13C-nuclear magnetic resonance studies of 85% 13C-enriched amino acids and small peptides. pH effects on the chemical shifts, coupling constants, kinetics of cis-trans isomerisation and conformation aspects.

The 13C chemical shifts of several 85% 13C-enriched amino acids and small peptides were studied as a function of pH. The results show that the chemical shifts of carbon atoms of ionizable groups vary significantly within the zone of their pK. Generally with the pH GOING FROM 7 to 1 all the deltaC are shifted more or less upfield with the exception of the carbonyl group carbon of the second last residue which is shifted slightly downfield. This suggests the formation of an hydrogen bond at acid pH involving in a seven-membered ring the C=O in question and the COOH terminal. The percentage of cis and trans conformers of glycyl-L-proline and glycyl-L-prolylglycine were studied as a function of pH. The trans form is always preponderant whatever the pH. The accessibility of the carbonyl group to protonation of the proline residue strongly influences the cis-trans equilibrium. Thus, with the pH varying from 7 to 1, the trans isomer changes from 61 to 85% for glycyl-L-proline and only from 77 to 80% for glycyl-L-prolylglycine. The proton NMR studies underline the important differences existing between the two molecular forms of glycyl-L-proline. The cis conformation is characterized with regard to the trans form by the non-equivalence of the alpha-protons of the glycine residue, by a lower pK(1) and by a larger deltadeltaHalpha of the proline residue as a function of pH. These results could suggest an end-to-end interaction in the cis form of the glycyl-L-proline molecule. The 13C-13C coupling constants were also studied as a function of pH. The results show that J(Co-Calpha) of a C-terminal residue, varying from 5 to 6 Hz and reflecting thhe pK of the carboxylate group, is a linear function of delta(Co) and delta(Calpha) as in the case of the amino acids. The total variation of the electron density of those two carbons in an amino acid is approximately 40% weaker than in a C-terminal residue. The charge distribution along the Calpha-C(o) bond, however, is practically the same in both cases. Finally the ratios of the conversion rate constants of the two isomers cis-trans of glycyl-proline were calculated at different pH values; the relations between the isomer percentages and delta(Co), delta(Calpha) on the one hand and the J(Co-Calpha) on the other were established.     

Real-time NMR studies on a transient folding intermediate of barstar.

The refolding of barstar, the intracellular inhibitor of barnase, is dominated by the slow formation of a cis peptidyl prolyl bond in the native protein. The triple mutant C40/82A P27A in which two cysteine residues and one trans proline were replaced by alanine was used as model system to investigate the kinetics and structural consequences of the trans/cis interconversion of Pro48. One- and two-dimensional real-time NMR spectroscopy was used to follow the trans/cis interconversion after folding was initiated by rapid dilution of the urea denatured protein. Series of 1H, 15N HSQC spectra acquired with and without the addition of peptidyl prolyl isomerase unambiguously revealed the accumulation of a transient trans-Pro48 intermediate within the dead time of the experiment. Subtle chemical shift differences between the native state and the intermediate spectra indicate that the intermediate is predominantly native-like with a local rearrangement in the Pro48 loop and in the beta-sheet region including residues Tyr47, Ala82, Thr85, and Val50.     

NMR analysis of cleaved Escherichia coli thioredoxin (1-73/74-108) and its P76A variant: cis/trans peptide isomerization

Inspection of high resolution three-dimensional (3D) structures from the protein database reveals an increasing number of cis-Xaa-Pro and cis-Xaa-Yaa peptide bonds. However, we are still far from being able to predict whether these bonds will remain cis upon single-site substitution of Pro or Yaa and/or cleavage of a peptide bond close to it in the sequence. We have chosen oxidized Escherichia coli thioredoxin (Trx), a member of the Trx superfamily with a single alpha/beta domain and cis P76 to determine the effect of single-site substitution and/or cleavage on this isomer. Standard two-dimensional (2D) NMR analysis were performed on cleaved Trx (1-73/74-108) and its P76A variant. Analysis of the NOE connectivities indicates remarkable similarity between the secondary and supersecondary structure of the noncovalent complexes and Trx. Analysis of the 2D version of the HCCH-TOCSY and HMQC-NOESY-HMQC and 13C-filtered HMQC-NOESY spectra of cleaved Trx with uniformly 13C-labeled 175 and P76 shows surprising conservation of both cis P76 and packing of 175 against W31. A similar NMR analysis of its P76A variant provides no evidence for cis A76 and shows only subtle local changes in both the packing of 175 and the interstrand connectivities between its most protected hydrophobic strands (beta2 and beta4). Indeed, a molecular simulation model for the trans P76A variant of Trx shows only subtle local changes around the substitution site. In conclusion, cleavage of R73 is insufficient to provoke cis/trans isomerization of P76, but cleavage and single-site substitution (P76A) favors the trans isomer.     

