NMR-spectra obtained at cryogenic temperatures below 150 K usually suffer from severe inhomogeneous line broadening, as flexible parts of molecules may be trapped in different conformations with different chemical shifts. While broad lines result in limited resolution and thus are considered an unwanted side-effect of low temperatures, they encode valuable information about conformational distributions of flexible molecules. In this contribution we combine dedicated isotope labeling techniques with DNP-enhanced MAS-NMR-spectroscopy of proteins in frozen solution to shed light onto different conformational ensembles sampled by protein backbone as well as by amino acid side chains.<br/>We exploit DNP-enhanced MAS NMR spectroscopy at low temperatures (~100K) to investigate conformational ensembles of intrinsically disordered proteins IDPs. Such proteins are not represented by a single well-defined structure but rather by a full conformational ensemble of structures which can interconvert rapidly. Traditional biophysical methods like solution NMR spectroscopy and FRET measurements usually determine ensemble averages and do not give direct insight into the conformational distributions. In frozen solution the full conformational ensemble is trapped and can be examined simultaneously, for example by (DNP-enhanced) solid-state NMR-spectroscopy. First, we studied the distribution of backbone conformations in the intrinsically disordered protein α-synuclein in frozen solution in different surroundings by evaluating the inhomogeneously broadened line-shapes of the Cα/Cβ cross peak. We could estimate the amount of disordered regions in fibrillar α-syn and delineate the membrane binding regions of α-syn in contact with membrane surfaces in in different protein to lipid ratios. We also found that secondary chemical shifts of neighboring amino acids tend to be correlated, a finding which suggests the formation of transient secondary structure elements.<br/>We also investigated the distribution of rotameric states sampled by amino acid side chains in well-folded as well as in intrinsically disordered proteins and model peptides. This distribution drastically depends on the local and global structure of the protein and exceeds the conformational space documented in the pdb database. Our experimental results match well the results from a combined DFT/solution NMR-study.

Shedding light on the disorder: Sensitive look into protein folding with hyperpolarized solid-state NMR

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.