Conformational analysis of the tetrapeptide Pro-D-Phe-Pro-Gly in aqueous solution

Conformational investigations of the tetrapeptide Pro-D-Phe-Pro-Gly in water solution were carried out by 1H and 13C NMR spectroscopy. The internal proline residue allows for the possibility of cis/trans isomerization about the D-Phe-Pro peptide bond resulting in two conformational isomers. The major isomer was identified as the trans isomer. The pH-dependence of the cis/trans equilibrium supports an additional stabilisation of the trans isomer by an intramolecular ionic interaction between the amino- and carboxy-terminus in the zwitterionic state. Based on 13C spin-lattice relaxation times (T1), different pyrrolidine ring conformations of Pro1 and Pro3 could be determined. By combination of several NMR data (vicinal coupling constants 3JN alpha, temperature dependence of the NH chemical shifts, differences in the chemical shifts between the beta and gamma carbons of the proline residues) and energy minimization calculations, a type II' beta-turn should contribute considerably to the overall structure of the trans isomer.     

NMR conformational analysis of cis and trans proline isomers in the neutrophil chemoattractant, N-acetyl-proline-glycine-proline

Alkaline hydrolysis of corneal proteins in the alkali-injured eye releases N-acetyl-proline-glycine-proline (Ac-Pro-Gly-Pro-OH) among other peptides. It has been shown that this tripeptide is a neutrophil chemoattractant. Existing data suggest that the release of this peptide is the catalytic event for early neutrophil invasion of the cornea leading to corneal ulcers. In order to design inhibitors of this tripeptide chemoattractant that would block neutrophil invasion and diminish corneal ulcers, we studied the solution properties of this tripeptide by NMR spectroscopy and compared this peptide to Ac-Pro-Gly-OH (a weaker chemoattractant), and to Ac-Pro-OH (inactive). The NMR data were consistent with Ac-Pro-Gly-Pro-OH existing in solution as a mixture of four isomers with different cis and trans conformations about the two X-proline amide bonds. The isomer with two trans conformations (trans-trans) was the most dominant (41%) in aqueous solution. This was followed by the isomers with mixed cis and trans conformations (trans-cis, 26% and cis-trans, 20%). The isomer with two cis conformations (cis-cis) was the least favored (13%). The populations of these isomers were investigated in DMSO and they were similar to those reported in aqueous solutions except that the ordering of the trans-cis and cis-trans isomers were reversed. NMR NH temperature coefficients and nuclear Overhauser effect (NOE) measurements as well as CD spectroscopy were used to demonstrate that the four isomers exist primarily in an extended conformation with little hydrogen bonding. The available (NOE) information was used with molecular dynamics calculations to construct a dominant solution conformation for each isomer of the tripeptide. This information will serve as a model for the design of peptide and nonpeptide inhibitors of the chemoattractant.     

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.     

Inhibition of rabbit lung angiotensin-converting enzyme by N alpha-[(S)-1-carboxy-3-phenylpropyl]L-alanyl-L-proline and N alpha-[(S)-1-carboxy-3-phenylpropyl]L-lysyl-L-proline

Two novel peptide analogs, N alpha-[(S)-1-carboxy-3-phenylpropyl]L-alanyl-L-proline and the corresponding L-lysyl-L-proline derivative, have been demonstrated to be potent competitive inhibitors of purified rabbit lung angiotensin-converting enzyme: Ki = 2 and 1 X 10(-10) M, respectively, at pH 7.5, 25 degrees C, and 0.3 M chloride ion. Second-order rate constants for addition of these inhibitors to enzyme under the same conditions are in the range 1-2 X 10(6) M-1 s-1; first-order rate constants for dissociation of the EI complexes are in the range 1-4 X 10(-4) s-1. The association rate constants are similar to those measured for D-3-mercapto-2-methylpropanoyl-L-proline, captopril, but the dissociation rate constants are severalfold slower and account for the higher affinity of these inhibitors for the enzyme. The dissociation constant for the EI complex containing N alpha-[(S)-1-carboxy-3-phenylpropyl]L-alanyl-L-proline is pH-dependent, and reaches a minimum at approximately pH 6: Ki = 4 +/- 1 X 10(-11) M. The pH dependence is consistent either with a model for which the protonation state of the secondary nitrogen atom in the inhibitor determines binding affinity, or one for which ionizations on the enzyme alone influence affinity for these inhibitors. The affinity of this inhibitor for the zinc-free apoenzyme is 2 X 10(4) times less than for the zinc-free apoenzyme is 2 X 10(4) times less than that for the holoenzyme. If considered as a "collected product" inhibitor, N alpha-[(S)-1-carboxy-3-phenylpropyl]L-alanyl-L-proline appears to derive an additional factor of 375 M in its affinity for the enzyme compared to that of the two products of its hypothetical hydrolysis, a consequence of favorable entropy effects.     

