Investigating proline puckering states in diproline segments in proteins

In the current study, the puckering states of the Proline ring occurring in diproline segments (^LPro‐^LPro) in proteins has been investigated with a segregation made on the basis of cis and trans states for the Pro‐Pro peptide bond and the conformational states for the diproline segment to investigate the effects of conformation of the diproline segment on the corresponding puckering state of the Proline ring in the segment if any. The value of the endocyclic ring torsional angles of the pyrrolidine ring has been used for calculating and visualizing various puckering states using a proposed new sign convention (+/?) nomenclature. The results have been compared to that obtained in a previous study on peptides from this group. In this study, quite interestingly, the Planar (G) conformation that was present in 14.3% of the cases in peptides, appears to be nearly a rare conformation in the case of proteins (1.9%). The present study indicates that the (C^γ‐exo/C^γ‐exo), (C^γ‐exo/Twisted C^γ‐exo‐C^β‐endo) and (Twisted C^γ‐endo‐C^β‐exo/Twisted C^γ‐endo‐C^β‐exo) categories are the most preferred combinations. For Proline rings in proteins, the states C^γ‐exo, Twisted C^γ‐exo‐C^β‐endo and Twisted C^γ‐endo‐C^β‐exo are the most preferred states. Within diproline segments, the pyrrolidine ring conformations do not show a strong co‐relation to the backbone conformation in which they are observed. It is likely that five‐membered rings have a considerable plasticity of structure and are readily deformed to accommodate a variety of energetically preferred backbone conformations. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 605–610, 2013.     

Characterization of a type of β‐bend ribbon spiral generated by the repeating (Xaa–Yaa–Aib–Pro) motif: The solution structure of harzianin HC IX,a 14‐residue peptaibol forming voltage‐dependent ion channels

The three‐dimensional solution structure of harzianin HC IX, a peptaibol antibiotic isolated from the fungus Trichoderma harzianum, was determined using CD, homonuclear, and heteronuclear two‐dimensional nmr spectroscopy combined with molecular modeling. This 14‐residue peptide, Ac Aib¹ Asn² Leu³ Aib⁴ Pro⁵ Ala⁶ Ile⁷ Aib⁸ Pro⁹ Iva¹⁰ Leu¹¹ Aib¹² Pro¹³ Leuol¹⁴ (Aib, α‐aminoisobutyric acid; Iva, isovaline; Leuol, leucinol), is a main representative of a short‐sequence peptaibol class characterized by an acetylated N‐terminus, a C‐terminal amino alcohol, and the presence of three Aib‐L ‐Pro motifs at positions 4–5, 8–9, and 12–13, separated by two dipeptide units. In spite of a lower number of residues, compared to the 18/20‐residue peptaibols such as alamethicin, harzianin HC IX exhibits remarkable membrane‐perturbing properties. It interacts with phospholipid bilayers, increasing their permeability and forming voltage‐gated ion channels through a mechanism slightly differing from that proposed for alamethicin. Sequence‐specific ¹H‐ and ¹³C‐nmr assignments and conformational nmr parameters (³J_NHCαH coupling constants, quantitative nuclear Overhauser enhancement data, temperature coefficients of amide and carbonyl groups, NH–ND exchange rates) were obtained in methanol solution. Sixty structures were calculated based on 98 interproton distance restraints and 6 Φ dihedral angle restraints, using high temperature restrained molecular dynamics and energy minimization. Thirty‐seven out of the sixty generated structures were consistent with the nmr data and were convergent. The peptide backbone consists in a ribbon of overlapping β‐turns twisted into a continuous spiral from Asn² to Leuol¹⁴ and forming a 26 Å long helix‐like structure. This structure is slightly amphipathic, with the three Aib–Pro motifs aligned on the less hydrophobic face of the spiral where the Asn² side chain is also present, while the more hydrophobic bulky side chains of leucines, isoleucine, isovaline, and leucinol are located on the concave side. The repetitive (Xaa–Yaa–Aib–Pro) tetrapeptide subunit, making up the peptide sequence, is characterized by four sets of (Φ,Ψ) torsional angles, with the following mean values: Φ_i = −90°, Ψ_i = −27°; Φ_i+1 = −98°, Ψ_i+1 = −17°; Φ_i+2 = −49°, Ψ_i+2 = −50°; Φ_i+3 = −78°, Ψ_i+3 = +3°. We term this particular structure, specifically occurring in the case of (Xaa–Yaa–Aib–Pro)_n sequences, the (Xaa–Yaa–Aib–Pro)‐β‐bend ribbon spiral. It is stabilized by 4 → 1 intramolecular hydrogen bonds and differs from both the canonical 3₁₀‐helix made of a succession of type III β‐turns and from the β‐bend ribbon spiral that has been described in the case of (Aib–Pro)n peptide segments. © 1999 John Wiley & Sons, Inc. Biopoly 50: 71–85, 1999     

