Solution Conformation Of Peptide Research Articles

Strategy for improving circular dichroism spectra deconvolution accuracy for macrocyclic peptides in drug discovery

Peptide therapeutics have emerged as an appealing modality in the pharmaceutical industry. Understanding peptide conformation in solution remains one of the most critical areas for peptide drug development. Circular dichroism (CD) spectroscopy is a useful technique to study the secondary structure of proteins and peptides, but the current approaches are limited to protein-focused models to predict high-order structures of peptides, and the models were built based on X-ray crystallography instead of solution-based technique, as a result, such models may have poor predictions for peptides. In this study, we present a novel CD deconvolution model to determine peptide conformation in solution. To quantitatively obtain secondary structure information using CD, a calibration model is needed beforehand to establish the relationship between each secondary structure feature and the corresponding CD response. A reference set containing the majority of cyclic peptides with known structures from solution-state NMR spectroscopy was used to build the calibration model for CD deconvolution. Improved prediction accuracy on the secondary structure determination for cyclic peptides was achieved by this model compared to the commercial standard model using commercially available platforms. This new CD deconvolution method is crucial for peptide conformational analysis in solution, and has the potential to greatly accelerate peptide drug candidate optimization in the pharmaceutical drug discovery field.

Mechanistic basis of the increased methylation activity of the SETD2 protein lysine methyltransferase towards a designed super-substrate peptide

Protein lysine methyltransferases have important regulatory functions in cells, but mechanisms determining their activity and specificity are incompletely understood. Naturally, SETD2 introduces H3K36me3, but previously an artificial super-substrate (ssK36) was identified, which is methylated >100-fold faster. The ssK36-SETD2 complex structure cannot fully explain this effect. We applied molecular dynamics (MD) simulations and biochemical experiments to unravel the mechanistic basis of the increased methylation of ssK36, considering peptide conformations in solution, association of peptide and enzyme, and formation of transition-state (TS) like conformations of the enzyme-peptide complex. We observed in MD and FRET experiments that ssK36 adopts a hairpin conformation in solution with V35 and K36 placed in the loop. The hairpin conformation has easier access into the active site of SETD2 and it unfolds during the association process. Peptide methylation experiments revealed that introducing a stable hairpin conformation in the H3K36 peptide increased its methylation by SETD2. In MD simulations of enzyme-peptide complexes, the ssK36 peptide approached TS-like structures more frequently than H3K36 and distinct, substrate-specific TS-like structures were observed. Hairpin association, hairpin unfolding during association, and substrate-specific catalytically competent conformations may also be relevant for other PKMTs and hairpins could represent a promising starting point for SETD2 inhibitor development.

Structure of whey protein hydrolysate used as emulsifier in wet and dried oil delivery systems: Effect of pH and drying processing

The secondary structure of whey protein concentrate hydrolysate (WPCH), used as an emulsifier in oil delivery systems, was investigated using Synchrotron Radiation Circular Dichroism (SRCD). The effect of pH on the conformation of peptides in solution and adsorbed at the oil/water interface, as well as the thermal stability of the systems was studied. Furthermore, oil-loaded microcapsules were produced by spray-drying or electrospraying to investigate the influence of encapsulating agents (glucose syrup, maltodextrin) and drying technique on the secondary structure of WPCH at the oil/water interface. Enzymatic hydrolysis resulted in peptides with a highly unordered structure (∼60% turns and unordered regions) in solution. However, WPCH adsorption onto the oil/water interface increased the α-helical content resulting in an improved thermal stability. The encapsulating agents and spray-drying process did not modify the conformation of WPCH at the oil/water interface. Nonetheless, electrospraying affected the SRCD spectra obtained for WPCH adsorbed at the oil/water interface.

Conformation and Morphology of 4-(NH2/OH)-Substituted l/d-Prolyl Polypeptides: Effect of Homo- and Heterochiral Backbones on Formation of β-Structures and Nanofibers.

