Influence of Dynamics on The Analysis of Solid-State NMR Data From Membrane-bound Peptides

Abstract

By isotope labeling of membrane-bound peptides, typically with 2H, 19F, or 15N, solid-state NMR experiments can yield data from which the orientation of peptides in a native membrane environment can be determined. Such an orientation is defined by a tilt angle and an azimuthal rotation angle.Here we show that to obtain correct values of the orientation angles, it is important to include dynamics in the analysis of the NMR data. Nevertheless the effects of dynamics are different depending on the type of isotope labeling and NMR experiment considered.To analyze the influence of dynamics in detail, we generated virtual NMR observables using a model peptide undergoing explicit Gaussian fluctuations of the orientation angles. For simulated 2H- or 19F-NMR data, even moderate motions were found to have a large influence, as calculated tilt values are consistently much too small, unless dynamics is properly considered. A simple dynamic model, including a molecular order parameter scaling factor, gives good results only for moderately mobile peptides, while for high mobility cases the correct tilt is only obtained by re-introducing the explicit Gaussian fluctuations in the fitting functions.In contrast, 15N-NMR data appear to be less sensitive to rigid-body peptide motions, and PISEMA spectra can give correct orientations even for highly mobile peptides, and assuming a static model for the analysis. The differences are due to the different orientation of the tensors of 2H- and 19F-labels, placed on peptide side chains, compared to the orientation of the 15N tensor, placed on amide backbone groups.We conclude that dynamics should be included in the analysis of solid-state NMR data of membrane-bound peptides. Not only does this give more accurate orientations, but it can also provide information about the dynamics of the peptide.