Qualitative and quantitative characterization of protein backbone heterogeneity by solid-state NMR spectroscopy

Abstract

Flexibility of the polypeptide chains plays the key role in protein functions, their un-, re- and misfolding pathways. Understanding the conformational landscape which protein chain occupies statically and dynamically is essential for understanding of cellular processes and ultimately, designing efficient drugs, safe pesticides, and industrial biotechnological processes. NMR spectroscopy is an indispensable technique studying disordered molecules site-specifically, both in solution and in the solid state. Protein disorder covers the continuum between the static set of defined states and dynamic ensembles. Dynamic disorder can be converted into static disorder by freeze-trapping and studied in the solid phase. In solid-state NMR, static disorder manifests itself as the presence of additional peaks or, in the general case, severe line broadening. Converting the distribution of the resonance frequencies into conformational ensembles is not a trivial task due to the multitude of factors that contribute to the nuclear resonance frequencies. This works proposes approaches to analyze residue-specific static disorder by interpretation and quantification of heterogeneously broadened peaks in multidimentsional solid-state NMR spectra. The engineered routines reconstruct the distributions of the backbone dihedral angles φ and ψ on the basis of database analyses and by help of dihedral-angle predictors. The workflows are tested on a model sample as well as on a naturally heterogeneous functional amyloid (EAS∆15 rodlet sample), where the obtained heterogeneity scores are compared to those formed by peak shape parameters (widths, intensities, and tilt). The analysis of the EAS ∆15 sample demonstrates the intrinsic power and weaknesses of the proposed analysis for rather challenging systems where the only available high-resolution physico-chemical data are the peak shapes in the solid-state NMR spectra.