Conformation of proline residues in bacteriorhodopsin

Proline, noted as a hydrophilic residue with helix-breaking potential, nevertheless occurs widely in putatively alpha-helical transmembrane segments of many transport proteins. Ligand-activated or enzyme-assisted trans/cis isomerization of an X-proline peptide bond (where X = any amino acid)--a dynamic, reversible event which could alter the orientation of a transmembrane alpha-helix--may provide the molecular basis for a protein channel regulatory process. Further elucidation of such a function requires knowledge of the isomeric status of the X-Pro bonds in native conformations of membrane proteins. We have used 13C nuclear magnetic resonance (NMR) spectroscopy to examine the conformation of intramembranous X-Pro peptide bonds in biosynthetically-labelled samples of a model transport protein, bacteriorhodopsin (bR) (purple membrane). Spectra of 13C-Tyr-carbonyl labelled bR (in the solvent system CHCl3:CD3OD (1:1) + 0.1 M LiClO4) first established that all 11 bR Tyr residues were sufficiently mobile for their resonances to be detected and resolved, independent of their domain location within the bR sequence. By taking advantage of the known diagnostic chemical shifts of the isomers of Pro-C gamma carbon resonances, spectra of bR labelled with 13C gamma-Pro were then used to demonstrate that all 11 bR X-Pro peptide bonds--including those within the protein's membrane domain (Pro50, Pro91, Pro186)--are in the trans conformation in resting state bR.     

Thermodynamic origin of cis/trans isomers of a proline-containing beta-turn model dipeptide in aqueous solution: a combined variable temperature 1H-NMR, two-dimensional 1H,1H gradient enhanced nuclear Overhauser effect spectroscopy (NOESY), one-dimensional steady-state intermolecular 13C,1H NOE, and molecular dynamics study

The cis/trans conformational equilibrium of the two Ac-Pro isomers of the beta-turn model dipeptide [13C]-Ac-L-Pro-D-Ala-NHMe, 98% 13C enriched at the acetyl carbonyl atom, was investigated by the use of variable temperature gradient enhanced 1H-nmr, two-dimensional (2D) 1H,1H nuclear Overhauser effect spectroscopy (NOESY), 13C,1H one-dimensional steady-state intermolecular NOE, and molecular dynamics calculations. The temperature dependence of the cis/trans Ala(NH) protons are in the region expected for random-coil peptides in H2O (delta delta/delta T = -9.0 and -8.9 ppb for the cis and trans isomers, respectively). The trans NH(CH3) proton indicates smaller temperature dependence (delta delta/delta T approximately -4.8 ppb) than that of the cis isomer (-7.5 ppb). 2D 1H,1H NOESY experiments at 273 K demonstrate significant NOEs between ProH alpha-AlaNH and AlaNH-NH(R) for the trans isomer. The experimental NOE data, coupled with computational analysis, can be interpreted by assuming that the trans isomer most likely adopts an ensemble of folded conformations. The C-CONH(CH3) fragment exhibits significant conformational flexibility; however, a low-energy conformer resembles closely the beta II-turn folded conformations of the x-ray structure of the related model peptide trans-BuCO-L-Pro-Me-D-Ala-NHMe. On the contrary, the cis isomer adopts open conformations. Steady-state intermolecular solute-solvent (H2O) 13C,1H NOE indicates that the water accessibility of the acetyl carbonyl carbons is nearly the same for both isomers. This is consistent with rapid fluctuations of the conformational ensemble and the absence of a highly shielded acetyl oxygen from the bulk solvent. Variable temperature 1H-nmr studies of the cis/trans conformational equilibrium indicate that the trans form is enthalpically favored (delta H degree = -5.14 kJ mole-1) and entropically (delta S degree = -5.47 J.K-1.mole-1) disfavored relative to the cis form. This demonstrates that, in the absence of strongly stabilizing sequence-specific interresidue interactions involving side chains and/or charged terminal groups, the thermodynamic difference of the cis/trans isomers is due to the combined effect of intramolecular and intermolecular (hydration) induced conformational changes.     

