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 ? to values of ?60 °± 30° 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–α are the most favorable conformational states for the diproline segment. The analysis on Xaa‐Pro‐Yaa sequences reveals that the Xaa‐Pro 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. © 2011 Wiley Periodicals, Inc. Biopolymers 97: 54–64, 2012.     

Local control of peptide conformation: Stabilization of cis proline peptide bonds by aromatic proline interactions

In the native state of proteins there is a marked tendency for an aromatic amino acid to precede a cis proline. There are also significant differences between the three aromatic amino acids with Tyr exhibiting a noticeably higher propensity than Phe or Trp to precede a cis proline residue. In order to study the role that local interactions play in these conformation preferences, a set of tetrapeptides of the general sequence acetyl-Gly-X-Pro-Gly-carboxamide (GXPG), where X = Tyr, Phe, Trp, Ala, or cyclohexyl alanine, were synthesized and studied by nmr. Analysis of the nmr data shows that none of the peptides adopt a specific backbone structure. Ring current shifts, the equilibrium constants, the Van't Hoff enthalpy, and the measured rate of cis-trans isomerization all indicate that the cis proline conformer is stabilized by favorable interactions between the aromatic ring and the proline residue. Analysis of the side chain conformation of the aromatic residue and analysis of the chemical shifts of the pyrrolidine ring protons shows that the aromatic side chain adopts a preferred conformation in the cis form. The distribution of rotamers and the effect of an aromatic residue on the cis-trans equilibrium indicate that the preferred conformation is populated to approximately 62% for the Phe containing peptide, 67% for the Tyr containing peptide, and between 75 and 80% for the Trp containing peptide. The interaction is unaffected by the addition of 8M urea. These local interactions favor an aromatic residue immediately preceding a cis proline, but they cannot explain the relative propensities for Phe-Pro, Tyr-Pro, and Trp-Pro cis peptide bonds observed in the native state of proteins. In the model peptides the percentage of the cis proline conformer is 21% GYPG while it is 17% for GFPG. This difference is considerably smaller than the almost three to one preponderance observed for cis Tyr-Pro peptide bonds vs cis Phe-Pro peptide bonds in the protein database. © 1998 John Wiley & Sons, Inc. Biopoly 45: 381–394, 1998     

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.     

Effect of the Phosphate Group with Different Negative Charges on the Conformation of Phosphorylated Ser/Thr-Pro Motif

Summary Ser/Thr-Pro motif is a widespread phosphorylated site in proteins, and its reversible phosphorylation is an important regulatory progress in many cell cycles and signal transduction. Recent research reveals that phosphorylation affects the local conformation of the peptide and its binding with the substrate through peptidyl--prolyl cis/trans isomerization. In order to further explore the effect of the phosphate group with different charges, four model peptides containing non- and phosphorylated Ser/Thr-Pro motif were synthesized using the classical solid-phase method. 1H-NMR, TOCSY, and ROESY were employed to characterize the conformation of the model peptides in solution with different pH value and analyze the peptidyl--prolyl isomerization at a molecular level. The results demonstrate that phosphorylation increases the cis conformation in the peptide and the maximum cis/trans ratio is obtained when the phosphate group has two negative charges. Furthermore, the experiments prove that the phosphorylation introduces a hydrogen bond between the phosphate and the NH of Ser/Thr residue, and the charges of the phosphate affect certain conformations of the phosphorylated Ser/Thr-Pro motif.     

The effect of phosphorylation on the conformation of oligo-peptides with Ser–Pro motif: a molecular dynamics simulation

Model tetrapeptide system was designed to investigate the cis/trans isomerization of peptidyl-prolyl imide bond of Ser–Pro motif. To establish the side-chain O-phosphorylation effect in regulating the peptides conformations, molecular dynamics (MD) simulations where carried out on the designed tetrapeptides and their corresponding phosphorylated forms by MD Insight II Discovery3 approach. The most stable configurations and the statistic cis/trans concentration distribution demonstrated that the phosphorylation evidently influences the peptidyl-prolyl imide bond isomerization and works as a key effect in regulating the peptide conformations. The charge state and the site provided for the charge of the phosphate moiety might be an important key. The results also demonstrated that phosphorylation changes the cis conformation ratio of the peptide and the maximum cis value is obtained when the phosphate group has no negative charge.     

Prediction ofcis/transisomerization in proteins using PSI-BLAST profiles and secondary structure information

Background  

The majority of peptide bonds in proteins are found to occur in thetransconformation. However, for proline residues, a considerable fraction of Prolyl peptide bonds adopt thecisform. Prolinecis/transisomerization is known to play a critical role in protein folding, splicing, cell signaling and transmembrane active transport. Accurate prediction of prolinecis/transisomerization in proteins would have many important applications towards the understanding of protein structure and function.     