The relative stereochemistry of C2 and C4 in 4-substituted prolyl polypeptides plays an important role in defining the derived conformation in solution. cis-(2S,4S)-Amino/hydroxy-l-prolyl polypeptide (lC-Amp9/lC-Hyp9) shows a PPII conformation in phosphate buffer and a β-structure in a relatively hydrophobic solvent, trifluoroethanol (TFE). It is now demonstrated that the homochiral enantiomeric cis-substituted d-prolyl polypeptide (dC-Amp9/dC-Hyp9) exhibits mirror image β-structures in TFE. In the case of alternating heterochiral prolyl peptides, it is the trans-substituted [lT(2S,4R)-dT(2R,4S)]n prolyl polypeptide that shows β-structures in TFE, while the cis-substituted [lC(2S,4S)-dC(2R,4R)]n prolyl polypeptide is disordered in both phosphate buffer and TFE. The results highlight the important chirality-specific structural requirements for β-structure formation. The observed conformation in solution (circular dichroism (CD)) is also correlated with the morphology of the self-assemblies (field emission scanning electron microscopy (FESEM)), with the PPII form leading to spherical nanoparticles and β-structures leading to nanofiber formation. The results shed light on the role of relative stereochemistry at C2 and C4 in defining the polyproline peptide conformation in solution and how different conformations drive self-assemblies of peptides toward specific nanostructures.

Dynamic elements and kinetics: Most favorable conformations of peptides in solution with measurements and simulations.

Small peptides in solution adopt a specific morphology as they function. It is of fundamental interest to examine the structural properties of these small biomolecules in solution and observe how they transition from one conformation to another and form functional structures. In this study, we have examined the structural properties of a simple dipeptide and a five-residue peptide with the application of far-UV circular dichroism (CD) spectroscopy as a function of temperature, fluorescence anisotropy, and all-atom molecular dynamics simulation. Analysis of the temperature dependent CD spectra shows that the simplest dipeptide N-acetyl-tryptophan-amide (NATA) adopts helical, beta sheet, and random coil conformations. At room temperature, NATA is found to have 5% alpha-helical, 37% beta sheet, and 58% random coil conformations. To our knowledge, this type of structural content in a simplest dipeptide has not been observed earlier. The pentapeptide (WK5) is found to have four major secondary structural elements with 8% 310 helix, 14% poly-L-proline II, 8% beta sheet, and 14% turns. A 56% unordered structural population is also present for WK5. The presence of a significant population of 310 helix in a simple pentapeptide is rarely observed. Fluorescence anisotropy decay (FAD) measurements yielded reorientation times of 45 ps for NATA and 120 ps for WK5. The fluorescence anisotropy decay measurements reveal the size differences between the two peptides, NATA and WK5, with possible contributions from differences in shape, interactions with the environment, and conformational dynamics. All-atom molecular dynamics simulations were used to model the structures and motions of these two systems in solution. The predicted structures sampled by both peptides qualitatively agree with the experimental findings. Kinetic modeling with optimal dimensionality reduction suggests that the slowest dynamic processes in the dipeptide involve sidechain transitions occurring on a 1 ns timescale. The kinetics in the pentapeptide monitors the formation of a distorted helical structure from an extended conformation on a timescale of 10 ns. Modeling of the fluorescence anisotropy decay is found to be in good agreement with the measured data and correlates with the main contributions of the measured reorientation times to individual conformers, which we define as dynamic elements. In NATA, the FAD can be well represented as a sum of contributions from representative conformers. This is not the case in WK5, where our analysis suggests the existence of coupling between conformational dynamics and global tumbling. The current study involving detailed experimental measurements and atomically detailed modeling reveals the existence of specific secondary structural elements and novel dynamical features even in the simplest peptide systems.

Probing Anti-inflammatory Properties Independent of NF-κB Through Conformational Constraint of Peptide-Based Interleukin-1 Receptor Biased Ligands.

