The Exact Nuclear Overhauser Enhancement: Recent Advances

Abstract

Although often depicted as rigid structures, proteins are highly dynamic systems, whose motions are essential to their functions. Despite this, it is difficult to investigate protein dynamics due to the rapid timescale at which they sample their conformational space, leading most NMR-determined structures to represent only an averaged snapshot of the dynamic picture. While NMR relaxation measurements can help to determine local dynamics, it is difficult to detect translational or concerted motion, and only recently have significant advances been made to make it possible to acquire a more holistic representation of the dynamics and structural landscapes of proteins. Here, we briefly revisit our most recent progress in the theory and use of exact nuclear Overhauser enhancements (eNOEs) for the calculation of structural ensembles that describe their conformational space. New developments are primarily targeted at increasing the number and improving the quality of extracted eNOE distance restraints, such that the multi-state structure calculation can be applied to proteins of higher molecular weights. We then review the implications of the exact NOE to the protein dynamics and function of cyclophilin A and the WW domain of Pin1, and finally discuss our current research and future directions.

Efficient Stereospecific Hβ2/3 NMR Assignment Strategy for Mid-Size Proteins

“…When 3 J Hα–Hβ couplings were added into the calculation, the iterative protocol yielded 66 stereospecific assignments. Next, we converted Pin1 conventional NOEs into eNOEs using our recently developed buildup series method to see if more precise distance restraints would impact the yield of stereospecific assignments [30]. Indeed, eNOEs were extremely helpful in defining rotameric states and bond angles, even without the 3 J Hα–Hβ couplings.…”

Extending the Applicability of Exact Nuclear Overhauser Enhancements to Large Proteins and RNA

“…For dense eNOE networks (an exceptional case is shown in Figure ), multistate structures can be calculated in CYANA that allow visualization of spatial sampling. This is due to the averaged nature of eNOEs, and consequently, an atom that samples multiple points in space will produce eNOE distances to nearby atoms that are the r −6 averages of all the distances sampled. If a sufficient amount of eNOEs define such an atom, then the combined network contains information about all of the sampled substates.…”

“…In addition, we showed that eNOEs contain similar and complementary information to both J couplings and RDCs . So far, we have only used eNOEs to calculate multistate ensembles of proteins up to about 150 resides . Currently, we are testing new directions for the applications of eNOE methodology.…”

NOE‐Derived Methyl Distances from a 360 kDa Proteasome Complex

“…[5] The distances are derived from exact measurements of the nuclear Overhauser enhancement (eNOE). [9][10][11] In our initial applications of the method, we used uniformly 13 C, 15 N-labeled samples to extract distances between all protons pairs. [12] However, diagonal peak overlap becomes an increasingly limiting factor for proteins larger than ca.…”

“…It consists of the homodimeric homoheptamer α rings referred to as α 7 α 7 . The distances are derived from exact measurements of the nuclear Overhauser enhancement (eNOE) . In our initial applications of the method, we used uniformly 13 C, 15 N‐labeled samples to extract distances between all protons pairs .…”