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, R 2 and R 1 ρ, 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, R ex, 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 R 1 ρ experiment further reduces the overall relaxation rate as compared with the usual R 1 ρ experiment because the protons relax as unlike spins, with rate R 1 ρ ,unlike, in the former experiment but as like spins, with rate R 1 ρ, in the latter. These reductions of the proton transverse-relaxation rate facilitated the detection of R ex contributions at several sites in the protein (1) from the B 1-field dependence of R 1 ρ ,unlike and (2) by comparing R 1 ρ ,unlike values with relaxation rates, R 2, 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 R 2 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.

Intramolecular domain–domain association/dissociation and phosphoryl transfer in the mannitol transporter of Escherichia coli are not coupled

“…R 1 -unlike spin relaxation rates in the rotating frame for backbone amide protons were measured on 2 H, 15 N-labeled phospho-IIBA Mt1 by using the pulse sequence described in ref. 20 with minor modifications. A 3-9-19 pulse train was used for water suppression in the WATERGATE scheme, and heat compensation was used during the relaxation delay (36).…”

“…where In the fast-exchange limit, (⌬ /k ex ) 2 Ͻ Ͻ 1, on the chemical-shift time scale, the R ex contribution to R 1 is given by (10,19,20) …”

“…The titration spectra obtained with the isolated IIA Mtl and IIB Mtl domains were used to estimate the lower limit of k ex and the range of ⌬ . Because the values of ⌬ were larger in the 1 H than 15 N dimension and stronger spin-lock field strengths can be applied for 1 H than 15 N to suppress the exchange contribution to R 1 , we made use of backbone amide proton ( 1 H N ) relaxation dispersion measurements in the rotating frame (20) by using perdeuterated phosphoIIBA Mtl . Among the titrating residues of IIBA Mtl , 10 residues with both well resolved cross-peaks in the 2D 1 H-15 N correlation spectrum and large 1 H N chemical-shift differences (⌬ H /2 Ͼ 60 Hz at a 1 H frequency of 750 MHz) relative to the free protein were selected for further analysis.…”

pH-triggered, activated-state conformations of the influenza hemagglutinin fusion peptide revealed by NMR

“…For this reason, attempts to measure the exchange rates by the commonly used 15 N relaxation dispersion experiment proved unsuccessful. However, carrying out relaxation dispersion measurements on the amide protons, where much stronger spin-lock fields are available (36), and cooling the sample temperature to 12°C, instead of the 32°C sample temperature used in all other experiments, allowed probing of the exchange process for the wild-type peptide (Fig. 7).…”

Correlated dynamics of consecutive residues reveal transient and cooperative unfolding of secondary structure in proteins

“…In principle, NMR spectroscopy offers the possibility to monitor conformational exchange processes at every position along the protein backbone and side chains, provided that suitable stable isotopes (e.g., 13 C and 15 N) have been incorporated (4,(11)(12)(13)(14)(15). Importantly, the chemical shifts of different nuclei are sensitive to different types of intramolecular motions, which provides a major motivation for pursuing multinuclear studies of conformational exchange (14)(15)(16)(17)(18)(19)(20)(21). Recent methods development has made possible measurement of differential multiple-quantum (MQ) relaxation rates that report on correlated dynamics of different spin interactions (22)(23)(24).…”