Interleukin-1β (IL-1β) binds to the IL-1 receptor (IL-1R) and is a key cytokine mediator of inflammasome activation. IL-1β signaling leads to parturition in preterm birth (PTB) and contributes to the retinal vaso-obliteration characteristic of oxygen-induced retinopathy (OIR) of premature infants. Therapeutics targeting IL-1β and IL-1R are approved to treat rheumatoid arthritis; however, all are large proteins with clinical limitations including immunosuppression, due in part to inhibition of NF-κB signaling, which is required for immuno-vigilance and cytoprotection. The all-D-amino acid peptide 1 (101.10, H-d-Arg-d-Tyr-d-Thr-d-Val-d-Glu-d-Leu-d-Ala-NH2) is an allosteric IL-1R modulator, which exhibits functional selectivity and conserves NF-κB signaling while inhibiting other IL-1-activated pathways. Peptide 1 has proven effective in experimental models of PTB and OIR. Seeking understanding of the structural requirements for the activity and biased signaling of 1, a panel of twelve derivatives was synthesized employing the various stereochemical isomers of α-amino-γ-lactam (Agl) and α-amino-β-hydroxy-γ-lactam (Hgl) residues to constrain the D-Thr-D-Val dipeptide residue. Using circular dichroism spectroscopy, the peptide conformation in solution was observed to be contingent on Agl, Hgl, and Val stereochemistry. Moreover, the lactam mimic structure and configuration influenced biased IL-1 signaling in an in vitro panel of cellular assays as well as in vivo activity in murine models of PTB and OIR. Remarkably, all Agl and Hgl analogs of peptide 1 did not inhibit NF-κB signaling but blocked other pathways, such as JNK and ROCK2 phosphorylation contingent on structure and configuration. Efficacy in preventing preterm labor correlated with a capacity to block IL-1β-induced IL-1β synthesis. Furthermore, the importance of inhibition of JNK and ROCK2 phosphorylation for enhanced activity was highlighted for prevention of vaso-obliteration in the OIR model. Taken together, lactam mimic structure and stereochemistry strongly influenced conformation and biased signaling. Selective modulation of IL-1 signaling was proven to be particularly beneficial for curbing inflammation in models of preterm labor and retinopathy of prematurity (ROP). A class of biased ligands has been created with potential to serve as selective probes for studying IL-1 signaling in disease. Moreover, the small peptide mimic prototypes are promising leads for developing immunomodulatory therapies with easier administration and maintenance of beneficial effects of NF-κB signaling.

Gd(3+) Spin Labels Report the Conformation and Solvent Accessibility of Solution and Vesicle-Bound Melittin.

Although Gd(3+)-based spin labels have been shown to be an alternative to nitroxides for double electron-electron resonance (DEER) distance measurements at high fields, their ability to provide solvent accessibility information, as nitroxides do, has not been explored. In addition, the effect of the label type on the measured distance distribution has not been sufficiently characterized. In this work, we extended the applicability of Gd(3+) spin labels to solvent accessibility measurements on a peptide in model membranes, namely, large unilamellar vesicles (LUVs) using W-band (2)H Mims electron-nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) techniques and Gd(3+)-ADO3A-labeled melittin. In addition, we carried out Gd(3+)-Gd(3+) DEER distance measurements to probe the peptide conformation in solution and when bound to LUVs. A comparison with earlier results reported for the same system with nitroxide labels shows that, although in both cases the peptide binds parallel to the membrane surface, the Gd(3+)-ADO3A label tends to protrude from the membrane into the solvent, whereas the nitroxide does the opposite. This can be explained on the basis of the hydrophilicity of the Gd(3+)-ADO3A labels in contrast with the hydrophobicity of nitroxides. The distance distributions obtained from different labels are accordingly different, with the Gd(3+)-ADO3A yielding consistently broader distributions. These discrepancies are most pronounced when the peptide termini are labeled, which implies that such labeling positions may be inadvisible.

