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|Titolo:||BACKBONE-ONLY PROTEIN SOLUTION STRUCTURES WITH A COMBINATION OF CLASSICAL AND PARAMAGNETISM-BASED CONSTRAINTS: A METHOD THAT CAN BE SCALED TO LARGE MOLECULES|
|Autori interni:||BARBIERI, RENATO|
|Data di pubblicazione:||2004|
|Abstract:||Herein, it is shown that a medium-resolution solution structure of a protein can be obtained with the sole assignment of the protein backbone and backbone-related constraints if a derivative with a firmly bound paramagnetic metal isavailab le. The proofof- concept isprovided on calbindin D9k, a calcium binding protein in which one of the two calcium ions can be selectively substituted by a paramagnetic lanthanide ion. The constraints used are HN (and Ha) nuclear Overhauser effects (NOEs), hydrogen bonds, dihedral angle constraints from chemical shifts, and the following paramagnetism-based constraints: 1) pseudocontact shifts, acquired by substituting one (or more) lanthanide(s) in the C-terminal calcium binding site; 2) NHN residual dipolar couplingsdue to self-orientation induced by the paramagnetic lanthanide( s); 3) cross-correlations between the Curie and internuclear dipole±dipole interactions; and 4) paramagnetism-induced relaxation rate enhancements. An upper distance limit for internuclear distances between any two backbone atoms was also given according to the molecular weight of the protein. For this purpose, the paramagnetism-based constraints were collectively implemented in the program CYANA for solution structure determinations, similarly to what was previously done for the program DYANA. The method is intrinsically suitable for large molecular weight proteins.|
|Appare nelle tipologie:||1a - Articolo su rivista|
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