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.     

Stereospecific pH-dependent degradation kinetics of R- and S-naproxen-beta-l-O-acyl-glucuronide

The hydrolysis and acyl migration of biosynthetic S-naproxen-beta-l-O-acyl glucuronide (I) and R-naproxen-beta-l-O-acyl glucuronide (II) was followed by HPLC. Nine first-order kinetic rate constants for the hydrolysis and acyl migration between the beta-l-O-acyl glucuronide, its alpha/beta-2, alpha/beta-3-, alpha/beta-4-, and alpha-1-O-acyl isomers and naproxen aglycone were determined for I and II at pH 7.00, 7.40 and 8.00 at 37 degrees C by kinetic simulation. For I the 3-O-acyl isomer was the most stable isomer as the pseudo-equilibrium ratio for the major acyl-migrated isomers was 1:1.5:0.9 (2-O-acyl isomer:3-O-acyl isomer:4-O-acyl isomer). The 3- and 4-O-acyl isomers of II were equally stable as the pseudo-equilibrium ratio for the major acyl-migrated isomers was 1:1.4:1.4 (2-O-acyl isomer:3-O-acyl isomer:4-O-acyl isomer). For both I and II, the pseudo-equilibrium ratio between the major 2-O-acyl isomer and the minor alpha-l-O-acyl isomer was 10:1 (2-O-acyl isomer:alpha-l-O-acyl isomer). The pseudo-equilibrium found for the major acyl-migrated isomers of I and II in the present study corresponds with the pattern previously published for R- and S-ketoprofen-beta-l-O-acyl glucuronide acyl-migrated isomers, suggesting that these findings may be general for acyl-migrated beta-l-O-acyl glucuronides of enantiomeric 2-arylpropionic acids.     

Stereoselectivity of interaction of phosphoenolpyruvate analogues with various phosphoenolpyruvate-utilizing enzymes

The halogenated phosphoenolpyruvate analogues (Z)-phosphoenol-3-fluoropyruvate, (E)-phosphoenol-3-fluoropyruvate, and (Z)-phosphoenol-3-bromopyruvate were synthesized and purified. The analogues were characterized by 1H and by 19F NMR where applicable. Absolute stereoselectivity of the fluorophosphoenolpyruvate isomers as substrates with the enzymes phosphoenolpyruvate carboxykinase, enolase, and pyruvate phosphate dikinase was observed. The Z isomer exhibited substrate activity with these enzymes while no substrate activity was measured with the E isomer. Both isomers exhibited substrate activity with the enzyme pyruvate kinase, however, with a substantial decrease in the Vmax/Km ratio compared to phosphoenolpyruvate as the substrate. A metal ion dependent stereoselectivity of inhibition was measured for these analogues with the enzymes phosphoenolpyruvate carboxykinase, enolase, and pyruvate kinase. The cation activator appears to affect the specificity and thus the catalytic site of these enzymes. Proton longitudinal relaxation rate titrations demonstrate that the dissociation constants, K3, of the fluorophosphoenolpyruvate isomers from the enzyme-Mn complex agree, in most cases, with the measured KI values and analogue binding resembles phosphoenolpyruvate binding. With the enzyme phosphoenolpyruvate carboxykinase, the KI not equal to K3 for (E)-fluorophosphoenolpyruvate which suggests that the binding of the E isomer is affected by the presence of the other substrates. The halogenated derivatives apparently undergo an enzyme-Mn catalyzed Michael-type addition reaction with the bromo-substituted analogue decomposing much faster than the fluoro analogues.     