Sodium versus potassium effects on the glutamic acid side-chains interaction on a heptapeptide

Equilibrium peptide conformations in solution, especially in the presence of salts, has been of interest for several decades. The fundamental interactions that determine the dominant peptide conformations in solution have been experimentally and computationally probed; however, a unified understanding has not yet emerged. In a previous study, we performed metadynamics simulations on the heptapeptide AEAAAEA in Sodium Chloride ( NaCl ) and Potassium Chloride ( KCl ) solutions at concentrations ranging from 0.5–2.0 M. Using a three-dimensional collective variable coordinate system, we computed the free energy landscapes in each saline environment as well as in pure water. We found that the presence of Na+and K+ions induces some changes in the stability of the conformers that define the state space, but does not alter the overall energetics between conformers and does not favor helical conformations. We investigate here, how the presence of salts ( NaCl and KCl ) affects the glutamic–glutamic interaction and its consequences on the stability of each equilibrium conformation. We perform this study through fixed backbone simulations for the most populated conformations identified in our previous work: the α-helix, 310-helix, π-helix, the extended polyproline II (PPII) and 2.51-helix conformations. It was found that for each conformation, there exists stable substates determined by the glutamic acid side-chains distance and orientation, and that Na+and K+cations (de)stabilize preferentially each conformation. It was also found that intramolecular single water mediated hydrogen bonds play a crucial role in the observed (de) stabilization of each equilibrium conformation.

Simulating the Amide I IR Signal of a Peptide in Solution using a Classical Implicit Water Approximation

Behavior of the amide I IR signal of peptides has been the object of much study because it can provide valuable information about the environments and conformations of peptides in solution. Numerous models have been proposed to predict the amide I band shape, the most successful of which may have limited utility in studying dynamic processes due to high computational cost. Here a new, simple method is employed to predict the effects that the aqueous environment and side chains have on the shape of the amide I IR signal for a polypeptide in water. Using GEPOL to generate a solute cavity, apparent charges at the solvent accessible surface are generated considering a conductor like response from the solvent to partial charges on atoms (AMBER, or CHARMM). Potentials on the atoms in amide groups resulting from the surface charges and from the side groups are then used along with an electrostatic potential map to predict how the amide I signals are perturbed from gas phase signals. Effects of cavity size and atomic partial charges on the integrity of predicted band shapes are examined. The method gives results comparable to much more computationally intensive methods.

Theoretical studies on the conformation of peptides in solution I. Conformation of N-acetyl glycine N-methyl amide in solution

A theoretical prediction of the most preferred conformation of N-acetyl glycine N-methyl amide in CCl4 is attempted. The calculation of the intrinsic or intramolecular interaction energies employs the method of partitioned energy potential. The intramolecular electrostatic energies include terms up to octopole segmental moments. It is pointed out that a truncation of the multipole interaction series at the monopole term is unjustified in this case. The calculation of intermolecular interactions with the environment is based on the method of continuum reaction field, including electrostatic, dispersion and cavity terms, which depend on the conformations through molecular dipole moments, molecular volume, and molecular exposed surface area. The most stable conformation predicted in this study (Φ = 255°, Φ = 105°) closely corresponds to the minimum in intramolecular electrostatic interactions. For other solvents intramolecular interactions and solvent effects may be of comparable importance in determining the most stable conformation of the solute molecule.

The importance of electrostatic charge and dielectric constant in conformational analysis of biologically active dipeptides