Polyproline II helix conformation in a proline-rich environment: a theoretical study

Interest centers here on whether a polyproline II helix can propagate through adjacent non-proline residues, and on shedding light on recent experimental observations suggesting the presence of significant PP(II) structure in a short alanine-based peptide with no proline in the sequence. For this purpose, we explored the formation of polyproline II helices in proline-rich peptides with the sequences Ac-(Pro)(3)-X-(Pro)(3)-Gly-Tyr-NH(2), with X = Pro (PPP), Ala (PAP), Gln (PQP), Gly (PGP), and Val (PVP), and Ac-(Pro)(3)-Ala-Ala-(Pro)(3)-Gly-Tyr-NH(2) (PAAP), by using a theoretical approach that includes a solvent effect as well as cis <--> trans isomerization of the peptide groups and puckering conformations of the pyrrolidine ring of the proline residues. Since (13)C chemical shifts have proven to be useful for identifying secondary-structure preferences in proteins and peptides, and because values of the dihedral angles (phi,psi) are the main determinants of their magnitudes, we have, therefore, computed the Boltzmann-averaged (13)C chemical shifts for the guest residues in the PXP peptide (X = Pro, Ala, Gln, Gly, and Val) with a combination of approaches, involving molecular mechanics, statistical mechanics, and quantum mechanics. In addition, an improved procedure was used to carry out the conformational searches and to compute the solvent polarization effects faster and more accurately than in previous work. The current theoretical work and additional experimental evidence show that, in short proline-rich peptides, alanine decreases the polyproline II helix content. In particular, the theoretical evidence accumulated in this work calls into question the proposal that alanine has a strong preference to adopt conformations in the polyproline II region of the Ramachandran map.     

1H-NMR studies of receptor-selective substance P analogues reveal distinct predominant conformations in DMSO-d6

Proton nmr parameters are reported for DMSO-d6 solutions of two receptor-selective substance P analogues: Ac[Arg6,Pro9]SP6-11, which is selective for the NK-1 (SP-P) receptor and [pGlu6,N-MePhe8]SP6-11, which selectively activates the NK-3 (SP-N) receptor. Full peak assignments of both analogues were obtained by COSY experiments. The chemical shifts, coupling constants, and temperature coefficients of amide proton chemical shifts as well as NOESY effects and calculated side-chain rotamer populations of Phe side chains are reported for both peptides. Analysis of coupling constants and temperature coefficients together with the nuclear Overhauser enhancement spectroscopy effects suggest that Ac[Arg6,Pro9]SP6-11 has a trans configuration about the Phe8-Pro9 amide bond and the preferred conformation of this analogue has a type I beta-turn. The nmr data for [pGlu6,N-MePhe8]SP6-11 suggest that this peptide exists as a mixture of cis-trans isomers in which the cis isomer can preferably adopt a type VI beta-turn conformation, and the trans isomer can adopt a gamma-turn conformation. There are indications that the two last turns are stabilized by a hydrogen bond between the syn carboxamide proton and the pGlu ring carbonyl.     

Structure of an elastin-mimetic polypeptide by solid-state NMR chemical shift analysis

The conformation of an elastin-mimetic recombinant protein, [(VPGVG)4(VPGKG)]39, is investigated using solid-state NMR spectroscopy. The protein is extensively labeled with 13C and 15N, and two-dimensional 13C-13C and 15N-13C correlation experiments were carried out to resolve and assign the isotropic chemical shifts of the various sites. The Pro 15N, 13Calpha, and 13Cbeta isotropic shifts, and the Gly-3 Calpha isotropic and anisotropic chemical shifts support the predominance of type-II beta-turn structure at the Pro-Gly pair but reject a type-I beta-turn. The Val-1 preceding Pro adopts mostly beta-sheet torsion angles, while the Val-4 chemical shifts are intermediate between those of helix and sheet. The protein exhibits a significant conformational distribution, shown by the broad line widths of the 15N and 13C spectra. The average chemical shifts of the solid protein are similar to the values in solution, suggesting that the low-hydration polypeptide maintains the same conformation as in solution. The ability to measure these conformational restraints by solid-state NMR opens the possibility of determining the detailed structure of this class of fibrous proteins through torsion angles and distances.     

