SOLID_NMR_DYNAMICS

Development of solid state NMR methods at 100 kHz magic angle spinning frequency for the study of internal protein dynamics and the application to membrane proteins

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 Obiettivo del progetto (Objective)

'This proposal concerns the development of new solid-state NMR methods using proton detection under Magic Angle Spinning (MAS) with frequencies up to 100 kHz. The 100 kHz MAS probe head is a prototype and will allow significant gains in sensitivity by scaling down the 1H-1H dipolar coupling interaction and resulting in narrow proton line width down to 10-20 Hz. Small quantities of protein samples in the range of 0.3 to 0.5 mg allow a signal-to noise ratio that exceeds that of tens of mg in a bigger rotor at lower MAS frequency. A focus of this proposal is the development and optimization of methods to reliably determine backbone dynamics in the solid state under 100 kHz MAS condition, based on 15N R1 and R1ρ relaxation rates as well as transverse 15N CSA/ NH dipolar cross-correlated relaxation rates and NH dipolar couplings that are sensitive up to the microsecond time scale. The experimental analysis will be supported by spin dynamics simulations. 15N T2 relaxation measurements remain a challenging task, as significant differences between bulk T2 measurements using a spin-echo sequence and T1ρ measurements show. We want to compare those measurements at 100 kHz MAS and rationalize systematic differences by spin dynamics simulations. Second, bulk T2 decay times will be estimated for different in- and anti-phase coherences, present in higher-dimensional backbone spectral assignment experiments. Scalar coupling-based INEPT and CP-based polarization transfers will be compared in terms of efficiency. Aim is to minimize loss of magnetization during transfer steps with unfavorable T2 decay properties or insufficient dipolar transfer efficiency and to design an optimized set of experiments for backbone assignment at fast MAS. Methods will be developed on the small protein ubiquitin, a well-studied model system, with the aim of transferring it to a membrane protein, e.g. the HIV viral protein envelope protein gp41.'