Rapid automated determination of chemical shift anisotropy values in the carbonyl and carboxyl groups of fd-y21m bacteriophage using solid state NMR

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Rapid automated determination of chemical shift anisotropy values in the carbonyl and carboxyl groups of fd-y21m bacteriophage using solid state NMR

Abstract

Determination of chemical shift anisotropy (CSA) in immobilized proteins and protein assemblies is one of several tools to determine protein dynamics on the timescales of microseconds and faster. The large CSA values of C=O groups in the rigid limit makes them in particular attractive for measurements of large amplitude motions, or their absence. In this study, we implement a 3D R-symmetry-based sequence that recouples the second spatial component of the 13C CSA with the corresponding isotropic 13Câ?²â??13C cross-peaks in order to probe backbone and sidechain dynamics in an intact fd-y21m filamentous phage viral capsid. The assignment of the isotropic cross-peaks and the analysis were conducted automatically using a new software named â??Ravenâ??. The software can be utilized to auto-assign any 2D 13Câ??13C or 15Nâ??13C spectrum given a previously-determined assignment table and generates simultaneously all intensity curves acquired in the third dimension. Here, all CSA spectra were automatically generated, and subsequently matched against a simulated set of CSA curves to yield their values. For the multi-copy, 50-residue-long protein capsid of fd-y21m, the backbone of the helical region is rigid, with reduced CSA values of ~â??12.5Â*kHz (~â??83Â*ppm). The N-terminus shows motionally-averaged CSA lineshapes and the carboxylic sidechain groups of four residues indicate large amplitude motions for D4, D5, D12 and E20. The current results further strengthen our previous studies of 15N CSA values and are in agreement with qualitative analysis of 13Câ??13C dipolar build-up curves, which were automatically obtained using our software. Our automated analysis technique is general and can be applied to study protein structure and dynamics, with data resulting from experiments that probe different variables such as relaxation rates and scaled anisotropic interactions.

Graphical abstract





Source: Journal of Biomolecular NMR