Bioactivity of folding intermediates studied by the recovery of enzymatic activity during refolding

The peptide bond preceding proline residues realizes a cis/trans conformational switch with high switching resistance in native proteins and folding intermediates. Therefore, individual isomers have the potential to differ in bioactivity. However, information about isomer-specific bioactivities is difficult to obtain because of the risk of affecting isomeric distribution by bioactivity assay components.Here we present an approach that allows for the measurement of the recovery of enzymatic activities of wild-type RNase T₁ and RNase T₁ variants during refolding under conditions where the population of enzyme-substrate or enzyme-product complexes is negligible. Recovery of enzymatic activity was continuously monitored within the visible range of the spectrum by addition of a fluorescence-labeled nucleotide substrate to the refolding sample. We found that a nonnative trans conformation at Pro39 renders the RNase T₁ almost completely inactive. A folding intermediate having a nonnative trans conformation at Pro55 shows about 46% of the enzymatic activity referred to the native state. Pro55, in contrast to the active site located Pro39, is situated in a solvent-exposed loop region remote from active-site residues. In both cases, peptidyl prolyl cis/trans isomerases accelerate the regain of nucleolytic activity. Our findings show that even if there is a considerable distance between the site of isomerization and the active site, conformational control of the bioactivity of proteins is likely to occur, and that the surface location of prolyl bonds suffices for the control of buried active sites mediated by peptidyl prolyl cis/trans isomerases.     

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.     

Solid-state geometry and conformation of linear,diastereoisomeric oligoprolines

We have carried out a systematic analysis of the solid-state conformational preferences of a number of linear homo-oligoprolines (to the tetramer) by ir absorption and x-ray diffraction. The peptides present different chiral sequences (tacticities), various types (urethane and amide) of N-protecting groups, and free and blocked C-termini (which imply different capabilities of forming H-bonds). The following conclusions can be drawn: (i) values for the geometry of the prolyl residue and the peptide bond in the cis and in the trans conformations are proposed; (ii) in general the conformational angles φ and ψ in the linear homo-oligoprolines have values appropriate for the polyproline II structure (conformation F); (iii) the pyrrolidine ring shows various types of puckering with no apparent relation to the backbone conformation; (iv) Pro-Pro peptide bonds generally take the trans conformation, the few cases of cis conformation being formed by Pro residues of different chirality; (v) the single H-bond donor — OH, when present, is always bonded to H-acceptors, which can be either the urethane or the amide or the peptide carbonyl but never the carbonyl group of the — COOH moiety.     

An examination of the involvement of proline peptide isomerization in protein folding.

The proline peptide isomerization model of protein folding predicts that the fraction of denatured polypeptide chains which rapidly fold should be quantitatively related to the numbers of cis and trans proline residues in the folded polypeptide conformation. However, we find that neither the comparative nor the absolute kinetic pattern for folding of the homologous proteins, tuna heart and horse heart ferricytochrome c which differ by one proline residue, is compatible with the quantitative predictions of the proline peptide isomerization model.     

Conformational energy studies on N-methylated analogs of thyrotropin releasing hormone, enkephalin, and luteinizing hormone-releasing hormone

Empirical conformational energy calculations have been carried out for N-methyl derivatives of alanine and phenylalanine dipeptide models and N-methyl-substituted active analogs of three biologically active peptides, namely thyrotropin-releasing hormone (TRH), enkephalin (ENK), and luteinizing hormone-releasing hormone (LHRH). The isoenergetic contour maps and the local dipeptide minima obtained, when the peptide bond (ω) preceding the N-methylated residue is in the trans configuration show that (1) N-methylation constricts the conformational freedom of both the ith and (i + 1)th residues; (2), the lowest energy position for both residues occurs around ? = ?135° ± 5° and ψ = 75° ± 5°, and (3) the α_L conformational state is the second lowest energy state for the (i + 1)th residue, whereas for the ith residue the C₅ (extended) conformation is second lowest in energy. When the peptide bond (ω_i) is in the cis configuration the ith residue is energetically forbidden in the range ? = 0° to 180° and ψ = ?180° to +180°. Conformations of low energy for ω_i = 0° are found to be similar to those obtained for the trans peptide bond. In all the model systems (irrespective of cis or trans), the α_R conformational state is energetically very high. Significant deviations from planarity are found for the peptide bond when the amide hydrogen is replaced by a methyl group. Two low-energy conformers are found for [(N-Me)His²]TRH. These conformers differ only in the ? and ψ values at the (N-Me)His² residue. Among the different low-energy conformers found for each of the ENK analogs [D -Ala²,(N-Me)Phe⁴, Met⁵]ENK amide and [D -Ala²,(N-Me)Met⁵]ENK amide, one low-energy conformer was found to be common for both analogs with respect to the side-chain orientations. The stability of the low-energy structures is discussed in the light of the activity of other analogs. Two low-energy conformers were found for [(N-Me)Leu⁷]LHRH. These conformations differ in the types of bend around the positions 6 and 7 of LHRH. One bend type is eliminated when the active analog [D -Ala⁶,(M-Me)Leu⁷]LHRH is considered.     