Isomer-specific proteolysis of model substrates: influence that the location of the proline residue exerts on cis/trans specificity

In an effort to further develop the technique of isomer-specific proteolysis, a number of proline-containing substrates were subjected to hydrolysis in the presence of chymotrypsin, trypsin, or prolidase. The objective was to determine whether direct hydrolysis of the cis form of the substrate could occur and, if so, the extent to which it is slower than the hydrolysis of the equivalent trans form. It is shown that for both peptide and amide substrates, which contain proline at the P2 position, the cis form can be hydrolyzed directly by either chymotrypsin or trypsin, in contrast to earlier suggestions in the literature. For similar amide substrates, it was found that chymotrypsin has a lower catalytic efficiency for the cis form, relative to the trans form, by a factor of 20 000 while, for trypsin and its substrate, the cis form was cleaved about 2000 times less efficiently. Results for a trypsin substrate with proline at the P2' position, rather than the P2 position, were quite different however, since there was no indication that the cis form could be directly cleaved even at the highest enzyme concentration. There was also no indication that prolidase could cleave the dipeptide Phe-Pro when the active bond itself is in the cis form. These collective results suggest that the ability of proteases to cleave a substrate with a cis peptide bond depends strongly on the location of the cis bond relative to the active bond that is being cleaved.     

Catalytic properties of the PepQ prolidase from Escherichia coli

The PepQ prolidase from Escherichia coli catalyzes the hydrolysis of dipeptide substrates with a proline residue at the C-terminus. The pepQ gene has been cloned, overexpressed, and the enzyme purified to homogeneity. The k(cat) and k(cat)/K(m) values for the hydrolysis of Met-Pro are 109 s(-1) and 8.4 x 10(5)M(-1)s(-1), respectively. The enzyme also catalyzes the stereoselective hydrolysis of organophosphate triesters and organophosphonate diesters. A series of 16 organophosphate triesters with a p-nitrophenyl leaving group were assessed as substrates for PepQ. The S(P)-enantiomer of methyl phenyl p-nitrophenyl phosphate was hydrolyzed with a k(cat) of 36 min(-1) and a k(cat)/K(m) of 710 M(-1)s(-1). The corresponding R(P)-enantiomer was hydrolyzed more slowly with a k(cat) of 0.4 min(-1) and a k(cat)/K(m) of 11 M(-1)s(-1). The PepQ prolidase can be utilized for the kinetic resolution of racemic phosphate esters. The PepQ prolidase was shown to hydrolyze the p-nitrophenyl analogs of the nerve agents GB (sarin), GD (soman), GF, and VX.     

NMR investigations of proline and its derivatives. 4-Proton magnetic resonance parameters and structure of acetyl-proline amide.

The proton magnetic resonance (PMR) spectrum of acetyl-proline amide in D2O solution has been analysed by computer simulation. The spectra of the cis and the trans isomers have been separated and their PMR parameters (chemical shift and coupling constants) are given. Vicinal coupling constants of the pyrrolidine ring are interpreted by means of a Karplus zone relation. The chemical shift effect of the anisotropy of both peptide planes is considered. It follows that both isomers are puckered with Cgamma in an endo position, but the cis isomer is more rigid than the trans isomer, which moreover undergoes a small interconversion of the Cgamma and Cdelta atoms between two extreme spatial positions. The dihedral angle phi has different values in both isomers. Thus, the dihedral angle between the two peptide planes is smaller in the trans isomer than in the cis isomer.     

Solution structure of a DNA duplex containing a formamide-adenine base pair

The N-(2-deoxy-beta3-D-erythro-pentofuranosyl) formamide residue results from a ring fragmentation product of thymine or cytosine. The presence of a formamide-adenine base pair in the sequence 5'd(AGGAACCACG).d(CGTGGFTCCT) has been studied by 1H and 31P nuclear magnetic resonance (NMR) and molecular dynamics. There are two possible isomers for the formamide side chain, either cis or trans. For each isomer, we observed an equilibrium in solution between two forms. First, a species where the formamide is intrahelical and paired with the facing adenine. For the cis isomer, the formamide is in a syn conformation and two hydrogen bonds with adenine are formed. The trans isomer is in an anti conformation and a single hydrogen bond is observed. In the second form, whatever the isomer, the formamide is rejected outside the helix, whereas the adenine remains inside.     