Cyclic peptides. XVIII. 13C spin-lattice relaxation times of (X-pro-Y)2 cyclic hexapeptides

¹³C spin-lattice relaxation times (T₁'s) of four cyclic hexapeptides of sequence, (X-L -Pro-Y)₂, are reported. The T₁'s of the protonated carbons, which undergo dipolar relaxation, are interpreted qualitatively in terms of the overall tumbling motion of the molecule and in terms of internal motion. It is found that three of the cyclic hexapeptides, those which adopt all-trans β-conformers, tumble isotropically and appear to lack internal motion in the peptide backbone. The method of Torchia and Lyerla was applied to these compounds in order to compare the mobility of the proline rings. The results show that the sequence and particular type of β-turn present affect the internal motion of the Pro ring. Data on a fourth cyclic hexapeptide, which occurs in a conformation with two-cis X-Pro bonds, suggests that internal motion of the backbone contributes an additional frequency component to the motion of the Y residue α-carbons. A consideration of the mobility of the proline rings in the conformer with two-cis peptide bonds revealed that they are significantly more rigid in the two-cis structure than in the all-trans.     

Comprehensive Chiroptical Study of Proline‐Containing Diamide Compounds

The continuously growing interest in the understanding of peptide folding led to the conformational investigation of methylamides of N‐acetyl‐amino acids as diamide models. Here we report the results of detailed conformational analysis on Ac‐Pro‐NHMe and Ac‐β‐HPro‐NHMe diamides. These compounds were analyzed by experimental and computational methods, the conformational distributions obtained by Density Functional Theory (DFT) calculations for isolated and solvated diamide compounds are discussed. The conformational preference of proline‐containing diamide compounds as a function of the ambience was observed by a number of chiroptical spectroscopic techniques, such as vibrational circular dichroism (VCD), electronic circular dichroism (ECD), Raman optical activity (ROA) spectroscopy, and additionally by single crystal X‐ray diffraction analyses. Based on a comparison between Ac‐Pro‐NHMe and Ac‐β‐HPro‐NHMe, one can conclude that due to the greater conformational freedom of the β‐HPro derivative, Ac‐β‐HPro‐NHMe shows different behavior in solid‐ and solution‐phase, as well. Ac‐β‐HPro‐NHMe tends to form cis Ac‐β‐HPro amide conformation in water, dichloromethane, and acetonitrile in contrast to its α‐Pro analog. On the other hand, the crystal structure of the β‐HPro compound cannot be related to any of the conformers obtained in vacuum and solution while the X‐ray structure of Ac‐Pro‐NHMe was identified as tα_L–, which is a trans Ac‐Pro amide containing conformer also predominant in polar solvents. Chirality 26:228–242, 2014. © 2014 Wiley Periodicals, Inc.     

Synthesis and diuretic activities of pseudoproline‐containing analogues of the insect kinin core pentapeptide

C‐2 dimethylated/unmethylated thiazolidine‐4‐carboxylic acid and C‐2 dimethylated oxazolidine‐4‐carboxylic acid were introduced into the insect kinin core pentapeptide in place of Pro³, yielding three new analogues. NMR analysis revealed that the peptide bond of Phe²‐pseudoproline (ΨPro)³ is practically 100% in cis conformation in the case of dimethylated pseudoproline‐containing analogues, about 50% cis for the thiazolidine‐4‐carboxylic acid analogue and about 33% cis for the parent Pro³ peptide. The diuretic activities are consistent with the population of cis conformation of the Phe²‐ΨPro³/Pro³ peptide bonds, and the results confirm a cis Phe‐Pro bond as bioactive conformation. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Structural role of a conserved active site cis proline in the Thermotoga maritima acetyl esterase from the carbohydrate esterase family 7

