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Defining the role of intrinsic disorder in proteins in the myriad of biological processes with which it is involved represents a significant goal in modern biophysics. Toward this end, NMR is uniquely suited for molecular studies of dynamic and disordered regions, but studying these regions in concert with their more structured domains and binding partners presents spectroscopic challenges. Here, we investigate the interactions between the structured and disordered regions of the human glucocorticoid receptor (GR). To do this, we developed an NMR strategy that relies on a novel relaxation filter for the simultaneous study of structured and unstructured regions. Using this approach, we conducted a comparative analysis of three translational isoforms of GR containing a folded DNA-binding domain (DBD) and two disordered regions that flank the DBD, one of which varies in size in the different isoforms. Notably, we were able to assign resonances that had previously been inaccessible because of the spectral complexity of the translational isoforms, which in turn allowed us to 1) identify a region of the structured DBD that undergoes significant changes in the local chemical environment in the presence of the disordered region and 2) determine differences in the conformational ensembles of the disordered regions of the translational isoforms. Furthermore, an ensemble-based thermodynamic analysis of the isoforms reveals conserved patterns of stability within the N-terminal domain of GR that persist despite low sequence conservation. These studies provide an avenue for further investigations of the mechanistic underpinnings of the functional relevance of the translational isoforms of GR while also providing a general NMR strategy for studying systems containing both structured and disordered regions., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Ionizable residues buried in hydrophobic environments in proteins are essential for many fundamental biochemical processes. These residues titrate with anomalous pK a values that are challenging to reproduce with structure-based calculations owing to the conformational reorganization coupled to their ionization. Detailed characterization of this conformational reorganization is of interest; unfortunately, the properties of buried Lys residues are difficult to study experimentally. Here we demonstrate the utility of 15 N NMR spectroscopy to gain insight into the protonation state, state of hydration and conformational dynamics of the Nζ amino group of buried Lys residues. The experiments were applied to five variants of staphylococcal nuclease, with internal Lys residues that titrate with pK a values ranging from 6.2 to 8.1. Direct detection of buried Lys residues with these NMR spectroscopy methods will enable correlation between thermodynamic and structural data as well as unprecedented examination of how conformational transitions coupled to their ionization affect their pK a values.