Dynamics of Ribonuclease H:  Temperature Dependence of Motions on Multiple Time Scales

Abstract

The temperature dependence of the backbone motions in Escherichia coli ribonuclease HI was studied on multiple time scales by 15N nuclear magnetic spin relaxation. Laboratory frame relaxation data at 285, 300, and 310 K were analyzed using the model-free and reduced spectral density approaches. The temperature dependence of the order parameters was used to define a characteristic temperature for the motions of the backbone N-H bond vectors on picosecond to nanosecond time scales. The characteristic temperatures for secondary structure elements, loops, and the C-terminus are approximately 1000, approximately 300, and approximately 170 K, respectively. The observed variation in the characteristic temperature indicates that the energy landscape, and thus the configurational heat capacity, is markedly structure dependent in the folded protein. The effective correlation times for internal motions do not show significant temperature dependence. Conformational exchange was observed for a large number of residues forming a contiguous region of the protein that includes the coiled coil formed by helices alpha A and alpha D. Exchange broadening in the CPMG experiments decreased with increased temperature, directly demonstrating that the microscopic exchange rate is faster than the pulse repetition rate of 1.2 ms. The temperature dependence of the exchange contributions to the transverse relaxation rate constant shows approximately Arrhenius behavior over the studied temperature range with apparent activation enthalpies of approximately 20-50 kJ/mol. Numerical calculations suggest that these values underestimate the activation barriers by at most a factor of 2. The present results obtained at 300 K are compared to those reported previously [Mandel, A. M., Akke, M., & Palmer, A. G., III (1995) J. Mol. Biol. 246, 144-163] to establish the reproducibility of the experimental techniques.

Solution NMR of Large Molecules and Assemblies

“…This can be highly effective for thermostable macromolecules, with the caveat that behavior at physiological temperatures should be † Authors supported by grants from the National Science Foundation (MCB-0092962) and National Institutes of Health (GM067807) *Contact information: foster.281@osu.edu, 614-292-1377, FAX: 614-292-6773. NIH Public Access [3][4][5][6]. Another ingenious approach to reduce tumbling rates involves encapsulating hydrated proteins in low-viscosity solvents (7); while promising, this approach…”

“…obtained by extrapolation (3)(4)(5)(6). Another ingenious approach to reduce tumbling rates involves encapsulating hydrated proteins in low-viscosity solvents (7); while promising, this approach has not yet met with widespread use, as the encapsulation process is technically challenging and system dependent.…”

Protein conformational flexibility prediction using machine learning

“…Experimental generalized order parameters have been reported for B3 domain of staphyloccocal protein G (GB3) [34], the villin headpiece domain (HP67) [35], the holo frenolicin acyl carrier protein (fren ACP) [36], and Escherichia coli ribonuclease H (RNaseH) [37][38][39][40]. Generalized order parameters were predicted using the structural coordinates from PDB files 1IGD, 1QQV, 1OR5, and 2RN2, respectively.…”

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

“…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) …”