A conserved cis proline residue located in the active site of Thermotoga maritima acetyl esterase (TmAcE) from the carbohydrate esterase family 7 (CE7) has been substituted by alanine. The residue was known to play a crucial role in determining the catalytic properties of the enzyme. To elucidate the structural role of the residue, the crystal structure of the Pro228Ala variant (TmAcE_P228A) was determined at 2.1 Å resolution. The replacement does not affect the overall secondary, tertiary, and quaternary structures and moderately decreases the thermal stability. However, the wild type cis conformation of the 227–228 peptide bond adopts a trans conformation in the variant. Other conformational changes in the tertiary structure are restricted to residues 222–226, preceding this peptide bond and are located away from the active site. Overall, the results suggest that the conserved proline residue is responsible for the cis conformation of the peptide and shapes the geometry of the active site. Elimination of the pyrrolidine ring results in the loss of van der Waals and hydrophobic interactions with both the alcohol and acyl moeities of the ester substrate, leading to significant impairment of the activity and perturbation of substrate specificity. Furthermore, a cis‐to‐trans conformational change arising out of residue changes at this position may be associated with the evolution of divergent activity, specificity, and stability properties of members constituting the CE7 family. Proteins 2017; 85:694–708. © 2016 Wiley Periodicals, Inc.     

Loss of intramolecular electrostatic interactions and limited conformational ensemble may promote self‐association of cis–tau peptide

Self‐association of proteins can be triggered by a change in the distribution of the conformational ensemble. Posttranslational modification, such as phosphorylation, can induce a shift in the ensemble of conformations. In the brain of Alzheimer's disease patients, the formation of intra‐cellular neurofibrillary tangles deposition is a result of self‐aggregation of hyper‐phosphorylated tau protein. Biochemical and NMR studies suggest that the cis peptidyl prolyl conformation of a phosphorylated threonine‐proline motif in the tau protein renders tau more prone to aggregation than the trans isomer. However, little is known about the role of peptidyl prolyl cis/trans isomerization in tau aggregation. Here, we show that intra‐molecular electrostatic interactions are better formed in the trans isomer. We explore the conformational landscape of the tau segment containing the phosphorylated‐Thr²³¹‐Pro²³² motif using accelerated molecular dynamics and show that intra‐molecular electrostatic interactions are coupled to the isomeric state of the peptidyl prolyl bond. Our results suggest that the loss of intra‐molecular interactions and the more restricted conformational ensemble of the cis isomer could favor self‐aggregation. The results are consistent with experiments, providing valuable complementary atomistic insights and a hypothetical model for isomer specific aggregation of the tau protein. Proteins 2015; 83:436–444. © 2014 Wiley Periodicals, Inc.     

Conformational Properties of the CalliFMRF-Amide Series Neuropeptides

Conformational properties of five neuropeptides belonging to the calliFMRF-amide series with the Xaa-Pro-Yaa-Gln-Asp-Phe-Met-Arg-Phe-NH₂ homologous sequences were studied by the method of theoretical conformational analysis. Three members of these group [(1) (Xaa = Thr, Yaa = Gln), (2) (Xaa = Thr, Yaa = Ser), and (3) (Xaa = Yaa = Ser)] can stimulate the saliva secretion from the separated salivary gland of the Calliphora vomitoria fly, whereas two other calliFMRF-amides [(4) (Xaa = Lys, Yaa = Asn) and (5) (Xaa = Ala, Yaa = Gly)] are inactive in this biological test. Low-energy spatial structures of the studied compounds were determined by a conformational analysis. A comparison of the stable structures of the biologically active and inactive neuropeptides revealed a similarity in their conformational properties and allowed determination of the role of separate residues in the peptide folding. The calculations demonstrated that the C-terminal hexapeptide fragment identical in all the five peptides tends to form -helical structure, whereas the variable N-terminal tripeptide regions of calliFMRF-amides (1)–(5) form more conformationally flexible structures.     