Conformation and mobility of tyrosine side chain in tetrapeptides. Specific effects of cis- and trans-proline in Tyr-Pro- and Pro-Tyr-segments

We examined the properties of tyrosine in four free tetrapeptides: Ala-Ala-Tyr-Ala (AATA), Ala-Pro-Tyr-Ala (APTA), Ala-Tyr-Ala-Ala (ATAA) and Ala-Tyr-Pro-Ala (ATPA) by CD, n.m.r. and energy calculations. Experimental data (the aromatic 1Lb signal, rotamer populations around the C alpha-C beta bond (chi 1), rotations around C beta-C gamma(chi 2), chemical shifts of ortho- and meta-protons in the phenolic ring (in aqueous and Me2SO solutions), NH proton temperature coefficients and vicinal coupling constants 3JNH-C alpha H in the backbone (Me2SO solution) were compared with calculated minimum energy conformations. We find qualitative agreement between the results of the different techniques with respect to global tendencies of conformational behaviour: we present experimental evidence showing that the presence of proline in the sequence has a more pronounced effect on the side chain organization of the residues preceding it than on one succeeding it. This steric influence of proline on its immediate neighbor is even stronger in the cis isomer than in the more common trans isomer. The strong preference for Rotamer II (chi 1 = 180 degrees) over Rotamer I (chi 1 = -60 degrees) in ATPA (cis-form) concomitant with a noticeable deviation of chi 2 is a striking example.     

Interaction of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pili strain PAK with a cross-reactive antibody: conformation of the bound peptide

The C-terminal receptor binding region of Pseudomonas aeruginosa pilin protein strain PAK (residues 128-144) has been the target for the design of a vaccine effective against P. aeruginosa infections. We have recently cloned and expressed a (15)N-labeled PAK pilin peptide spanning residues 128-144 of the PAK pilin protein. The peptide exists as a major (trans) and minor (cis) species in solution, arising from isomerization around a central Ile(138)-Pro(139) peptide bond. The trans isomer adopts two well-defined turns in solution, a type I beta-turn spanning Asp(134)-Glu-Gln-Phe(137) and a type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142). The cis isomer adopts only one well-defined type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142) but displays evidence of a less ordered turn spanning Asp(132)-Gln-Asp-Glu(135). These turns have been implicated in cross-reactive antibody recognition. (15)N-edited NMR spectroscopy was used to study the binding of the (15)N-labeled PAK pilin peptide to an Fab fragment of a cross-reactive monoclonal antibody, PAK-13, raised against the intact PAK pilus. The results of these studies are as follows: the trans and cis isomers bind with similar affinity to the Fab, despite their different topologies; both isomers maintain the conformational integrity of their beta-turns when bound; binding leads to the preferential stabilization of the first turn over the second turn in each isomer; and binding leads to the perturbation of resonances within regions of the trans and cis backbone that undergo microsecond to millisecond motions. These slow motions may play a role in induced fit binding of the first turn to Fab PAK-13, which would allow the same antibody combining site to accommodate either trans or cis topology. More importantly for vaccine design, these motions may also play a role in the development of a broad-spectrum vaccine capable of generating an antibody therapeutic effective against the multiple strains of P. aeruginosa.     