Our aim was to use molecular modeling to determine the solution conformations of small peptides (2–6 residues), which are substrates for both peptide transporters and peptidases, and can be clinically important as the basis of various therapeutic agents including β-lactams and ACE inhibitors. We evaluated two conformational search strategies and, in particular, the influence of electrostatic charge and dielectric constant on the results. AlaAla (uncharged), AlaAla (charged), N-acetyl-Ala-Ala-O-methyl and N-acetyl-Ala-Ala-methylamide were modeled using grid search and random search as implemented within SYBYL 6.4 using distance-dependent dielectric constants of between 1 (in vacuo) and 80 (water). The two search procedures located similar energy minima for both forms of AlaAla at any given dielectric constant, indicating that random searches sample conformational space sufficiently well for dipeptides at least. Analyses of the minimum-energy conformers computed for each molecule showed that, whereas the dielectric constant had minimal effect for AlaAla (uncharged), N-acetyl-Ala-Ala-O-methyl and N-acetyl-Ala-Ala-methylamide, for AlaAla (charged), as the dielectric constant approached 1 (in vacuo), this conformer had a cis peptide bond and was the only conformer present. We conclude that it is essential to model peptides in their charged forms at dielectric constants of approximately 80 to be able to determine the solution conformations of peptides recognized by peptidases and peptide transporters.

Structural investigations of basic amphipathic model peptides in the presence of lipid vesicles studied by circular dichroism, fluorescence, monolayer and modeling

A cationic amphiphilic peptide made of 10 leucine and 10 lysine residues, and four of its fluorescent derivatives in which leucines were substituted by Trp residues at different locations on the primary sequence have been synthesized. The interactions of these five peptides with neutral anionic or cationic vesicles were investigated using circular dichroism, steady state and time-resolved fluorescence with a combination of Trp quenching by brominated lipid probes, monolayers, modeling with minimization and simulated annealing procedures. We show that all the five peptides interact with neutral and anionic DMPC, DMPG, DOPC or egg yolk PC vesicles. The binding takes place whatever the peptide conformation in solution is. In the case of DMPC bilayers the binding free energy Δ G is estimated at −8 kcal mole −1 and the number of phospholipid molecules involved is about 20–25 per peptide molecule. Peptides are bound as single-stranded α helices orientated parallel to the bilayer surface. In the anchoring of phospholipid head groups around the peptides, the lipid molecules are not smeared out in a plane parallel to the membrane surface but are organized around the hydrophilic face of the α helices like `wheat grains around an ear' and protrude outside the bilayer towards the solvent. We suggest that such a lipid arrangement generates transient structural defects responsible for the membrane permeability enhancement. When an electrical potential is applied, the axis of the peptide helices remains parallel to the membrane surface and does not reorient to give rise to a bundle of helix monomers that forms transmembrane channels via a `barrel stave' mechanism. The penetration depth of α helices in relation to the position of phosphorus atoms in the unperturbed lipid leaflet is estimated at 3.2 Å.

Solution conformations of peptides representing the sequence of the toxin pardaxin and analogues in trifluoroethanol‐water mixtures: Analysis of CD spectra

Solution conformations of peptides representing the sequence of the toxin pardaxin and analogues in trifluoroethanol‐water mixtures: Analysis of CD spectra

Studies on Rubia akane(RA) derivatives. Part 10. Backbone transformation of RA-VII, an antitumour cyclic hexapeptide, through thionation. X-Ray crystal structure of [Tyr-3-Ψ(CH2NH)-Ala-4]RA-VII

RA-VII 1 was thionated with Davy reagent methyl 6 or Davy reagent p-tolyl 7 to afford novel thionopeptides, [Tyr-6-Ψ(CS-NH)-D-Ala-1]RA-VII 8, [D-Ala-1-Ψ(CS-NH)-Ala-2; Tyr-3-Ψ(CS-NH)-Ala-4]RA-VII 9 and [Ala-2-Ψ(CS-NH)-Tyr-3; Tyr-3-Ψ(CS-NH)-Ala-4]RA-VII 10 in addition to known analogues [Tyr-3-Ψ(CS-NH)-Ala-4]RA-VII 4 and [Tyr-3-Ψ(CS-NH)-Ala-4; Tyr-6-Ψ(CS-NH)-D-Ala-1]RA-VII 5. Thionopeptide 4 was reduced with nickel borohydride to give reduced peptide 11. An X-ray analysis and an NMR study revealed that compound 11 adopts a different conformation within the 18-membered-ring moiety from the predominant solution conformation of peptide 1 and of thionopeptides 4, 5, 8–10, which would explain the loss in activity of compound 11.