Noncollagenous region of the streptococcal collagen‐like protein is a trimerization domain that supports refolding of adjacent homologous and heterologous collagenous domains

Proper folding of the (Gly‐Xaa‐Yaa)_n sequence of animal collagens requires adjacent N‐ or C‐terminal noncollagenous trimerization domains which often contain coiled‐coil or beta sheet structure. Collagen‐like proteins have been found recently in a number of bacteria, but little is known about their folding mechanism. The Scl2 collagen‐like protein from Streptococcus pyogenes has an N‐terminal globular domain, designated V_sp, adjacent to its triple‐helix domain. The V_sp domain is required for proper refolding of the Scl2 protein in vitro. Here, recombinant V_sp domain alone is shown to form trimers with a significant α‐helix content and to have a thermal stability of T_m = 45°C. Examination of a new construct shows that the V_sp domain facilitates efficient in vitro refolding only when it is located N‐terminal to the triple‐helix domain but not when C‐terminal to the triple‐helix domain. Fusion of the V_sp domain N‐terminal to a heterologous (Gly‐Xaa‐Yaa)_n sequence from Clostridium perfringens led to correct folding and refolding of this triple‐helix, which was unable to fold into a triple‐helical, soluble protein on its own. These results suggest that placement of a functional trimerization module adjacent to a heterologous Gly‐Xaa‐Yaa repeating sequence can lead to proper folding in some cases but also shows specificity in the relative location of the trimerization and triple‐helix domains. This information about their modular nature can be used in the production of novel types of bacterial collagen for biomaterial applications.     

High‐resolution structures of collagen‐like peptides [(Pro‐Pro‐Gly)4‐Xaa‐Yaa‐Gly‐(Pro‐Pro‐Gly)4]: Implications for triple‐helix hydration and Hyp(X) puckering

Structures of (Pro‐Pro‐Gly)₄‐Xaa‐Yaa‐Gly‐(Pro‐Pro‐Gly)₄ (ppg9‐XYG) where (Xaa, Yaa) = (Pro, Hyp), (Hyp, Pro) or (Hyp, Hyp) were analyzed at high resolution using synchrotron radiation. Molecular and crystal structures of these peptides are very similar to those of the (Pro‐Pro‐Gly)₉ peptide. The results obtained in this study, together with those obtained from related compounds, indicated the puckering propensity of the Hyp in the X position: (1) Hyp(X) residues involved in the Hyp(X):Pro(Y) stacking pairs prefer the down‐puckering conformation, as in ppg9‐OPG, and ppg9‐OOG; (2) Hyp(X) residues involved in the Hyp(X):Hyp(Y) stacking pairs prefer the up‐puckering conformation if there is no specific reason to adopt the down‐puckering conformation. Water molecules in these peptide crystals are classified into two groups, the 1st and 2nd hydration waters. Water molecules in the 1st hydration group have direct hydrogen bonds with peptide oxygen atoms, whereas those in the 2nd hydration group do not. Compared with globular proteins, the number of water molecules in the 2nd hydration shell of the ppg9‐XYG peptides is very large, likely due to the unique rod‐like molecular structure of collagen model peptides. In the collagen helix, the amino acid residues in the X and Y positions must protrude outside of the triple helix, which forces even the hydrophobic side chains, such as Pro, to be exposed to the surrounding water molecules. Therefore, most of the waters in the 2nd hydration shell are covering hydrophobic Pro side chains by forming clathrate structures. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 361–372, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Steric and electronic interactions controlling the cis/trans isomer equilibrium at X‐pro tertiary amide motifs in solution