Evidence that the substrate backbone conformation is critical to phosphorylation by p42 MAP kinase

The effect of prolyl bond isomers on the substrate recognition capabilities of various endoproteases may be investigated in a reaction where both cis/trans isomers co-exist. Here we address the question of whether enzyme reactions at the side chain of an amino acid preceding proline proceed through an isomer specific pathway. The proline-directed p42 mitogen-activated protein kinase (ERK2) was used to phosphorylate the serine side chain in Pro-Arg-Ser-Pro-Phe-4-nitroanilide under conditions where different amounts of cis prolyl isomer of the substrate were present. Initial phosphorylation rates were calculated ranging between zero at 100% cis isomer and around 60 pM/min at the equilibrium content of 83.5% trans isomer. In the presence of the peptidyl-prolyl cis/trans isomerase human hFKBP12 (500 nM), cis/trans isomerization proceeds rapidly, permitting the maximal phosphorylation rate to be observed in the dead time of the experiment. Results show that correct signature sequences are not sufficient to render potential substrates reactive to proline-directed enzymatic phosphorylations, but that the conformational state of the peptide bond following serine (threonine) is a critical determinant. Therefore, catalysis by peptidyl-prolyl cis/trans isomerases may add a new level of control to intracellular protein phosphorylations.     

Enhanced stability of cis Pro-Pro peptide bond in Pro-Pro-Phe sequence motif

Identification of sequence motifs that favor cis peptide bonds in proteins is important for understanding and designing proteins containing turns mediated by cis peptide conformations. From (1)H NMR solution studies on short peptides, we show that the Pro-Pro peptide bond in Pro-Pro-Phe almost equally populates the cis and trans isomers, with the cis isomer stabilized by a CHc...pi interaction involving the terminal Pro and Phe. We also show that Phe is over-represented at sequence positions immediately following cis Pro-Pro motifs in known protein structures. Our results demonstrate that the Pro-Pro cis conformer in Pro-Pro-Phe sequence motifs is as important as the trans conformer, both in short peptides as well as in natively folded proteins.     

A conserved cis peptide bond is necessary for the activity of Bowman-Birk inhibitor protein

The Bowman-Birk inhibitor (BBI) family of protease inhibitors has an inhibitory region comprising a disulfide-linked nine-residue loop that adopts the characteristic canonical motif found in many serine protease inhibitors. A unique feature of the BBI loop is the presence of a cis peptide bond at the edge of the inhibitory loop. BBI-related protein fragments that encapsulate this loop retain the structure and inhibitory activity of the parent protein. The most common BBI loop sequence has a proline-proline element with a cis-trans geometry at P3'-P4'. We have examined this element by analysis of the inhibitory activity and structure for a series of synthetic fragments where each of these proline residues has been systematically replaced with alanine. The results show that only when a proline is present at P3' are potent inhibition and a cis peptide bond at that position in the solution structure observed, suggesting that this conformation is required for biological activity. Though a P4' proline is not essential for activity, it effectively stabilizes the cis conformation at P3' by suppressing alternative conformations. This is most evident from the Pro-Ala variant, which comprises a 1:1 mixture of slowly exchanging and structurally different cis and trans isomers. Monitoring the action of trypsin on this mixture by NMR shows that this protease interacts selectively with the cis P3' structure, providing direct evidence for the link between activity and the nativelike structure of the cis isomer. This is, to the best of our knowledge, the first example where cis isomer selectivity can be demonstrated for a proteinase.     

Complete determination of the Pin1 catalytic domain thermodynamic cycle by NMR lineshape analysis

The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the peptide bond preceding a proline residue between cis and trans isomers. To best understand the mechanisms of Pin1 regulation, rigorous enzymatic assays of isomerization are required. However, most measures of isomerase activity require significant constraints on substrate sequence and only yield rate constants for the cis isomer, [Formula: see text] and apparent Michaelis constants, [Formula: see text]. By contrast, NMR lineshape analysis is a powerful tool for determining microscopic rates and populations of each state in a complex binding scheme. The isolated catalytic domain of Pin1 was employed as a first step towards elucidating the reaction scheme of the full-length enzyme. A 24-residue phosphopeptide derived from the amyloid precurser protein intracellular domain (AICD) phosphorylated at Thr668 served as a biologically-relevant Pin1 substrate. Specific (13)C labeling at the Pin1-targeted proline residue provided multiple reporters sensitive to individual isomer binding and on-enzyme catalysis. We have performed titration experiments and employed lineshape analysis of phosphopeptide (13)C-(1)H constant time HSQC spectra to determine [Formula: see text], [Formula: see text], [Formula: see text], and [Formula: see text] for the catalytic domain of Pin1 acting on this AICD substrate. The on-enzyme equilibrium value of [E·trans]/[E·cis]?=?3.9 suggests that the catalytic domain of Pin1 is optimized to operate on this substrate near equilibrium in the cellular context. This highlights the power of lineshape analysis for determining the microscopic parameters of enzyme catalysis, and demonstrates the feasibility of future studies of Pin1-PPIase mutants to gain insights on the catalytic mechanism of this important enzyme.     

