Conformational distributions of unfolded polypeptides from novel NMR techniques

Abstract

How the information content of an unfolded polypeptide sequence directs a protein towards a well-formed three-dimensional structure during protein folding remains one of the fundamental questions in structural biology. Unfolded proteins have recently attracted further interest due to their surprising prevalence in the cellular milieu, where they fulfill not only central regulatory functions, but also are implicated in diseases involving protein aggregation. The understanding of both the protein folding transition and these often natively unfolded proteins hinges on a more detailed experimental characterization of the conformations and conformational transitions in the unfolded state. This description is intrinsically very difficult due to the very large size of the conformational space. In principle, solution NMR can monitor unfolded polypeptide conformations and their transitions at atomic resolution. However, traditional NMR parameters such as chemical shifts, J couplings, and nuclear Overhauser enhancements yield only rather limited and often qualitative descriptions. This situation has changed in recent years by the introduction of residual dipolar couplings and paramagnetic relaxation enhancements, which yield a high number of well-defined, quantitative parameters reporting on the averages of local conformations and long-range interactions even under strongly denaturing conditions. This information has been used to obtain plausible all-atom models of the unfolded state at increasing accuracy. Currently, the best working model is the coil model, which derives amino acid specific local conformations from the distribution of amino acid torsion angles in the nonsecondary structure conformations of the protein data bank. Deviations from the predictions of such models can often be interpreted as increased order resulting from long-range contacts within the unfolded ensemble.

Comprehensive structural and dynamical view of an unfolded protein from the combination of single-molecule FRET, NMR, and SAXS

“…The timescales derived from correlation spectroscopy agree with the observation that (with the exception of the first β-hairpin) the NMR spectra of unfolded ubiquitin are mostly in fast chemical exchange (39,71), that is, the interconversion between all conformations is considerably faster than the microsecond timescale of chemical shifts. The NMR observables (i.e., chemical shifts, scalar couplings, RDCs, PREs, and other relaxation rates) are thus averages over all conformations (8).…”

“…Distance and angular information can be obtained from nuclear Overhauser enhancements (14), three-bond scalar couplings (15), paramagnetic relaxation enhancements (PREs) (16), pseudo contact shifts (17), residual dipolar couplings (RDCs) (8,18), chemical shifts (19), and hydrogen bond scalar couplings (20). Thus, on the order of 10 geometric parameters per amino acid can be obtained with relative ease on unfolded polypeptides (21).…”

“…Due to the very large number of conformational degrees of freedom of the unfolded polypeptide ensemble, it is of utmost importance to obtain as many independent experimental parameters as possible for a quantitative description of its behavior. Three experimental methods have been particularly informative in this respect: NMR spectroscopy (2,5,(7)(8)(9), small angle X-ray scattering (SAXS) (10,11), and single-molecule Förster resonance energy transfer spectroscopy (single-molecule FRET) (12,13).…”

Introducing Protein Intrinsic Disorder

“…population and location) and hence provide important insights into the structures populated in the conformational ensemble in IDPs. 51,55,56 One potential problem associated with this kind of approach concerns incorrect frequency referencing, which can result in systematic errors in the secondary shifts. In order to address this problem, the Cα and Cβ chemical shifts (that shift in opposing directions for α-helical segments) can be used simultaneously to estimate the level of secondary structure in disordered proteins.…”

“…61,63 As compared to chemical shifts and NOEs, RDCs present a powerful tool that provides a far more extensive and quantitative description of local, and possibly, global order in the unfolded state. 55 Indeed, RDCs measured between pairs of nuclei in partially aligned proteins, are very sensitive probes of time and ensemble-averaged conformational equilibria exchanging on timescales up to the millisecond and can therefore be used to characterize both the structure and the dynamics of unfolded proteins. 59 An example taken from ref.…”