A systematic understanding of the noncovalent interactions that influence the structures of the cis conformers and the equilibrium between the cis and the trans conformers, of the X‐Pro tertiary amide motifs, is presented based on analyses of ¹H‐, ¹³C‐NMR and FTIR absorption spectra of two sets of homologous peptides, X‐Pro‐Aib‐OMe and X‐Pro‐NH‐Me (where X is acetyl, propionyl, isobutyryl and pivaloyl), in solvents of varying polarities. First, this work shows that the cis conformers of any X‐Pro tertiary amide motif, including Piv‐Pro, are accessible in the new motifs X‐Pro‐Aib‐OMe, in solution. These conformers are uniquely observable by FTIR spectroscopy at ambient temperatures and by NMR spectroscopy from temperatures as high as 273 K. This is made possible by the persistent presence of n_i‐1→π_i* interactions at Aib, which also influence the disappearance of steric effects at these cis X‐Pro rotamers. Second, contrary to conventional understanding, the energy contribution of steric effects to the cis/trans equilibrium at the X‐Pro motifs is found to be nonvariant (0.54 ± 0.02 kcal/mol) with increase in steric bulk on the X group. Third, the current studies provide direct evidence for the weak intramolecular interactions namely the n_i‐1→π_i*, the N_Pro???H_i+1 (C₅a), and the C₇ hydrogen bond that operate and influence the structures, stabilities, and dynamics between different conformational states of X‐Pro tertiary amide motifs. NMR and IR spectral data suggest that the cis conformers of X‐Pro motifs are ensembles of short‐lived rotamers about the C′_X–N_Pro bond. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 66–77, 2014.     

N‐terminal diproline and charge group effects on the stabilization of helical conformation in alanine‐based short peptides: CD studies with water and methanol as solvent

Protein folding problem remains a formidable challenge as main chain, side chain and solvent interactions remain entangled and have been difficult to resolve. Alanine‐based short peptides are promising models to dissect protein folding initiation and propagation structurally as well as energetically. The effect of N‐terminal diproline and charged side chains is assessed on the stabilization of helical conformation in alanine‐based short peptides using circular dichroism (CD) with water and methanol as solvent. A1 (Ac–Pro–Pro–Ala–Lys–Ala–Lys–Ala–Lys–Ala–NH₂) is designed to assess the effect of N‐terminal homochiral diproline and lysine side chains to induce helical conformation. A2 (Ac–Pro–Pro–Glu–Glu–Ala–Ala–Lys–Lys–Ala–NH₂) and A3 (Ac–d Pro–Pro–Glu–Glu–Ala–Ala–Lys–Lys–Ala–NH₂) with N‐terminal homochiral and heterochiral diproline, respectively, are designed to assess the effect of Glu...Lys (i , i  + 4) salt bridge interactions on the stabilization of helical conformation. The CD spectra of A1 , A2 and A3 in water manifest different amplitudes of the observed polyproline II (PPII) signals, which indicate different conformational distributions of the polypeptide structure. The strong effect of solvent substitution from water to methanol is observed for the peptides, and CD spectra in methanol evidence A2 and A3 as helical folds. Temperature‐dependent CD spectra of A1 and A2 in water depict an isodichroic point reflecting coexistence of two conformations, PPII and β‐strand conformation, which is consistent with the previous studies. The results illuminate the effect of N‐terminal diproline and charged side chains in dictating the preferences for extended‐β, semi‐extended PPII and helical conformation in alanine‐based short peptides. The results of the present study will enhance our understanding on stabilization of helical conformation in short peptides and hence aid in the design of novel peptides with helical structures. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

The Structural Basis of a Conserved P2 Threonine in Canonical Serine Proteinase Inhibitors

Abstract

Bowman-Birk inhibitors (BBIs) are a well-studied family of canonical inhibitor proteins of serine proteinases. In nature, the active region of BBIs possesses a highly conserved Thr at the P2 position. The importance of this residue has been reemphasized by synthetic BBI reactive site loop proteinomimetics. In particular, this residue was exclusively identified for active chymotrypsin inhibitors selected from a BBI template-assisted combinatorial peptide library. A further kinetic analysis of 26 P2 variant peptides revealed that Thr provides both optimal binding affinity and optimal resistance against enzymatic turnover by chymotrypsin.

