Mark Rance

Improved spectral resolution in COSY 1H NMR spectra of proteins via double quantum filtering

A double quantum filter is inserted into a two-dimensional correlated (COSY) 1H NMR experiment to obtain phase-sensitive spectra in which both cross peak and diagonal peak multiplets have anti-phase fine structure, and in which the cross peaks and the major contribution to the diagonal peaks have absorption lineshapes in both dimensions. The elimination of the dispersive character of the diagonal peaks in phase-sensitive, double quantum-filtered COSY spectra allows identification of cross peaks lying immediately adjacent to the diagonal, which represents a significant improvement over the conventional COSY experiment.

Sensitivity improvement in proton-detected two-dimensional heteronuclear correlation NMR spectroscopy

Abstract The sensitivity of proton-detected two-dimensional heteronuclear correlation NMR spectroscopy can be increased by as much as a factor of √2 relative to that of the conventional methods, for heteronuclei with a single attached proton. The enhanced sensitivity is obtained by refocusing and detecting two orthogonal in-phase proton magnetization components, rather than the single component recorded conventionally. The two magnetization components are deconvoluted to produce two pure-phase spectra that are added together to produce a spectrum with an enhanced signal-to-noise ratio. Methods for improving the sensitivity of the main classes of heteronuclear correlation experiments are presented and the effect of relaxation during the new experiments on the sensitivity enhancement is discussed. The new methods are demonstrated by comparing conventional and sensitivity-enhanced 1H-15N heteronuclear correlation spectra of bovine pancreatic trypsin inhibitor at 15N natural abundance.

Folding of immunogenic peptide fragments of proteins in water solution: I. Sequence requirements for the formation of a reverse turn

A systematic examination by 1H nuclear magnetic resonance of the population of beta-turn-containing conformers in several series of short linear peptides in water solution has demonstrated a dependence on amino acid sequence which has important implications for initiation of protein folding. The peptides consist of a number of variants of the sequence Tyr-Pro-Tyr-Asp, the trans isomer of which was previously shown to contain a reverse turn in water. Two-dimensional rotating-frame nuclear Overhauser effect spectroscopy provides unequivocal evidence that substantial populations of reverse turn conformations occur in water solutions of certain of these peptides. In the unfolded state, the peptides adopt predominantly extended chain (beta) conformations in water. It appears probable from the nuclear Overhauser effect connectivities observed that the reverse turns in the trans isomers are predominantly type II. The low temperature coefficient of the amide proton resonance of the residue at position 4 of the turn suggests the presence of an intramolecular hydrogen bond. The presence of the beta-turn conformation has been confirmed for certain peptides by circular dichroism measurements. Substitutions at positions 3 and 4 in the sequence Tyr-Pro-Tyr-Asp-Val can enhance or abolish the beta-turn population in the trans peptide isomers. The residue at position 3 of the turn is the primary determinant of its stability. A small amount of additional stabilization appears to result from an electrostatic interaction between the side-chain of residue 4 and the unblocked amino terminus. For peptides of the series Tyr-Pro-X-Asp-Val, where X represents all L-amino acid except Trp and Pro, the temperature coefficient of the Asp4 amide proton resonance provides a measure of the beta-turn population. The beta-turn populations in water solution measured in this way correlate with the beta-turn probabilities determined from protein crystal structures. This indicates that it is frequently the local amino acid sequence, rather than medium- to long-range interactions in the folded protein, that determines the beta-turn conformation in the folded state. Such sequences are excellent candidates for protein folding initiation sites. A high population of structured forms appears to be present in the cis isomer of certain of the peptides, as shown by a considerable increase in the proportion of the cis isomer and by measurement of nuclear Overhauser effects and 3JN alpha coupling constants.(ABSTRACT TRUNCATED AT 400 WORDS)

Obtaining high-fidelity spin-12 powder spectra in anisotropic media: Phase-cycled Hahn echo spectroscopy☆

In the spectroscopy of isolated spin-1 nuclei in anisotropic media, the spectral distortion resulting from the inability to record the initial part of the free-induction decay was eliminated by using the quadrupolar echo technique. Wideline spin-12 spectroscopy is subject to the same problems associated with finite receiver recovery time as is spin-1 spectroscopy; however, these problems have been generally ignored in the past. Through the use of a very simple and well-known pulse sequence (90°-τ-180°) the chemical shift interaction, among others, can be refocused to generate a so-called Hahn echo. If magnetic dipolar interactions with like nuclei and fluctuating interactions with other nuclei may be neglected, then the refocusing of the magnetization will be complete and an undistorted spin-12 powder spectrum can be obtained by Fourier transforming the signal starting at the peak of the echo. The practical aspects of implementing the Hahn echo technique are discussed. A phase-cycling scheme is proposed to prevent spectral distortion due to misset pulse lengths. The effects of finite pulse lengths are investigated, and the theoretical single pulse and echo distortion factors are demonstrated to differ from those which were determined for quadrupolar echo spectroscopy. Experimental verification of some of the theoretical predictions is provided.