Synthesis of Cyclobutane Nucleosides 2-Preparation of Thymine and Uracil Analogues

1-(2-Oxocyclobutyl-4-benzoyloxymethyl)-2,4(1H,3H)-pyrimidinedione and 1-(2-oxocyclobutyl-4-benzoyloxymethyl)-5-methyl-2,4(1H,3H)-pyrimidinedione can be prepared by reaction of uracil and thymine, respectively, with 3-benzoyloxymethyl-2-bromocyclobutanone. The N-alkylation gave both cis and trans isomers with the trans isomer predominating for uracil whereas the trans isomer was the only product which could be isolated for thymine. Both series were subjected to borohydride reduction followed by transesterification with methoxide giving the corresponding uracil and thymine nucleoside analogues. The uracil derivative 1-(2-oxocyclobutyl-4-benzoyloxymethyl)-2,4(1H,3H)-pyrimidinedione was irradiated in aqueous acetonitrile to generate isonucleoside analogues.     

Proton NMR characterization of isomeric sulfmyoglobins: preparation, interconversion, reactivity patterns, and structural features

The preparations of sulfmyoglobin (sulf-Mb) by standard procedures have been found heterogeneous by 1H NMR spectroscopy. Presented here are the results of a comprehensive study of the factors that influence the selection among the three dominant isomeric forms of sperm whale sulf-Mb and their resulting detailed optical and 1H NMR properties as related to their detectability and structural properties of the heme pocket. A single isomer is formed initially in the deoxy state; further treatment in any desired oxidation/ligation state can yield two other major isomers. Acid catalysis and chromatography facilitate formation of a second isomer, particularly in the high-spin state. At neutral pH, a third isomer is formed by a first-order process. The processes that alter oxidation/ligation state are found to be reversible and are judged to affect only the metal center, but the three isomeric sulf-Mbs are found to exhibit significantly different ligand affinity and chemical stability. The present results allow, for the first time, a rational approach for preparing a given isomeric sulf-Mb in an optimally pure state for subsequent characterization by other techniques. While optical spectroscopy can distinguish the alkaline forms, only 1H NMR clearly distinguishes all three ferric isomers. The ring current shifts in the carbonyl complexes of reduced sulf-Mb complexes support saturation for a pyrrole in each isomer. The hyperfine shift patterns in the various oxidation/spin states of sulf-Mbs indicate relatively small structural alteration, and the proximal and distal sides of the heme suggest that peripheral electronic effects are responsible for the differentially reduced ligand affinities for the three isomeric sulf-Mbs. The first 1H NMR spectra of sulfhemoglobins are presented, which indicate a structure similar to that of the initially formed sulf-Mb isomer but also suggest the presence of a similar molecular heterogeneity as found for sulf-Mb, albeit to a smaller extent.     

Proline-valine pseudo peptide enol lactones. Effective and selective inhibitors of chymotrypsin and human leukocyte elastase

Pro-Val pseudo dipeptides incorporating protio and halo enol lactones were tested for inhibitory activity against the serine proteases human leukocyte elastase (HLE), porcine pancreatic elastase, alpha-chymotrypsin, trypsin, thrombin, and urokinase. The protio enol lactones 1a-c were found to be HLE substrates but were poor alternate substrate inhibitors. The bromo enol lactone trans isomer 2a was found to be a very effective inhibitor of HLE and chymotrypsin, as shown by the binding constants (KI), acylation rates (ka), inactivation rates, and partition ratios determined for each enzyme. This inhibitor shows better specificity toward its target enzyme HLE than monosubstituted halo enol lactones; we attribute this to a pseudo dipeptide acyl enzyme whose structure is similar to that adopted by good peptide substrates of HLE. Inactivation of chymotrypsin by the bromo enol lactone 2a is permanent, but inactivation of HLE is partially recoverable upon treatment with the nucleophile hydrazine, indicating that lactone 2a produces two species of inactivated HLE. The more stable of these species could be the result of alkylation of His-57 by the electrophilic bromomethyl ketone revealed in the acyl enzyme, and the less stable, hydrazine-reactivatable species could be the result of alkylation of Asp-102 or the hydrolysis of the bromomethyl ketone group in the initially formed acyl enzyme to form a new, more stable acyl enzyme.     