Herein, we report the H-NMR spectroscopic study of a 5-membered sub-set of these reactive site loop peptides representing a stepwise elimination of the Thr side-chain functionalities and inversion of its side-chain chirality. The P2 Thr variant adopts a three-dimensional structure that closely mimics the one of the corresponding region of the complete protein. This validates the use of this template for the investigation of structure-function relationships. While the overall backbone geometry is similar in all studied variants, conformational changes induced by the modification of the P2 side chain have now been identified and provide a rational explanation of the kinetically observed functional differences. Eliminating the γ-methyl group has little structural effect, whereas the elimination of the γ-oxygen atom or the inversion of the side-chain chirality results in characteristic changes to the intramolecular hydrogen bond network. We conclude that the transannular hydrogen bond between the P2 Thr side-chain hydroxyl and the P5′ backbone amide is an important conformational constraint and directs the hydrophobic contact of the P2 Thr side chain with the enzyme surface in a functionally optimal geometry, both in the proteinomimetic and the native protein.

In at least four canonical inhibitor protein families similar structural arrangements for a conserved P2 Thr have been observed, which suggests an analogous functional role. Substitutions at P2 of the proteinomimetic also affect the conformational balance between cis and trans isomers at a distant Pro-Pro motif (P3′-P4′). Presented with a mixture of cis/trans isomers chymotrypsin appears to interact preferably with the conformer that retains the cis-P3′ Pro-trans-P4′ Pro geometry found in the parent BBI protein.     

Determination of an unusual secondary structural element in the immunostimulating tetrapeptide rigin in aqueous environments: insights via MD simulations, 1H NMR and CD spectroscopic studies

An immunomodulating tetrapeptide, rigin (H‐Gly‐Gln‐Pro‐Arg‐OH), has been examined for its secondary structure preferences through combined use of high‐temperature unrestrained MD simulations in implicit water and 1D and 2D ¹H NMR spectroscopy. The distribution of backbone torsion angles revealed the predominance of trans conformers across the Xaa‐Pro peptide bond. The results of MD simulations revealed that of the five predicted families A–E, the predominant families, family A (92 structures), family C (63 structures) and family D (31 structures), could be complemented by extensive 1D and 2D ¹H NMR parameters acquired in aqueous PBS solution. A survey of specific inter‐ and intraresidue NOEs substantiated the predominance of an unusual type VII β‐turn structure, defined by two torsion angles, i.e. ψ_Gln ～ 155° and ?_Pro ～ ? 65° across the Gln‐Pro segment. The proposed semi‐folded kinked topology precluded formation of any intramolecular interaction, i.e. hydrogen bond or electrostatic interaction. Far‐UV CD spectral characteristics of rigin in aqueous PBS solution and non‐aqueous structure‐promoting organic solvents, TFE and TMP, revealed its strong solvent dependence. However, in aqueous PBS solution, the presence of a weak negative shoulder at nm could be ascribed to a small population with ordered, semi‐folded topology. We propose that the plausible structural attributes may be exploited for design and rigidification of the bioactive conformation of this immunomodulator toward improved immunopharmacological properties. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Cis-Trans equilibrium and kinetic studies of acetyl-L-proline and glycyl-L-proline

By means of carbon-13 nmr (at 25 MHz) the trans/cis conformer ratio in glycyl-L -proline has been measured in aqueous (D₂O) solution over the temperature range 33–96°C. It is found that ΔH⁰ = ?4.2 kJ/mole and ΔS⁰ = ?9.7 J/mole/K. Measurements of the T₁ values for the proline ring carbons yielded values consistent with a fast puckering process involving both the β- and γ-carbons. Measurements of the rate of cis-trans conformational interconversion in glycyl-L -proline, using complete line-shape analysis for the glycyl α-carbon resonance, gave values for the trans → cis isomerization as follows: ΔH^≠ = 83.5 ± 0.2 kJ/mole; ΔS^≠ = 0.0 ± 10 J/mole/K. A more approximate determination from coalescence temperature observations gave a value of ΔG^≠ of 82.0 ± 0.4 kJ/mole for this process in acetyl-L -proline in aqueous solution. The presence of 12M NaSCN lowered this barrier by ca. 2.6 kJ/mole. Such measurements are relevant to present theoretical models of the denaturation-renaturation processes in proteins, in which proline residues may play a key role.     

