A new amide proton R1rho experiment permits accurate characterization of microsecond time-scale conformational exchange

• Published in: Journal of biomolecular NMR Vol. 32; no. 4; pp. 281 - 293
• Main Authors: Eichmüller, Christian, Skrynnikov, Nikolai R
• Format: Journal Article
• Language: English
• Published: Netherlands 01.08.2005
• Subjects: Amides - chemistry; Carbon Isotopes; Deuterium; Myocardium - chemistry; Nitrogen Isotopes; Nuclear Magnetic Resonance, Biomolecular; Protein Conformation; Protein Structure, Tertiary; Protons; Troponin C - chemistry
• ISSN: 0925-2738

A new off-resonance spin-lock experiment to record relaxation dispersion profiles of amide protons is presented. The sensitivity-enhanced HSQC-type sequence is designed to minimize the interference from cross-relaxation effects and ensure that the dispersion profiles in the absence of micros-ms time-scale dynamics are flat. Toward this end (i) the proton background is eliminated by sample deuteration (Ishima et al., 1998), (ii) 1H spin lock is applied to two-spin modes 2(H(x)Sin theta + H(z)Cos theta) N(z), and (iii) the tilt angle theta approximately 35 degrees is maintained throughout the series of measurements (Desvaux et al. Mol. Phys., 86 (1995) 1059). The relaxation dispersion profiles recorded in this manner sample a wide range of effective rf field strengths (up to and in excess of 20 kHz) which makes them particularly suitable for studies of motions on the time scale < or = 100 micros. The new experiment has been tested on the Ca2+-loaded regulatory domain of cardiac troponin C. Many residues show pronounced dispersions with remarkably similar correlation times of approximately 30 micros. Furthermore, these residues are localized in the regions that have been previously implicated in conformational changes (Spyracopoulos et al. Biochemistry, 36 (1997) 12138).