Domain-domain motions in proteins from time-modulated pseudocontact shifts.

In recent years paramagnetic NMR derived structural constraints have become increasingly popular for the study of biomolecules. Some of these are based on the distance and angular dependences of pseudo contact shifts (PCSs). When modulated by internal motions PCSs also become sensitive reporters on molecular dynamics. We present here an investigation of the domain-domain motion in a two domain protein (PA0128) through time-modulation of PCSs. PA0128 is a protein of unknown function from Pseudomonas aeruginosa (PA) and contains a Zn(2+) binding site in the N-terminal domain. When substituted with Co(2+) in the binding site, several resonances from the C-terminal domain showed severe line broadening along the (15)N dimension. Relaxation compensated CPMG experiments revealed that the dramatic increase in the (15)N linewidth came from contributions of chemical exchange. Since several sites with perturbed relaxation are localized to a single beta-strand region, and since extracted timescales of motion for the perturbed sites are identical, and since the magnitude of the chemical exchange contributions is consistent with PCSs, the observed rate enhancements are interpreted as the result of concerted domain motion on the timescale of a few milliseconds. Given the predictability of PCS differences and the easy interpretation of the experimental results, we suggest that these effects might be useful in the study of molecular processes occurring on the millisecond to microsecond timescale.