Probing mutation-induced structural perturbations by refinement against residual dipolar couplings: application to the U4 spliceosomal RNP complex.

Confident interpretation of biochemical experiments performed with mutated proteins relies on verification of the integrity of the mutant structures. We present a simple and rapid refinement protocol for comparing the structures of mutated and wild-type proteins. Our approach involves measurement of residual dipolar couplings, and only requires assignment of the backbone resonances of the mutant species. We demonstrate application of the protocol to a mutant of the 15.5K protein, a core component of the U4 spliceosomal ribonucleoprotein (RNP) complex. Confirmation of the unperturbed structure of the mutated protein prompted re-examination of a previous mutagenesis study and indicated that the interpretation of mutant binding affinities in terms of direct interfacial contacts should be applied with caution.