Sensitivity-Enhanced NMR Reveals Alterations in Protein Structure by Cellular Milieus

Abstract

Biological processes occur in complex environments containing a myriad of potential interactors. Unfortunately, limitations on the sensitivity of biophysical techniques normally restrict structural investigations to purified systems, at concentrations that are orders of magnitude above endogenous levels. Dynamic nuclear polarization (DNP) can dramatically enhance the sensitivity of NMR spectroscopy and enable structural studies in biologically complex environments. Here we applied DNP NMR to investigate the structure of a protein containing both an environmentally sensitive folding pathway and an instrinsically disordered region, the yeast prion protein Sup35. We added an exogenously-prepared isotopically-labeled protein to deuterated lysates, rendering the biological environment “invisible” and enabling highly efficient polarization transfer for DNP. In this environment, structural changes occurred in a region known to influence biological activity but intrinsically disordered in purified samples. Thus, DNP makes structural studies of proteins at endogenous levels in biological contexts possible and such contexts can influence protein structure.

Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

“…Short-range TEDOR spectra of these fibers revealed that whereas the majority of alanine sites had chemical shifts consistent with β-sheet or random-coil secondary structure (Fig. 4A), a quarter of the alanine sites had chemical shifts consistent with α-helical conformations, in line with previous observations of the M domain upon cryoprotection (47). This establishes that alanine takes on a variety of secondary structures in NM fibrils, consistent with its representation across the primary sequence of NM.…”

Insights into protein misfolding and aggregation enabled by solid-state NMR spectroscopy

“…Dynamic nuclear polarization (DNP) methods are under continuing development as a means to achieve dramatic increases in sensitivity. A number of published studies have demonstrated the applicability to protein aggregate studies in particular [128–132]. One interesting application is to study smaller amounts of labeled protein aggregates under dilute conditions, for instance in cellular extracts [132].…”

“…A number of published studies have demonstrated the applicability to protein aggregate studies in particular [128–132]. One interesting application is to study smaller amounts of labeled protein aggregates under dilute conditions, for instance in cellular extracts [132]. Current DNP methods are typically reliant on cryogenic temperatures, which enhance the performance of dipolar-recoupling experiments, but also limit the ability to measure highly valuable dynamics data and often result in reductions in spectral resolution [82, 133].…”

Structured States of Disordered Proteins from Genomic Sequences

“…However, except for a tiny percentage of cases (~ 1%), we do not know whether apparently disordered proteins can take on ordered states in vivo (Frederick et al, 2015) and the conditions for functional and structural experiments are unlikely to be known a priori for the vast majority. In the extreme, so-called fuzzy complexes have functional disordered regions that have been confirmed even when in their bound states (Tompa and Fuxreiter, 2008).…”