Using Amide ¹H and ¹⁵N Transverse Relaxation To Detect Millisecond Time-Scale Motions in Perdeuterated Proteins: Application to HIV-1 Protease

Abstract

Measurements of proton transverse relaxation rates, R2 and R1ρ, have not been commonly performed for proteins because cross correlations among the numerous 1H−1H dipolar interactions complicate analysis of the data. In addition, these interactions make large contributions to the relaxation of the amide protons, making it difficult to detect if an exchange of chemical shifts also makes a contribution, Rex, to relaxation. To overcome these problems, we have investigated proton relaxation of a perdeuterated protein, HIV-1 protease, bound to a small protonated inhibitor DMP323. Perdeuteration significantly reduces the contributions of 1H−1H dipolar interactions to the relaxation of the amide protons. The ROESY R1ρ experiment further reduces the overall relaxation rate as compared with the usual R1ρ experiment because the protons relax as unlike spins, with rate R1ρ,unlike, in the former experiment but as like spins, with rate R1ρ, in the latter. These reductions of the proton transverse-relaxation rate facilitated the detection of Rex contributions at several sites in the protein (1) from the B1-field dependence of R1ρ,unlike and (2) by comparing R1ρ,unlike values with relaxation rates, R2, obtained from Carr−Purcell−Meiboom−Gill (CPMG) and Hahn-echo experiments. The significant reduction of the proton spin-flip rate in the perdeuterated protein enabled measurement of 15N R2 values using the CPMG method and the same large duration between 180° pulses as used in the 1H CPMG experiments. Hence, relaxation data of both nuclei were utilized to obtain complementary information about sites experiencing exchange of chemical shifts in the protein.