Cis-trans isomerization of the Epstein-Barr virus determinant peptide EENLLDFVRF after the DM1 TCR recognition of the HLA-B*4405/peptide complex

The Epstein–Barr virus determinant peptide EENLLDFVRF shows high immunogenicity when presented by HLA-B*4405 allotype. This fact is accompanied by a cis–trans isomerization of the Leu5-Asp6 peptide bond upon TCR binding of the pMHC complex. Molecular dynamics simulations of pMHC/TCR structures, with the EENLLDFVRF peptide in cis and trans conformations have been employed in order to examine the structure and dynamics of the pMHC complex with such an unusual conformation. The results, based on MM-PBSA free energy computations as well as buried surface area analysis and interactions at the pMHC/TCR interface, indicate that the TCR binds preferably the pMHC complex with the Leu5-Asp6 peptide bond in cis conformation. It is the first time that this notable conformational feature of T-cell epitope is investigated.     

Effect of proline residues on protein folding

Conformational energy calculations have been used to study the role of the proline residues in the folding of bovine pancreatic trypsin inhibitor. In the calculation, each of the four proline residues of this small protein is forced from the trans to cis peptide isomer while still part of the native folded structure. The cis proline residue can always be accommodated by small changes of the native conformation (< 1 Å root-mean-square deviation). For three of the four proline residues, Pro2, Pro9 and Pro 13, being in the cis form is calculated to destabilize the folded conformation by less than 11 kcal/mol, suggesting that rapid folding to a stable native-like conformation can occur with either isomeric form. For one of these three, Pro13, the destabilization is only 1 kcal/mol, suggesting the existence of an alternative folded native conformation with Pro13 cis. The fourth proline residue, Pro8, is calculated to destabilize the native conformation by so much (33 kcal/mol) that it will block folding in the manner proposed by Brandts et al. (1975).     

Structure of the cyclic peptide [W8S]contryphan Vn: effect of the tryptophan/serine substitution on trans–cis proline isomerization

The structural characterization of [W8S]contryphan Vn, an analogue of Contryphan Vn with tryptophan 8 substituted with a serine residue (W8S), was performed by NMR spectroscopy, molecular dynamics simulations and fluorescence spectroscopy. Contryphan Vn, a bioactive cyclic peptide from the venom of the cone snail Conus ventricosus, contains an S–S bridge between two cysteines and a d-tryptophan. Like other Contryphans, [W8S]contryphan Vn has proline 7 isomerized trans, while the proline 4 has nearly equivalent populations of cis and trans configurations. The thermodynamic and kinetic parameters of the trans–cis isomerization of proline 4 were measured. The isomers of [W8S]contryphan Vn with proline 4 in cis and trans show structural differences. The absence of the salt bridge between the same Asp2 and Lys6, present in Contryphan Vn, may be attributed to the lack of the hydrophobic side chain of Trp8 where it likely protects the electrostatic interactions. These results may contribute to identifying, in these cyclic peptides, the structural determinants of the mechanism of proline trans–cis isomerization, this being also an important step in protein folding.     

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.     

Green‐lighting green fluorescent protein: Faster and more efficient folding by eliminating a cis–trans peptide isomerization event

Wild‐type green fluorescent protein (GFP) folds on a time scale of minutes. The slow step in folding is a cis–trans peptide bond isomerization. The only conserved cis‐peptide bond in the native GFP structure, at P89, was remodeled by the insertion of two residues, followed by iterative energy minimization and side chain design. The engineered GFP was synthesized and found to fold faster and more efficiently than its template protein, recovering 50% more of its fluorescence upon refolding. The slow phase of folding is faster and smaller in amplitude, and hysteresis in refolding has been eliminated. The elimination of a previously reported kinetically trapped state in refolding suggests that X‐P89 is trans in the trapped state. A 2.55 Å resolution crystal structure revealed that the new variant contains only trans‐peptide bonds, as designed. This is the first instance of a computationally remodeled fluorescent protein that folds faster and more efficiently than wild type.     

