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Narrowing of protein NMR spectral lines broadened by chemical exchange.

ABSTRACT: Broadening of spectral lines is a signature of chemical exchange phenomena on microsecond to millisecond time scales but has deleterious effects on spectral resolution and sensitivity. A multipulse method based on chemical shift scaling that reduces chemical exchange broadening during frequency-encoding periods of liquid-state multidimensional NMR experiments is described. The proposed scheme utilizes low-power radiofrequency pulses, which offer the advantages of short cycle times and minimal sample heating. The method is suitable for biological macromolecules, as relaxation not resulting from chemical exchange is reduced by placing the magnetization along the z axis for part of the evolution trajectory. The resolution and sensitivity enhancement for resonances broadened by chemical exchange is demonstrated on the protein ribonuclease A. The work demonstrates the feasibility of applying coherent averaging techniques, which were originally developed in solid-state NMR spectroscopy, to biological NMR spectroscopy in the liquid state for resolution enhancement and facilitates the detection of resonances that are severely broadened by chemical exchange processes.

SUBMITTER: Li Y

PROVIDER: S-EPMC2921271 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Narrowing of protein NMR spectral lines broadened by chemical exchange.

Li Ying Y Palmer Arthur G AG

Journal of the American Chemical Society 20100701 26

Broadening of spectral lines is a signature of chemical exchange phenomena on microsecond to millisecond time scales but has deleterious effects on spectral resolution and sensitivity. A multipulse method based on chemical shift scaling that reduces chemical exchange broadening during frequency-encoding periods of liquid-state multidimensional NMR experiments is described. The proposed scheme utilizes low-power radiofrequency pulses, which offer the advantages of short cycle times and minimal sa ...[more]

PMID: 20550111

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Project description:BackgroundNuclear Magnetic Resonance (NMR) spectroscopy offers a variety of experiments to study protein-ligand interactions at atomic resolution. Among these experiments, 15N Heteronuclear Single Quantum Correlation (HSQC)experiment is simple, less time consuming and highly informative in mapping the binding site of the ligand. The interpretation of 15N HSQC becomes ambiguous when the chemical shift perturbations are caused by non-specific interactions like allosteric changes and local structural rearrangement. Under such cases, detailed chemical exchange analysis based on chemical shift perturbation will assist in locating the binding site accurately.Methodology/principal findingsWe have automated the mapping of binding sites for fast chemical exchange systems using information obtained from 15N HSQC spectra of protein serially titrated with ligand of increasing concentrations. The automated program Auto-FACE (Auto-FAst Chemical Exchange analyzer) determines the parameters, e.g. rate of change of perturbation, binding equilibrium constant and magnitude of chemical shift perturbation to map the binding site residues.Interestingly, the rate of change of perturbation at lower ligand concentration is highly sensitive in differentiating the binding site residues from the non-binding site residues. To validate this program, the interaction between the protein hBcl(XL) and the ligand BH3I-1 was studied. Residues in the hydrophobic BH3 binding groove of hBcl(XL) were easily identified to be crucial for interaction with BH3I-1 from other residues that also exhibited perturbation. The geometrically averaged equilibrium constant (3.0 x 10(4)) calculated for the residues present at the identified binding site is consistent with the values obtained by other techniques like isothermal calorimetry and fluorescence polarization assays (12.8 x 10(4)). Adjacent to the primary site, an additional binding site was identified which had an affinity of 3.8 times weaker than the former one. Further NMR based model fitting for individual residues suggest single site model for residues present at these binding sites and two site model for residues present between these sites. This implies that chemical shift perturbation can represent the local binding event much more accurately than the global binding event.Conclusion/significanceDetail NMR chemical shift perturbation analysis enabled binding site residues to be distinguished from non-binding site residues for accurate mapping of interaction site in complex fast exchange system between small molecule and protein. The methodology is automated and implemented in a program called "Auto-FACE", which also allowed quantitative information of each interaction site and elucidation of binding mechanism.

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Narrowing of protein NMR spectral lines broadened by chemical exchange.

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Narrowing of protein NMR spectral lines broadened by chemical exchange.

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