Rotational diffusion anisotropy of proteins from simultaneous analysis of 15N and 13C alpha nuclear spin relaxation.

Current methods of determining the rotational diffusion tensors of proteins in solution byNMR spectroscopy exclusively utilize relaxation rate constants for backbone amide 15N spins.However, the distributions of orientations of N-H bond vectors are not isotropic in manyproteins, and correlations between bond vector orientations reduce the accuracy and precisionof rotational diffusion tensors extracted from 15N spin relaxation data. The inclusion of both13Cα and 15N spin relaxation rate constants increases the robustness of the diffusiontensor analysis because the orientations of the Cα-Hα bond vectors differ from theorientations of the N-H bond vectors. Theoretical and experimental results for calbindin D9k,granulocyte colony stimulating factor, and ubiquitin, three proteins with different distributionsof N-H and Cα-Hα bond vectors, are used to illustrate the advantages of thesimultaneous utilization of 13Cα and 15N relaxation data.

Suppression of the effects of cross-correlation between dipolar and anisotropic chemical shift relaxation mechanisms in the measurement of spin-spin relaxation rates

Cross correlation between dipolar and anisotropic chemical shift relaxation mechanisms complicates measurements of heteronuclear spin-spin relaxation rate constants by the Carr-Purcell-Meiboom-Gill (CPMG) technique if the magnitudes of the chemical shift anisotropy and the dipolar interaction are comparable. Experimental schemes are described that attenuate the effects of cross correlation and permit accurate measurements of spin-spin relaxation rate constants for heteronuclei with significant chemical shift anisotropies. The theoretical analysis is confirmed by measurements of 15N and 13C spin-spin relaxation in a peptide. Application of 180° pulses to the protons directly attached to the heteronuclei in synchrony with the even echoes of the heteronuclear spins yields more accurate results than continuous irradiation of the protons with a composite pulse decoupling sequence or application of a single 180° pulse to the protons in the middle of the CPMG pulse train.

Sensitivity improvement in isotropic mixing (TOCSY) experiments

Abstract A simple modification of the two-dimensional TOCSY experiment which leads to a √2 improvement in sensitivity is described. This improvement is achieved by retaining the magnetization transfer pathways from both of the orthogonal, in-phase magnetization components which are present during the isotropic mixing period. In addition, a simple baseline correction procedure which can greatly improve the quality of NMR spectra is described.

A systematic approach to the suppression of J cross peaks in 2D exchange and 2D NOE spectroscopy

Abstract An optimization procedure is described for NMR techniques which require signal averaging of experiments with a variable time delay, either for the suppression of artifacts or for the optimization of coherence transfer over a given frequency range. The procedure is applied to 2D exchange spectroscopy, where artifacts due to zero-quantum coherence evolving in the mixing period (J cross peaks) can be canceled by coaddition of signals from experiments with a refocusing pulse inserted at suitably chosen points in the mixing interval. It is shown that experiments must be averaged over a sizeable number of τi values to obtain satisfactory cancellation. Furthermore, suppression schemes may fail in systems with more than two spins if two or more of the chemical shifts are nearly degenerate.

Ligand and Receptor Dynamics Contribute to the Mechanism of Graded PPARγ Agonism

Ligand binding to proteins is not a static process, but rather involves a number of complex dynamic transitions. A flexible ligand can change conformation upon binding its target. The conformation and dynamics of a protein can change to facilitate ligand binding. The conformation of the ligand, however, is generally presumed to have one primary binding mode, shifting the protein conformational ensemble from one state to another. We report solution nuclear magnetic resonance (NMR) studies that reveal peroxisome proliferator-activated receptor γ (PPARγ) modulators can sample multiple binding modes manifesting in multiple receptor conformations in slow conformational exchange. Our NMR, hydrogen/deuterium exchange and docking studies reveal that ligand-induced receptor stabilization and binding mode occupancy correlate with the graded agonist response of the ligand. Our results suggest that ligand and receptor dynamics affect the graded transcriptional output of PPARγ modulators.

NMR ANALYSIS OF CARDIAC TROPONIN C-TROPONIN I COMPLEXES: EFFECTS OF PHOSPHORYLATION

Phosphorylation of the cardiac specific amino‐terminus of troponin I has been demonstrated to reduce the Ca2+ affinity of the cardiac troponin C regulatory site. Recombinant N‐terminal cardiac troponin I proteins, cardiac troponin I(33–80), cardiac troponin I(1–80), cardiac troponin I(1–80)DD and cardiac troponin I(1–80)pp, phosphorylated by protein kinase A, were used to form stable binary complexes with recombinant cardiac troponin C. Cardiac troponin I(1–80)DD, having phosphorylated Ser residues mutated to Asp, provided a stable mimetic of the phosphorylated state. In all complexes, the N‐terminal domain of cardiac troponin I primarily makes contact with the C‐terminal domain of cardiac troponin C. The non‐phosphorylated cardiac specific amino‐terminus, cardiac troponin I(1–80), was found to make additional interactions with the N‐terminal domain of cardiac troponin C.