Mechanistic information on the reaction of cis- and trans-[RuCl2(DMSO)4] with d(T2GGT2) derived from MALDI-TOF and HPLC studies

Reactions of trans and cis isomers of the Ru(II) complex [RuCl(2)(DMSO)(4)] with single-stranded hexanucleotide d(T(2)GGT(2)) were studied in aqueous solutions in the absence and presence of excess chloride by high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS). Despite the different reactive species formed from the two isomers in aqueous solution, similar reaction products are obtained in their interaction with d(T(2)GGT(2)). Both [RuCl(2)(DMSO)(4)] isomers bind to the oligonucleotide in the bidentate mode to form thermodynamically stable bis-guanosine adducts, Ru(G-N7)(2). Significant differences were observed in the reaction rates, however the reaction with trans- [RuCl(2)(DMSO)(4)] is ca. 5-10 times faster in comparison to that observed for the cis analogue. This difference is interpreted in terms of different rate-limiting steps for the trans and cis complexes, respectively. It is suggested that the rate of the reaction with the trans isomer is controlled by dissociation of a Cl(-) ligand from the initially formed trans,cis,cis-[RuCl(2)(DMSO)(2)(H(2)O)(2)]. In the contrast, release of a dimethyl sulfoxide molecule from the reactive species cis,fac-[RuCl(2)(DMSO)(3)(H(2)O)] is likely to be rate limiting for the cis analogue. Significant influence of electrostatic interactions on the reaction rate was observed for the trans isomer. Mechanistic interpretation of the observed reactivity trends based on data obtained from UV-Vis spectroscopy, HPLC and MALDI-TOF MS studies is presented and discussed within the paper.     

Conformational analysis of cyclic hexapeptides designed as constrained ligands for the SH2 domain of the p85 subunit of phosphatidylinositol-3-OH kinase

The structures of the cyclic hexapeptide cyclo(-Gly-Tyr-Val-Pro-Met-Leu-) ( 1 ) and its phosphotyrosyl (pTyr) derivative cyclo[-Gly-Tyr(PO₃H₂)-Val-Pro-Met-Leu-] ( 2 ), designed as constrained models of a sequence that interacts with the src homology 2 (SH2) region of the p85 subunit of phosphatidylinositol-3-OH kinase (PI-3 kinase), were studied in methanol/water solutions by 500 MHz nmr spectroscopy. Compound 1 was found to exist as a 2:1 mixture of isomers about the Val-Pro bond (trans and cis prolyl) between 292–330 K in 75% CD₃O (D,H)/(D,H)₂O solutions. A third species of undetermined structure (ca. 5%) was also observed. Compound 2, a model of phosphorylated peptide ligand that binds to the PI-3 kinase SH2 domain, exhibited similar conformational isomerism. When either compound was dissolved in pure solvent [i.e., 100% CD₃O(H,D) or (H,D)₂O] the ratio of cis to trans isomers was ca 1:1. A battery of one- and two-dimensional nmr experiments at different temperatures and solvent compositions allowed a complete assignment of both the cis and trans forms of 1 and indicated the trans compound to be the major isomer. The spectral properties of the phosphorylated derivative 2 paralleled those of 1 , indicating like conformations for the two compounds. Analysis of rotating frame Overhauser spectroscopy data, coupling constants, amide proton temperature dependence, and amide proton exchange rates generated a set of constraints that were employed in energy minimization and molecular dynamics calculations using the CHARMM force field. The trans isomer exists with the tyrosine and C-terminal Tyr(+3) (Met) residues at opposite corners of the 18-membered ring separated by a distance of 16–18 Å, in contrast with the cis isomer where the side chains of these residues are much closer in space (7–14 Å). It was previously shown that the pTyr and the third amino acid C-terminal to this residue are the critical recognition elements for pTyr-peptide binding to the PI-3 kinase SH2 domain. Such cyclic structures may offer appropriate scaffolding for positioning important amino acid side chains of pTyr-containing peptides as a means of increasing their binding affinities to SH2 domains, and in turn provide a conceptual approach toward the design of SH2 domain directed peptidomimetics. © 1995 John Wiley & Sons, Inc.     

