Insights into Protein Dynamics from ¹⁵N-¹H HSQC

Abstract. Protein dynamic information is customarily extracted from ¹⁵N NMR spin-relaxation experiments. These experiments can only be applied to (small) proteins that can be dissolved to high concentrations. However, most proteins of interest to the biochemical and biomedical community are large and relatively insoluble. These proteins often have functional conformational changes, and it is particularly regretful that these processes cannot be supplemented by dynamical information from NMR. We ask here whether (some) dynamic information can be obtained from the ¹H line widths in ¹⁵N-¹H HSQC spectra. Such spectra are widely available, also for larger proteins. We developed computer programs to predict amide proton line widths from (crystal) structures. We aim to answer the following basic questions: is the ¹H linewidth of a HSQC cross peak smaller than average because its ¹H nucleus has few dipolar neighbors, or because the resonance is motionally narrowed? Is a broad line broad because of conformational exchange, or because the ¹H nucleus resides in a dense proton environment? We calibrate our programs by comparing computational and experimental results for GB1 (58 residues). We deduce that GB1 has low average ¹HN order parameters (0.8), in broad agreement with what was found by others from ¹⁵N relaxation experiments (Idiyatullin et al., 2003). We apply the program to the BPTI crystal structure and compare the results with a ¹⁵N-¹H HSQC spectrum of BPTI (56 residues) and identify a cluster of conformationally broadened ¹HN resonances that belong to an area, for which millisecond dynamics has been previously reported from ¹⁵N relaxation data (Szyperski et al., J. Biomol. NMR 3, 151-164, 1993). We feel that our computational approach is useful to glean insights into the dynamical properties of larger biomolecules for which high-quality ¹⁵N relaxation data cannot be recorded.