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.     

Single proline residues can dictate the oxidative folding pathways of cysteine-rich peptides

The cysteine-rich N and C-terminal domains of minicollagen-1 from Hydra nematocysts fold with excesses of oxidized/reduced glutathione (10:1) into globular structures with distinct cystine frameworks despite their identical cysteine sequence pattern. An additional main difference is the cis conformation of a conserved proline residue in the N-terminal and the trans conformation of this residue in the C-terminal domain. Comparative analysis of the oxidative folding revealed for the C-terminal domain a fast and highly cooperative formation of a single disulfide isomer. Conversely, oxidation of the N-terminal domain proceeds mainly via an intermediate that results from the fast quasi-stochastic disulfide formation according to the proximity rule. The rate of conversion of the bead-like isomer into the globular end-product is largely dominated by the trans-to-cis isomerization of the critical proline residue as well assessed by its replacement with (4R)- and (4S)-fluoroproline known to exhibit distinct propensities for the trans and cis conformation, respectively. Independently, whether the trans or cis conformation is favored by these substitutions, both analogues retain sufficient sequence-encoded information to fold almost quantitatively into the identical cystine framework and thus spatial structure of the parent peptide with the critical proline residue as cis isomer, but at rates significantly lower for the (4R) than for the (4S)-fluoroproline analogue. Correspondingly, other sequence-encoded structural elements have to act as a driving force for these unidirectional folding pathways despite the rather simple sequence composition consisting only of aliphatic residues, some proline and only one aromatic residue (tyrosine) in the core parts of the C and N-terminal domains. The two cysteine-rich domains of minicollagen-1 may well represent ideal targets for ab initio structure calculations in order to learn more about the elementary information encoded in such primordial molecules.     

Parallel channels and rate-limiting steps in complex protein folding reactions: prolyl isomerization and the alpha subunit of Trp synthase,a TIM barrel protein

A kinetic folding mechanism for the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, involving four parallel channels with multiple native, intermediate and unfolded forms, has recently been proposed. The hypothesis that cis/trans isomerization of several Xaa-Pro peptide bonds is the source of the multiple folding channels was tested by measuring the sensitivity of the three rate-limiting phases (tau(1), tau(2), tau(3)) to catalysis by cyclophilin, a peptidyl-prolyl isomerase. Although the absence of catalysis for the tau(1) (fast) phase leaves its assignment ambiguous, our previous mutational analysis demonstrated its connection to the unique cis peptide bond preceding proline 28. The acceleration of the tau(2) (medium) and tau(3) (slow) refolding phases by cyclophilin demonstrated that cis/trans prolyl isomerization is also the source of these phases. A collection of proline mutants, which covered all of the remaining 18 trans proline residues of alphaTS, was constructed to obtain specific assignments for these phases. Almost all of the mutant proteins retained the complex equilibrium and kinetic folding properties of wild-type alphaTS; only the P217A, P217G and P261A mutations caused significant changes in the equilibrium free energy surface. Both the P78A and P96A mutations selectively eliminated the tau(1) folding phase, while the P217M and P261A mutations eliminated the tau(2) and tau(3) folding phases, respectively. The redundant assignment of the tau(1) phase to Pro28, Pro78 and Pro96 may reflect their mutual interactions in non-random structure in the unfolded state. The non-native cis isomers for Pro217 and Pro261 may destabilize an autonomous C-terminal folding unit, thereby giving rise to kinetically distinct unfolded forms. The nature of the preceding amino acid, the solvent exposure, or the participation in specific elements of secondary structure in the native state, in general, are not determinative of the proline residues whose isomerization reactions can limit folding.     

