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-   -   Pulse EPR-enabled interpretation of scarce pseudocontact shifts induced by lanthanide binding tags (http://www.bionmr.com/forum/journal-club-9/pulse-epr-enabled-interpretation-scarce-pseudocontact-shifts-induced-lanthanide-binding-tags-22902/)

nmrlearner 11-23-2015 06:58 PM

Pulse EPR-enabled interpretation of scarce pseudocontact shifts induced by lanthanide binding tags
 
Pulse EPR-enabled interpretation of scarce pseudocontact shifts induced by lanthanide binding tags

Abstract

Pseudocontact shifts (PCS) induced by tags loaded with paramagnetic lanthanide ions provide powerful long-range structure information, provided the location of the metal ion relative to the target protein is known. Usually, the metal position is determined by fitting the magnetic susceptibility anisotropy (Î?Ï?) tensor to the 3D structure of the protein in an 8-parameter fit, which requires a large set of PCSs to be reliable. In an alternative approach, we used multiple Gd3+-Gd3+ distances measured by double electronâ??electron resonance (DEER) experiments to define the metal position, allowing Î?Ï?-tensor determinations from more robust 5-parameter fits that can be performed with a relatively sparse set of PCSs. Using this approach with the 32Â*kDa E. coli aspartate/glutamate binding protein (DEBP), we demonstrate a structural transition between substrate-bound and substrate-free DEBP, supported by PCSs generated by C3-Tm3+ and C3-Tb3+ tags attached to a genetically encoded p-azidophenylalanine residue. The significance of small PCSs was magnified by considering the difference between the chemical shifts measured with Tb3+ and Tm3+ rather than involving a diamagnetic reference. The integrative sparse data approach developed in this work makes poorly soluble proteins of limited stability amenable to structural studies in solution, without having to rely on cysteine mutations for tag attachment.



Source: Journal of Biomolecular NMR


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