Molecular design and optimization of hepatic cancer SLP76‐derived PLCγ1 SH3‐binding peptide with the systematic N‐substitution of peptide PXXP motif

The phospholipase Cγ1 (PLCγ1) is essential for T‐cell signaling and activation in hepatic cancer immune response, which has a regulatory Src homology 3 (SH3) domain that can specifically recognize and interact with the PXXP‐containing decapeptide segment (¹⁸⁵QP P VP P QRPM¹⁹⁴, termed as SLP76^185–194 peptide) of adaptor protein SLP76 following T‐cell receptor ligation. The isolated peptide can only bind to the PLCγ1 SH3 domain with a moderate affinity due to lack of protein context support. Instead of the traditional natural residue mutagenesis that is limited by low structural diversity and shifted target specificity, we herein attempt to improve the peptide affinity by replacing the two key proline residues Pro187 and Pro190 of SLP76^185–194 PXXP motif with nonnatural N‐substituted amino acids, as the proline is the only endogenous N‐substituted amino acid. The replacement would increase peptide flexibility but can restore peptide activity by establishing additional interactions with the domain. Structural analysis reveals that the domain pocket can be divided into a large amphipathic region and a small negatively charged region; they accommodate hydrophobic, aromatic, polar, and moderate‐sized N‐substituted amino acid types. A systematic replacement combination profile between the peptide residues Pro187 and Pro190 is created by structural modeling, dynamics simulation, and energetics analysis, from which six improved and two reduced N‐substituted peptides as well as native SLP76^185–194 peptide are identified and tested for their binding affinity to the recombinant protein of the human PLCγ1 SH3 domain using fluorescence‐based assays. Two N‐substituted peptides, SLP76^185–194(N‐Leu187/N‐Gln190) and SLP76^185–194(N‐Thr187/N‐Gln190), are designed to have high potency (K_d = 0.67 ± 0.18 and 1.7 ± 0.3 μM, respectively), with affinity improvement by, respectively, 8.5‐fold and 3.4‐fold relative to native peptide (K_d = 5.7 ± 1.2 μM).     

A method to detect nonproline cis peptide bonds in proteins

Based on the geometrical parameters around seventeen incorrectly assigned trans conformations of peptide bonds in protein structures and their correct cis counterparts, we have devised an algorithm that is capable of detecting these sites. The algorithm was optimized to reliably find all of the seventeen test cases. It can be used to quickly scan an atomic coordinate file or the complete Brookhaven Protein Data Base for more likely candidates for non‐Pro cis peptide bonds. Also, it can be of help to guide the crystallographer in intermediate stages of structure determination towards suspect areas. © 1999 John Wiley & Sons, Inc. Biopoly 50: 536–544, 1999     

Nmr studies of the molecular conformations in the linear oligopeptides H-(L-Ala)n-L-Pro-OH

The molecular conformations of the linear oligopeptides H-(L -Ala)_n-L -Pro-OH, with n = 1,2 and 3, have been investigated. ¹³C nmr observation of the equilibrium between the cis and trans forms of the Ala-Pro peptide bond indicated the occurrence of nonrandom conformations in solutions of these flexible peptides. The formation of the nonrandom species containing the cis form of the Ala-Pro bond was found to depend on the deprotonation of the carboxylic acid group of proline, the solvent, and the ionic strength in aqueous solution. The influence of intramolecular hydrogen bonding on the relative conformational energies of the species containing the cis and trans Ala-Pro peptide bond was studied by comparison of the peptides H-(Ala)_n-Pro-OH with analogous molecules where hydrogen bond formation was excluded by the covalent structure. In earlier work a hydrogen bond between the protonated terminal carboxylic acid group and the carbonyl oxygen of the penultimate amino acid residue had been suggested to stabilize conformations including trans proline. For the systems described here this hypothesis can be ruled out, since the cis:trans ratio is identical for molecules with methyl ester protected and free protonated terminal carboxylic acid groups of proline. Direct evidence for hydrogen bond formation between the deprotonated terminal carboxylic acid group and the amide proton of the penultimate amino acid residue in the molecular species containing cis proline was obtained from ¹H nmr studies. However, the cis:trans ratio of the Ala-Pro bond was not affected by N-methylation of the penultimate amino acid residue, which prevents formation of this hydrogen bond. Overall the experimental observations lead to the conclusion that the relative energies of the peptide conformations including cis or trans proline are mainly determined by intramolecular electrostatic interactions, whereas in the molecules considered, intramolecular hydrogen bonding is a consequence of specific peptide backbone conformations rather than a cause for the occurrence of energetically favored species. Independent support for this conclusion was obtained from model consideration which indicated that electrostatic interactions between the terminal carboxylic acid group and the carbonyl oxygen of the penultimate amino acid residue could indeed account for the observed relative conformational energies of the species containing cis and trans proline, respectively.     