Synthesis and immunosuppressive activity of new cyclolinopeptide A analogs modified with beta-prolines

Immune response suppressors are used in the medical praxis to prevent graft rejection after organ transplantation and in the therapy of some autoimmune diseases. As a continuation of our previous work searching for new, effective suppressors devoid of toxicity, we present the synthesis, conformational analysis, and biological activity of nonapeptides 1-6, analogs of naturally existing immunomodulatory peptide CLA. New CLA analogs were modified with (S)-beta(2)-iso-proline 7 or (S)-beta(3)-homo-proline 8, respectively. The conformational influence of the beta-iso-proline and beta-homo-proline building blocks was analyzed by NMR spectroscopy. Peptides 1-6 exist as a mixture of four isomers due to cis/trans isomerization of the Xxx-Pro peptide bond. The major isomers of peptides 1, 3, and 4 contain all peptide bonds of the trans geometry. The geometry of the proline-proline bond of the second populated isomer of peptides 3 and 4 is cis. The proline-proline peptide bond is cis for the major isomers of peptides 2, 5, and 6. The peptides were tested for their ability to suppress the proliferative response of mouse splenocytes to T- and B-cell mitogens and the secondary humoral immune response to sheep erythrocytes in vitro in parallel with a reference drug-cyclosporine A. The immunoregulatory actions of the peptides depended on the position and content of proline isomers and were, with some exceptions, strongly inhibitory at the highest dose tested (100 microg/ml). In addition, the peptides were practically devoid of toxicity at that dose. In conclusion, the replacement of Pro by beta-Pro may be useful for fine-tuning CLA immunosuppressive potency and undesirable toxicity.     

Effects of i and i+3 residue identity on cis-trans isomerism of the aromatic(i+1)-prolyl(i+2) amide bond: implications for type VI beta-turn formation

Cis-trans isomerization of amide bonds plays critical roles in protein molecular recognition, protein folding, protein misfolding, and disease. Aromatic-proline sequences are particularly prone to exhibit cis amide bonds. The roles of residues adjacent to a tyrosine-proline residue pair on cis-trans isomerism were examined. A short series of peptides XYPZ was synthesized and cis-trans isomerism was analyzed. Based on these initial studies, a series of peptides XYPN, X = all 20 canonical amino acids, was synthesized and analyzed by NMR for i residue effects on cis-trans isomerization. The following effects were observed: (a) aromatic residues immediately preceding Tyr-Pro disfavor cis amide bonds, with K(trans/cis)= 5.7-8.0, W > Y > F; (b) proline residues preceding Tyr-Pro lead to multiple species, exhibiting cis-trans isomerization of either or both X-Pro amide bonds; and (c) other residues exhibit similar values of K(trans/cis) (= 2.9-4.2), with Thr and protonated His exhibiting the highest fraction cis. beta-Branched and short polar residues were somewhat more favorable in stabilizing the cis conformation. Phosphorylation of serine at the i position modestly increases the stability of the cis conformer. In addition, the effect of the i+3 residue was examined in a limited series of peptides TYPZ. NMR data indicated that aromatic residues, Pro, Asn, Ala, and Val at the i+3 residue all favor cis amide bonds, with aromatic residues and Asn favoring more compact phi at Tyr(cis) and Ala and Pro favoring more extended phi at Tyr(cis). D-Alanine at the i+3 position particularly disfavors cis amide bonds.     

Conformational studies of N-Tyr-MIF-1 in aqueous solution by 1H nuclear magnetic resonance spectroscopy.

N-Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) is an endogenous brain peptide with multiple effects on animal behavior. However, there have been no studies on the conformation of this tetrapeptide. In this report, we studied the conformation of N-Tyr-MIF-1 in aqueous solution by conventional one-dimensional and two-dimensional (COSY and NOESY) 1H nuclear magnetic resonance spectroscopy at 300 MHz. A complete set of assignments for the resolved resonances and approximate assignments for the overlapping resonances were made. The results demonstrate that N-Tyr-MIF-1 is in slow exchange between two conformers, most likely determined by the cis and trans states of the proline residue. The minor conformation represents 30 +/- 3% of the population over the temperature range from 3 degrees to 73 degrees. In the major conformation, the tyrosine aromatic ring appears to be close enough to interact directly with the proline pyrrolidine ring, as indicated by a strong temperature dependence of the proline C beta H, C delta H and C delta H' chemical shifts. In contrast, this interaction of the tyrosine and proline rings is not present in the minor conformation.