The trans-to-cis proline isomerization in E. coli Trx folding is accelerated by trans prolines

The conserved fold of thioredoxin (Trx)-like thiol/disulfide oxidoreductases contains an invariant cis-proline residue (P76 in Escherichia coli Trx) that is essential for Trx function and that is responsible for the folding rate-limiting step. E. coli Trx contains four additional prolines, which are all in the trans conformation in the native state. Notably, a recent study revealed that replacement of all four trans prolines in Trx by alanines (Trx variant Trx1P) further slowed the rate-limiting step 25-fold, indicating that one or several of the four trans prolines accelerate the trans-to-cis transition of P76 in Trx wild-type (wt). Here, we characterized the folding kinetics of Trx variants containing cisP76 and one or several of the natural trans prolines of Trx wt with NMR spectroscopy. First, we demonstrate that the isomerization reaction in Trx1P is a pure two-state transition between two distinct tertiary structures, in which all observed NMR resonances changes follow the same first-order kinetics. Moreover, we show that trans-P68 is the critical residue responsible for the faster folding of wt Trx relative to the single-proline (P76) variant Trx1P, as the two-proline variant Trx2P(P76P68) already folds seven times faster than Trx1P. trans-P34 also accelerates trans-to-cis isomerization of P76, albeit to a smaller extent. Overall, the results demonstrate that trans prolines can significantly modulate the kinetics of rate-limiting trans-to-cis proline isomerization in protein folding. Finally, we discuss possible mechanisms of acceleration and the potential significance of a protein-internal folding acceleration mechanism for Trx in a living cell.     

Intricacies of interactions,dynamics and solvent effects in enzyme catalysis: a computational perspective on cyclophilin A

The essential role of enzymes in biological processes has continually ignited sparks of interest in their mechanism of action. Fully understanding the mechanism of enzymes has broad implications in protein engineering and drug design. The more than five order of magnitude speed-up in the rate of peptidyl–prolyl cis–trans isomerisation by cyclophilin A (CypA) has been the target of intense research. CypA serves as a tractable model system, because it reversibly catalyses the rotation around peptidyl–prolyl bonds without any bond breakage or formation. Here, we discuss the results of recent computational approaches used to study the mechanism of CypA. We highlight the critical role of enzyme and substrate conformational dynamics in the developing interactions as the substrate approaches the transition state that results in an astonishing enhancement of isomerisation rate. The rate of isomerisation is affected by the intricate coupling between the dynamics of the substrate, enzyme and solvent. CypA binds its substrates via conformational selection, where rearrangements of key active site residues are necessary for substrate recognition. The conformational plasticity of the active site allows the enzyme to accommodate the most favourable interactions with the transition state that can be exploited for structure-based drug design.     

A theoretical estimate of the energy barriers between stable conformations of the proline dimer.

Semi-empirical energy calculations for an internal Pro-Pro dimer are presented that take into account the nature of the flexibility of the proline ring due to its puckering. Calculations show that three stable conformations are available for the dimer: the cis (ω = 0°, ψ = 160°); the trans (ω = 180°, ψ = 160°, also referred to as trans′); and the cis′ (ω = 180°, ψ = ?40°) conformations. The best conformational pathways between these stable conformations are determined. Calculations also show that the barrier for cis′–trans′ conversion is of the same order of magnitude as that for cis–trans conversion.     

A statistical analysis of the PPII propensity of amino acid guests in proline-rich peptides

There has been considerable debate about the intrinsic PPII propensity of amino-acid residues in denatured polypeptides. Experimentally, the propensity scale is based on the behavior of guest amino-acid residues placed in the middle of polyproline hosts. We have used classical molecular dynamics simulations, with state-of-the-art force fields to carry out a comprehensive analysis of the conformational equilibria of the proline-based host oligopeptides with single guests. The tracked structural characteristics include the PPII content, the cis/trans isomerization of the prolyl bonds, the puckering of the pyrrolidine rings of the proline residues, and the secondary structural motifs. We find no evidence for an intrinsic PPII propensity in any of the guest amino acids other than proline. Instead, the PPII content as derived from experiments may be explained in terms of: 1), a local correlation between the dihedral angles of the guest amino acid and the proline residue immediately preceding it; and 2), a nonlocal correlation between the cis/trans states of the peptide bonds. In terms of the latter, we find that the presence of a guest (other than proline, tyrosine, or tryptophan) increases the trans content of most of the prolyl bonds, which results in an effective increase of the peptide PPII content. With respect to the local dihedral correlations, we find that these are well described in terms of the so-called odds-ratio statistic. Expressed in terms of free energy language, the PPII content based on the odds-ratio of the relevant residues correlate well with the experimentally measured PPII content.