Targeting the SH3 domain of human osteoclast‐stimulating factor with rationally designed peptoid inhibitors

Human osteoclast‐stimulating factor (hOSF) is an intracellular protein produced by osteoclasts that induces osteoclast formation and bone resorption. The protein contains a modular Src homology 3 (SH3) domain that mediates the intermolecular recognition and interaction of hOSF with its biological partners. Here, we proposed targeting the hOSF SH3 domain to disrupt hOSF–partner interactions for bone disease therapy by using SH3 inhibitors. In the procedure, the primary sequences of three known hOSF‐interacting proteins (c‐Src, SMN and Sam68) were parsed, from which totally 31 octapeptide segments that contain the core SH3‐binding motif PXXP were extracted, and their binding behavior to hOSF SH3 domain was investigated at structural level using a biomolecular modeling protocol. Several SH3‐binding candidates were identified theoretically and then determined to have high or moderate affinity for the domain using fluorescence spectroscopy assays. One potent peptide ⁴²⁵APP ARP VK⁴³² (K_d = 3.2 μM), which corresponds to the residues 425–432 of Sam68 protein, was used as template to derive N substitution of peptides (peptoids). Considering that proline is the only endogenous N‐substituted amino acid that plays a critical role in SH3–peptide binding, the substitution was addressed at the two key proline residues (Pro427 and Pro430) of the template peptide with nine N‐substituted amino acid types. By systematically evaluating the structural and energetic effects of different N‐substituted amino acids presenting at the two proline sites on peptide binding, we rationally designed five peptoid inhibitors and then determined in vitro their binding affinity to hOSF SH3 domain. Consequently, two designed peptoids APP AR( N ‐Clp) VK and APP AR( N ‐Ffa) VK with Pro430 replaced by N‐Clp and N‐Ffa were confirmed to have increased (K_d = 0.87 μM) and comparable (K_d = 2.9 μM) affinities relative to the template, respectively. In addition, we also found that the Pro427 residue plays an essential role in restricting peptide/peptoid conformations to polyproline II (PPII) helix as the basic requirement of SH3 binding so that the residue cannot be modified. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Interruptions in the collagen repeating tripeptide pattern can promote supramolecular association

The standard collagen triple‐helix requires a perfect (Gly‐Xaa‐Yaa)_n sequence, yet all nonfibrillar collagens contain interruptions in this tripeptide repeating pattern. Defining the structural consequences of disruptions in the sequence pattern may shed light on the biological role of sequence interruptions, which have been suggested to play a role in molecular flexibility, collagen degradation, and ligand binding. Previous studies on model peptides with 1‐ and 4‐residue interruptions showed a localized perturbation within the triple‐helix, and this work is extended to introduce natural collagen interruptions up to nine residue in length within a fixed (Gly‐Pro‐Hyp)_n peptide context. All peptides in this set show decreases in triple‐helix content and stability, with greater conformational perturbations for the interruptions longer than five residue. The most stable and least perturbed structure is seen for the 5‐residue interruption peptide, whose sequence corresponds to a Gly to Ala missense mutation, such as those leading to collagen genetic diseases. The triple‐helix peptides containing 8‐ and 9‐residue interruptions exhibit a strong propensity for self‐association to fibrous structures. In addition, a small peptide modeling only the 9‐residue sequence within the interruption aggregates to form amyloid‐like fibrils with antiparallel β‐sheet structure. The 8‐ and 9‐residue interruption sequences studied here are predicted to have significant cross‐β aggregation potential, and a similar propensity is reported for ～10% of other naturally occurring interruptions. The presence of amyloidogenic sequences within or between triple‐helix domains may play a role in molecular association to normal tissue structures and could participate in observed interactions between collagen and amyloid.