Untitled

Abstract

Current methods of determining the rotational diffusion tensors of proteins in solution by NMR spectroscopy exclusively utilize relaxation rate constants for backbone amide 15N spins. However, the distributions of orientations of N-H bond vectors are not isotropic in many proteins, and correlations between bond vector orientations reduce the accuracy and precision of rotational diffusion tensors extracted from 15N spin relaxation data. The inclusion of both 13C alpha and 15N spin relaxation rate constants increases the robustness of the diffusion tensor analysis because the orientations of the C alpha-H alpha bond vectors differ from the orientations of the N-H bond vectors. Theoretical and experimental results for calbindin D9k, granulocyte colony stimulating factor, and ubiquitin, three proteins with different distributions of N-H and C alpha-H alpha bond vectors, are used to illustrate the advantages of the simultaneous utilization of 13C alpha and 15N relaxation data.

A further investigation of the cytochrome b 5–cytochrome c complex

“…The existence of different interaction regions on cytochrome b 5 has already been proposed [15,57]. The above findings are also in agreement with the diffusional properties of the molecules in the adduct, obtained from 15 N relaxation data using well-documented approaches [38,39,58]. The diffusion tensor obtained for cytochrome c in the complex is in agreement with binding by cytochrome b 5 occurring at only one site, while the tensor for cytochrome b 5 suggests that binding by the partner occurs at more than one site.…”

“…Intensities at 10 different delays were acquired for both R 1 and R 2 measurements. The diffusion anisotropy of the molecules in solution was calculated from the local s m values derived from the R 2 /R 1 ratio of each individual residue [38], using the program quadric_diffusion [39]. Inertia moments were calculated with the program pdb_inertia [39].…”

“…The diffusion anisotropy of the molecules in solution was calculated from the local s m values derived from the R 2 /R 1 ratio of each individual residue [38], using the program quadric_diffusion [39]. Inertia moments were calculated with the program pdb_inertia [39]. CLEANEX experiments [40] were performed in order to monitor the accessibility of amide moieties to the solvent in the two separate proteins and in equimolar mixtures of them.…”

A copper(I) protein possibly involved in the assembly of Cu A center of bacterial cytochrome c oxidase

“…15 N R 1 , R 2 , and steady-state heteronuclear NOEs were measured with pulse sequences as described by Farrow et al (22), with the water signal suppressed with ''water flipback'' scheme (23). All spectra were processed with the NMRPIPE program (24) and analyzed with SPARKY 3 (26,27).…”

A redox switch in CopC: An intriguing copper trafficking protein that binds copper(I) and copper(II) at different sites

“…R 2 were also measured with longer refocusing times ( CPMG ) to determine the occurrence of exchange processes (27,28). The overall rotational correlation time m value and the rotational diffusion tensor were estimated from the R 2 ͞R 1 ratio with the program QUADRIC DIFFUSION (29,30), by using the 3D structure of Cu(I)-CopC.…”