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FP5

HSFFC NMR Résumé de rapport

Project ID: HPRI-CT-2001-50028
Financé au titre de: FP5-HUMAN POTENTIAL
Pays: Italy

Protocol for analysis of protein proton relaxation profiles

Software tools have been developed for the analysis of the collective relaxation of protein proton spins in order to develop a model for the interpretation of the magnetization decays. The model is used for a quantitative analysis of the relaxation profiles. Such analysis can provide direct information on the lack of rigidity, through the order parameter, which is directly related to foldedness down to the extreme situation of a totally unfolded protein, and on protein aggregation, through a safe estimate of the re-orientational time of the protein.

Many dynamic properties of proteins can thus be accessed through the analysis of the so-called spectral density of their magnetic nuclei, now directly accessible by relaxometric measurements. With respect to water nuclei dispersions, the analysis is not complicated by the unknown number of long-lived water molecules and exchangeable protein protons, or by the presence of water protons that exchange faster than the re-orientational time. The methods were tested on data acquired for the proteins lysozyme (at different pH), human serum albumin and a-synuclein.

We found that this protocol can provide information on protein dynamics and protein aggregation, as it reveals an excellent sensitivity to oligomerization equilibria. Furthermore, it allows a straightforward definition of an effective order parameter, which does not coincide with, but rather complements, that derived from high resolution NMR relaxation analysis. A fast method for the estimation of the order parameter has been described, based on one CORMA calculation at low magnetic field, besides the fit of the experimental data. The method has been used for evaluating the fraction of protein protons that undergo local motions with time scales shorter than the rotational correlation times for well-folded proteins.

Using this method, on the other hands, unfolded proteins can be readily identified by a very low order parameter value. Finally, the fact that no field-independent high field plateau is theoretically expected in the dispersion profiles permits the straightforward assignment of any residual relaxation plateau at high field to the presence of local motions and, in principle, even their quantification.

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Claudio LUCHINAT, (Full Professor)
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