Solid‐State NMR Measurements of Asymmetric Dipolar Couplings Provide Insight into Protein Side‐Chain Motion

Abstract

Understanding conformational flexibility is of critical importance for understanding protein function, folding, and interactions with other proteins and ligands. NMR spectroscopy is an important tool for such investigations in solution [1] and increasingly also in the solid state [2] since it allows siteresolved studies of dynamic processes. An experimental characterization of all motional modes of a protein is a great challenge and simplified models are necessary. In NMR studies of dynamics, motional amplitudes are generally expressed in terms of a single order parameter, [3] discarding the details of the motion, such as the motional asymmetry. Herein, we show a significant extension of this description, by detecting asymmetric motion of side chains in a protein in the solid state.Dipolar couplings are particularly powerful probes of local molecular dynamics in the solid state. In the absence of motion, the tensor describing the dipolar interaction between two nuclei is a traceless axially symmetric second-rank tensor. It can be characterized by a single parameter, namely its anisotropy d D,rigid which depends only on the internuclear distance and isotope type of the nuclei involved (for the definition see the Supporting Information). In the presence of "fast" motional processes, in other words, processes with a correlation time shorter than approximately 1/d D,rigid , (typically 10-100 ms), the dipolar coupling tensor becomes partially averaged. In the case of a motional process with threefold (C 3 ) or higher symmetry, for example, an isotropic motion within a cone, the averaged tensor remains axially symmetric and is fully characterized by the effective anisotropy d D which has a reduced value compared to d D,rigid . In this case, the motional amplitude can be expressed by a single order parameter [4] However, in the case of a general fast motion, the characterization solely by S is incomplete because the averaged dipolar tensor is no longer axially symmetric [5] and one additional tensor parameter, the asymmetry h D , is needed for a complete description (for the definition, see the Supporting Information). The asymmetry h D varies between zero (symmetric tensor) and one.[5a]In solution-state NMR spectroscopy, dipolar couplings can be measured as residual couplings (RDCs) in anisotropic media. The evaluation of motional amplitudes from RDCs is challenging because RDCs also depend on the (a priori unknown) degree of molecular alignment and the orientation of a given vector relative to the alignment frame and usually data from different alignment media must be combined.[6] The situation is much simplified in solid-state magic-angle-spinning (MAS) NMR, where overall molecular tumbling is absent, allowing the direct measurement of dipolar couplings that depend only on the interatomic distance and dynamics. For the case of one-bond dipolar couplings (CÀH, NÀH, or CÀN) the rigid-limit dipolar coupling tensor is known from the bond lengths. Thus, measurements of the dipolar coupling tensor provide direct access t...

Studying dynamics by magic-angle spinning solid-state NMR spectroscopy: Principles and applications to biomolecules

“…One notable exception is the REDOR experiment that has a built-in normalization, which eliminates the need to use ad hoc empirical fit parameters. In a recent application, REDOR has been applied to characterize non-symmetric side-chain motions [154].…”

“…Is such a fit possible -in other words, is the solution one obtains unique and unambiguous, or is it ill-defined? This question has been addressed in detail and by different means by a few recent studies [110,154,178,180,182], and we aim to summarise the main conclusions in Figure 32g and h. To this end, we assume a certain motional model, i.e. we assume an N-H site that undergoes motion described by an order parameter (S 2 = 0.85) and a time scale of either 100 ps (panel g) or 50 ns (panel h).…”

Dynamics in the solid-state: perspectives for the investigation of amyloid aggregates, membrane proteins and soluble protein complexes

“…Obviously, the long-term goal should be to obtain model dependent information on dynamics. The quantification of the asymmetry parameter g of 2 H tensors (Hologne et al 2005(Hologne et al , 2006 or of dipolar coupling (Schanda et al 2011) allows to get information on motional models, such as jump angle and population of side chain rotameric states. Additional information might come from selectively side chain protonated samples in an otherwise deuterated matrix (Asami et al 2010;Asami and Reif 2013).…”

Biological Solid-state NMR at ETH Zurich

“…[1][2][3][4][5][6][7][8] Globular proteins can often be characterized by X-ray crystallography and liquid-state NMR spectroscopy, but solid-state NMR adds additional und unique possibilities, e.g. in the context of dynamical investigations, [9,10] or in the structural characterization of sedimented instead of crystallized proteins. [11,12] Protein misfolding and subsequent aggregation is at the origin of over 20 human diseases termed 'conformational' diseases including Alzheimer's, Parkinson's and the prion diseases.…”