Assignment of Pseudo-contact-shifted 1H NMR resonances in the EF site of Yb3 +-substituted rabbit parvalbumin through a combination of 2D techniques and magnetic susceptibility tensor determination
✍ Scribed by Francesco Capozzi; Mauro Andrea Cremonini; Claudio Luchinat; Marco Sola
- Publisher
- John Wiley and Sons
- Year
- 1993
- Tongue
- English
- Weight
- 1021 KB
- Volume
- 31
- Category
- Article
- ISSN
- 0749-1581
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✦ Synopsis
Abstract
Pseudo‐contact‐shifted ^1^H NMR resonances of rabbit parvalbumin substituted with Yb^3+^ at the Ca^2+^ EF site have been sequence‐specific assigned by a combination of 2D techniques and magnetic susceptibility tensor determination. By taking the X‐ray structure of the homologous carp protein as a starting point, the sequence‐specific assignment proceeded in a stepwise fashion. The first resonances assigned on the basis of their unique connectivity pattern were used to make a first coarse determination of the X‐tensor parameters; the latter were used to guess the identity of further hyperfine‐shifted resonances, which were then checked through 2D connectivities and in turn allowed us to improve the reliability of the X‐tensor parameters. This cyclic procedure was repeated until virtually all pseudo‐contact‐shifted resonances were assigned. In the last steps the ^X^‐tensor parameters did not change appreciably. The orientation of the tensor was found to be very similar to that previously proposed for the homologous Yb^3+^−substituted protein from carp. Modest discrepancies between some calculated and experimental shift values in the present system were found to be due mainly to structural differences between the present protein and the homologous protein whose x‐ray structure was used as model and, to a lesser extent, to indetermination in the diamagnetic shift values. The success of the analysis and the univocal solution for the X‐tensor parameters under these conditions show that the above procedure is robust enough to permit both signal assignment and X‐tensor determination even when the structural data are only a rough approximation of the actual structure. These findings may open the way to the development of a systematic use of pseudo‐contact shifts as constraints for MD refinement of solution structures of paramagnetic metalloproteins.