Kinetics of Fast Tetramerization of the Huntingtin Exon 1 Protein Probed by Concentration-Dependent On-Resonance R1ρ Measurements

An approach for the quantitative description of the kinetics of very fast exchange processes (tau(ex) < 50-100 mu s) associated with transient, reversible protein oligomerization, is presented. We show that on-resonance N-15-R-1 rho. measurements conducted as a function of protein concentration at several spin-lock radio frequency field strengths are indispensable for unambiguous determination of the rate constants for interconversion between monomeric and higher order oligomeric species. The approach is experimentally demonstrated on the study of fast, reversible tetramerization of the full-length Huntingtin exon 1 protein, htt(ex1), responsible for Huntington's disease. Incorporation of concentration-dependent N-15-R-2,R-eff data, obtained from on-resonance R-1 rho measurements performed at three spin-lock field strengths, into analysis of the kinetic scheme describing reversible tetramerization of htt(ex1) allowed us to uniquely determine the rate constants of interconversion between the various species. This approach serves as a valuable complement to the existing array of NMR techniques for studying early, transient oligomerization events in protein aggregation pathways.