Strategies for acquisition of resonance assignment spectra of highly dynamic membrane proteins: a GPCR case study

[nmrlearner]

Strategies for acquisition of resonance assignment spectra of highly dynamic membrane proteins: a GPCR case study

Abstract

In protein nuclear magnetic resonance (NMR), chemical shift assignment provides a wealth of information. However, acquisition of high-quality solid-state NMR spectra depends on protein-specific dynamics. For membrane proteins, bilayer heterogeneity further complicates this observation. Since the efficiency of cross-polarization transfer is strongly entwined with protein dynamics, optimal temperatures for spectral sensitivity and resolution will depend not only on inherent protein dynamics, but temperature-dependent phase properties of the bilayer environment. We acquired 1-, 2-, and 3D homo- and heteronuclear experiments of the chemokine receptor CCR3 in a 7:3 phosphatidylcholine:cholesterol lipid environment. 1D direct polarization, cross polarization (CP), and T2â?? experiments indicate sample temperatures belowâ??â??â??25Â*°C facilitate higher CP enhancement and longer-lived transverse relaxation times. T1rho experiments indicate intermediate timescales are minimized below a sample temperature ofâ??â??â??20Â*°C. 2D DCP NCA experiments indicated optimal CP efficiency and resolution at a sample temperature ofâ??â??â??30Â*°C, corroborated by linewidth analysis in 3D NCACX atâ??â??â??30Â*°C compared toâ??â??â??5Â*°C. This optimal temperature is concluded to be directly related the lipid phase transition, measured to be betweenâ??â??â??20 and 15Â*°C based on rINEPT signal of all-trans and trans-gauche lipid acyl conformations. Our results have critical implications in acquisition of SSNMR membrane protein assignment spectra, as we hypothesize that different lipid compositions with different phase transition properties influence protein dynamics and therefore the optimal acquisition temperature.



Source: Journal of Biomolecular NMR