NMR studies of carbohydrates and carbohydrate-mimetic peptides recognized by an anti-group B Streptococcus antibody

As part of a program to investigate the origins of peptide-carbohydrate mimicry, the conformational preferences of peptides that mimic the group B streptococcal type III capsular polysaccharide have been investigated by NMR spectroscopy. Detailed studies of a dodecapeptide, FDTGAFDPDWPA, a molecular mimic of the polysaccharide antigen, and two new analogs, indicated a propensity for beta-turn formation. Different beta-turn types were found to be present in the trans and cis (Trp-10-Pro-11) isomers of the peptide: the trans isomer favored a type I beta-turn from residues Asp-7-Trp-10, whereas the cis isomer exhibited a type VI beta-turn from residues Asp-9-Ala-12. The interaction of the dodecapeptide FDTGAFDPDWPA with a protective anti-group B Streptococcus monoclonal antibody has also been investigated, by transferred nuclear Overhauser effect NMR spectroscopy and saturation-transfer difference NMR spectroscopy (STD-NMR). The peptide was found to adopt a type I beta-turn conformation on binding to the antibody; the peptide residues (Asp-7-Trp-10) forming this turn are recognized by the antibody, as demonstrated by STD-NMR experiments. STD-NMR studies of the interactions of oligosaccharide fragments of the capsular polysaccharide have also been performed and provide evidence for the existence of a conformational epitope.     

Peptidyl-prolyl cis-trans isomerase activity as studied by dynamic proton NMR spectroscopy

Recently the identity of the peptidyl-prolyl cis-trans isomerase (PPIase), which accelerates the cis/trans isomerization of prolyl peptide bonds and cyclophilin, the binding protein for the immunosuppressive drug Cyclosporin A (CsA), was discovered. The PPIase catalysis toward the substrate Suc-Ala-Phe-Pro-Phe-pNA has been studied by 1H NMR spectroscopy. Using the bandshape analysis technique the rate of interconversion between the cis and trans isomers of the substrate could be measured in the presence of PPIase and under equilibrium conditions. The acceleration is inhibited by equimolar amounts of CsA. The results provide evidence that the PPIase catalysis is more complex than a simple exchange between two states.     

Dihydrolipoyl transacetylase of Escherichia coli. Formation of 8-S-acetyldihydrolipoamide

The dihydrolipoyl transacetylase component (E2) of the pyruvate dehydrogenase complex catalyzes the reaction of acetyl coenzyme A (acetyl-CoA) with dihydrolipoamide, producing coenzyme A and S-acetyldihydrolipoamide. The acetyl group is shown by experiments reported herein to be bonded to S8 in the enzymatic product. 1H NMR analysis of synthetic samples of both structural isomers of S-acetyl-S-(phenylmercurio)dihydrolipoamide enabled structural assignments to be made. Reaction of 8-S-acetyl-6-S-(phenylmercurio)dihydrolipoamide with 3-mercaptopropionic acid in chloroform produced 8-S-acetyldihydrolipoamide which contained a small amount (5%) of the 6-S isomer. Reaction of 6,8-di-S-acetyldihydrolipoamide with NH2OH produced a 4:1 mixture of 6-S-acetyldihydrolipoamide and the 8-S isomer. These compounds did not isomerize at significant rates in chloroform but rapidly isomerized to the equilibrium mixture in aqueous solution (Keq = 3.4). The second-order rate constants for the hydroxide-catalyzed isomerization were found to be kf = (1.15 +/- 0.07) X 10(6) M-1 X s-1 and kr = (3.36 +/- 0.20) X 10(5) M-1 X s-1 in the direction of the formation of the 8-S isomer. The enzymatic product was trapped by addition of phenylmercuric hydroxide within 15 s-30 min after starting the reaction. 1H NMR analysis of the products obtained at various times showed that the enzymatic product was 8-S-acetyldihydrolipoamide, which underwent progressive isomerization to the mixture of isomers within a few minutes. In the reaction of acetyl-CoA with dihydrolipoamide, the latter substrate reacts in place of enzyme-bound dihydrolipoyl moieties. Therefore, acetylation occurs at the 8-S position of bound lipoyl groups.