigated using deletion and substitution analogs of the synthetic peptide. Residues 5, 23, and 24 were not required for activity since the 17-residue PKI-(6-22)-amide retained full potency. Sequential removal of the first seven amino acids from the NH2 terminus of PKI-(5-24)-amide caused a progressive 50-fold loss of inhibitory potency. In contrast, substitution of either Thr6, Asp9, or Ile11 with alanine, or Ala8 by leucine, in PKI-(5-22)-amide produced less than 3-fold decreases in potency. Of the 2 aromatic residues in PKI-(5-22)-amide, the individual substitution of Phe10 and Tyr7 by alanine caused, respectively, 90- and 5-fold decreases in inhibitory potency, demonstrating important roles for each. This NH2-terminal portion of the peptide is believed to contain a significant portion of alpha-helix. Many recognition or structural determinants are also essential in the COOH-terminal portion of PKI-(5-22)-amide. In addition to the basic subsite provided by the three arginines, several other of the residues are critical for full inhibitory potency. Substitution of Ile22 by glycine in either PKI-(5-22)-amide or PKI-(14-22)-amide lowered the inhibitory potency by 150- and 50-fold, respectively. Separate replacement of Gly17 or Asn20, in either PKI-(5-22)-amide or PKI-(14-22)-amide, caused 7-15-fold decreases in potency. Substitution of both Gly17 and Asn20 together (in PKI-(14-22)-amide) produced a synergistic loss of inhibitory activity. [Leu13,Ile14]PKI-(5-22)-amide, a doubly substituted analog exhibited a 42-fold increase in Ki value. We conclude that Ser13 and/or Gly14, Gly17, Asn20, and Ile22 each contribute important features to the binding of these inhibitory peptides to the protein kinase, either by providing recognition determinants, inducing structure, and/or allowing essential peptide backbone flexibility.(ABSTRACT TRUNCATED AT 400 WORDS)

Conformation‐Determining role for the N‐acetyl group in the O‐glycosidic linkage, α‐GalNAc‐Thr

AbstractAn 1H‐nmr study of 2‐acetamido‐2‐deoxy‐3,4,6‐tri‐O‐acetyl‐D‐galactopyranose (AcGalNAc) glycosylated Thr‐containing tripeptides in Me2SO‐d6 solution reveals two mutually exclusive intramolecular hydrogen bonds. In Z‐Thr(AcGalNAc)‐Ala‐Ala‐OMe, there is an intramolecular hydrogen bond between the Thr amide proton and the sugar N‐acetyl carbonyl oxygen. The strength of this hydrogen bond will be dependent on the amino acid residues on the Thr C terminal side to some undetermined distance. In Ac‐Thr(AcGalNAc)‐Ala‐Ala‐OMe, a different intramolecular hydrogen bond between the sugar N‐acetyl amide proton and the Thr carbonyl oxygen exists. The choice of hydrogen bonds seems dependent on the bulkiness of the residues on the Thr N terminal side. The consequence of such strong hydrogen bonds is a clearly defined orientation of the sugar moiety with respect to the peptide backbone. In the former, the plane of the sugar pyranose ring is roughly oriented perpendicularly to the peptide backbone. The latter orientation is where the plane of the sugar ring is roughly in line with the peptide backbone. In both orientations, the sugar moiety can increase the shielding of the neighboring amino acid residues from the solvent. The idea that the amino acid residues near the glycosylated Thr influence orientation of the sugar moiety with respect to the peptide backbone and in turn possibly hinder peptide backbone flexibility has interesting implications in the conformational as well as the biological role of O‐glycoproteins.