Heterodetic bicyclic decapeptide cyclo (Glu1 ‐Leu2 ‐Pro3‐Gly4‐Ser5‐Ile6‐Pro7‐Ala8) cyclo‐(1γ‐5β) Phe9‐Gly10

Bicyclic peptides are useful model molecules that can mimic the constrained local folding of a great number of natural peptides and proteins, such as ionophoric peptides, enzyme active site, and ligand-receptor active site. The synthesis of the bicyclic title compound with the liquid phase method is described with experimental details. Of particular interest is the heterodetic closure of the second ring. The peptide showed a complexing activity with metal cations like Ba2+, Ca2+, and Mg2+. The free bicyclic peptide conformation in solution has been studied by means of NMR spectroscopy and a plausible structure model worked out with model building on NMR constraints is proposed.

Combined use of spectroscopic and energy calculation methods for the determination of peptide conformation in solution.

This paper describes the combined use of energy calculations and spectroscopic data for the determination of peptide conformations in solution. The approach involves (i) experimental measurements of spectroscopic parameters for a molecule, (ii) calculation of these parameters for low-energy conformers previously determined with regard to local fluctuations in conformation and (iii) a random search for statistically weighted combinations of conformers which provide a good agreement between the calculated and experimental data. The above approach was used to study the conformation of a spin-labelled angiotensin molecule (SL-AT). It appears that the C-terminal hexapeptide of SL-AT possesses two geometrically different spatial forms of the backbone in aqueous solution, with mean values for the statistical weight of 0.78 and 0.22, respectively. In contrast, the N-terminal part of the molecule is conformationally labile. The data obtained can be used to describe the conformation of angiotensin in solution.

Conformation of peptides in solution. Circular dichroism studies of N-acetyl-L-alanine N-methylamide and N-acetyl-L-serine N-methylamide

Conformation of peptides in solution. Circular dichroism studies of N-acetyl-L-alanine N-methylamide and N-acetyl-L-serine N-methylamide

Temperature dependence of peptide NH chemical shifts in benzene: Delineation of solvent‐shielded and exposed amide protons

BiopolymersVolume 20, Issue 3 p. 625-628 Communications To The EditorFree Access Temperature dependence of peptide NH chemical shifts in benzene: Delineation of solvent-shielded and exposed amide protons Y. V. Venkatachalapathi, Y. V. Venkatachalapathi Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012, IndiaSearch for more papers by this authorP. Balaram, P. Balaram Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012, IndiaSearch for more papers by this author Y. V. Venkatachalapathi, Y. V. Venkatachalapathi Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012, IndiaSearch for more papers by this authorP. Balaram, P. Balaram Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012, IndiaSearch for more papers by this author First published: March 1981 https://doi.org/10.1002/bip.1981.360200313Citations: 15AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 Craig, L. C., Cowburn, D. & Bleich, H. (1975) Annu. Rev. Biochem. 44, 477– 490. 2 Stern, A., Gibbons, W. A. & Craig, L. C. (1968) Proc. Natl. Acad. Sci. USA 61, 734– 741. 3 Pitner, T. P. & Urry, D. W. (1972) J. Am. Chem. Soc. 94, 1399– 1400. 4 Kopple, K. D. & Schamper, T. J. (1972) J. Am. Chem. Soc. 94, 3644– 3646. 5 Pitner, T. P., Glickson, J. D., Dadok, J. & Marshall, G. R. (1974) Nature 250, 582– 584. 6 Kopple, K. D., Ohnishi, M. & Go, A. (1969) J. Am. Chem. Soc. 91, 4264– 4272. 7 Hruby, V. J. (1974) Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 3, B. Weinstein, Ed., Dekker, New York, pp. 1– 188. 8 Ohnishi, M. & Urry, D. W. (1969) Biochem. Biophys. Res. Commun. 36, 194– 202. 9 Pysh, E. S. & Toniolo, C. (1977) J. Am. Chem. Soc. 99, 6211– 6219. 10 Nagaraj, R., Shamala, N. & Balaram, P. (1979) J. Am. Chem. Soc. 101, 16– 20. 11 Klotz, I. M. & Franzen, J. S. (1962) J. Am. Chem. Soc. 84, 3461– 3466. 12 Rao, C. P., Nagaraj, R., Rao, C. N. R. & Balaram, P. (1980) Biochemistry 19, 425– 431. 13 Prasad, B. V. V., Shamala, N., Nagaraj, R., Chandrasekaran, R. & Balaram, P. (1979) Biopolymers 18, 1635– 1646. 14 Laszlo, P. (1967) Progress in Nuclear Magnetic Resonance Spectroscopy, Vol. 3, J. W. Emsley, J. Feeney & L. H. Sutcliffe, Eds., Pergamon, Oxford, pp. 231– 402. 15 Suzuki, I., Tsuboi, M., Shimanouchi, T. & Mizushima, S. (1959) J. Chem. Phys. 31, 1437– 1438. 16 Hatton, J. V. & Richards, R. E. (1962) Mol. Phys. 5, 139– 152. Citing Literature Volume20, Issue3March 1981Pages 625-628 ReferencesRelatedInformation

