Paramagnetic relaxation enhancement‐assisted structural characterization of a partially disordered conformation of ubiquitin

Abstract

Nuclear magnetic resonance (NMR) is a powerful tool to study three‐dimensional structures as well as protein conformational fluctuations in solution, but it is compromised by increases in peak widths and missing signals. We previously reported that ubiquitin has two folded conformations, N1 and N2 and plus another folded conformation, I, in which some amide group signals of residues 33–41 almost disappeared above 3 kbar at pH 4.5 and 273 K. Thus, well‐converged structural models could not be obtained for this region owing to the absence of distance restraints. Here, we reexamine the problem using the ubiquitin Q41N variant as a model for this locally disordered conformation, I. We demonstrate that the variant shows pressure‐induced loss of backbone amide group signals at residues 28, 33, 36, and 39–41 like the wild‐type, with a similar but smaller effect on CαH and CβH signals. In order to characterize this I structure, we measured paramagnetic relaxation enhancement (PRE) under high pressure to obtain distance restraints, and calculated the structure assisted by Bayesian inference. We conclude that the more disordered I conformation observed at pH 4.0, 278 K, and 2.5 kbar largely retained the N2 conformation, although the amide groups at residues 33–41 have more heterogeneous conformations and more contact with water, which differ from the N1 and N2 states. The PRE‐assisted strategy has the potential to improve structural characterization of proteins that lack NMR signals, especially for relatively more open and hydrated protein conformations.

Dynamic aspects of pressure and temperature‐stabilized intermediates of outer surface protein A

“…Information regarding localized conformational dynamics and thermodynamic stability might be useful for the rational design of effective OspA vaccines. Moreover, since PRE observation under high‐pressure is only limited in ubiquitin, 27 the present is a confirmation of PRE under high‐pressure. A combination of PRE‐assisted high‐pressure NMR and MD simulation is, in general, useful for characterizing the dynamic structures of functionally essential protein intermediates.…”

“…Although previous studies have reported that the C‐terminal half of the protein (β9‐C‐terminus) exhibits lower stability than the N‐terminal half, our understanding of how the central β‐sheet and C‐terminal domain are disordered remains limited. In this study, we further characterized the pressure‐ and temperature‐stabilized intermediates of OspA using a paramagnetic relaxation enhancement (PRE)‐assisted NMR 23‐27 . The PRE effect arises from dipole‐dipole interactions between unpaired electrons of the paramagnetic agent and the nucleus of interest, and thus, spin relaxation contributions show r −6 dependence of distance between the paramagnetic center and nucleus.…”

Nonthermal excitation effects mediated by sub-terahertz radiation on hydrogen exchange in ubiquitin

“…Error (Δ R ) was defined using the sum of each Error at two time points t and 3 min as follows: where each Error ( R t ) was estimated from the signal/noise ratio (SNR) considering error propagation according to ( 53 ): …”

Dynamic aspects of pressure and temperature-stabilized intermediates of outer surface protein A

“…Moreover, since PRE observation under high pressure is only limited in ubiquitin, the present is a confirmation of PRE under high pressure. 24 A combination of PRE-assisted high-pressure NMR and MD simulation is, in general, useful for characterizing the dynamic structures of functionally essential protein intermediates.…”

“…Here, we further characterized the pressure-and temperature-stabilized intermediates of OspA using a paramagnetic relaxation enhancement (PRE)-assisted NMR. [20][21][22][23][24] The PRE effect arises from dipole-dipole interactions between unpaired electrons of the paramagnetic agent and the nucleus of interest, and thus, spin relaxation contributions show r -6 dependence of distance between the paramagnetic center and nucleus. Accordingly, when the paramagnetic agent was conjugated to a portion of the central domain, we were able to collect information on the distance between the central β-sheet and the N-and C-terminal domains.…”