Solution Conformation of Peptides through 1H Nuclear Magnetic Double Resonance Measurement of Amide Hydrogen Exchange Rates: Study of the Active Pentapeptide Fragment of Thymopoietin

The amide hydrogen exchange rates of peptides (and other biomolecules) can be related to their secondary and tertiary structures. The exchange rates in the range ∼0.1 to ∼100 s−1 can be conveniently measured in H2O by a double resonance technique involving a combination of two separate 1H NMR experiments: (1) the transfer of solvent saturation and (2) the selective saturation recovery of the labile hydrogen. When a peptide exists in a dynamic equilibrium among several conformations, the interpretation of the above two experiments in terms of intrinsic exchange rates is not straightforward in general. The effect of a multistate conformational averaging is considered in the interpretation of the above two experiments, and analytical expressions have been presented for a few limiting cases of conformational and chemical equilibria. The amide hydrogen exchange rates of the pentapeptide Arg-Lys-Asp-Val-Tyr, the active fragment of the thymic hormone thymopoietin, have been measured in H2O through the transfer of solvent saturation and saturation recovery experiments. The results suggest that this fragment exists in a highly motile dynamic equilibrium among several conformations. In some of these, the Val4 NH hydrogen is shielded significantly from the solvent.

Solution conformation of peptides by the intramolecular nuclear Overhauser effect experiment. Study of valinomycin-K+

This study demonstrates how the intramolecular nuclear Overhauser effect (NOE) experiment can be employed quantitatively to select from a set of possible conformations for a peptide or a protein the particular conformation (or a group of conformations) most consistent with the data. This procedure is demonstrated on a model depsipeptide system--valinomycin K+ in CDCl3--for which the solution conformation has been inferred by other methods. The NOE enhancements are very sensitive to the conformations assumed by this antibiotic. It is shown that the set of conformations, collectively labeled as A2 (including the X-ray crystallographic structure) gives a very good description of the NOE enhancements. The structure proposed by Bystrov et al. (1977. Eur. J. Biochem. 78:63) for the uncomplexed valinomycin in nonpolar solvents is also consistent with the experimental data on the potassium complex. Using statistical hypothesis testing involving the Hamilton R-factor ratio criterion, all the other models have been rejected as inconsistent with the experimental data. A general formalism is presented for describing the NOE effects in isotropically reorienting molecules. The formalism is not restricted to the extreme narrowing limit of the rotational correlation times and hence applies to both small and large molecules. Some of the factors that can influence the NOE measurements, viz. anisotropic rotational diffusion, conformational averaging, and nuclear spin diffusion, have been considered in this study.