| dc.description.abstract | Disorder in proteins, or in extended protein regions, is more commonplace and functionally relevant than was conventionally assumed. Until a few decades ago, well-defined structure was considered as the only prerequisite for properly functional proteins. Since the development of disorder predictors, it has become apparent that intrinsically disordered proteins (IDPs) comprise significant proportions of eukaryotic genomes. An increasing number of investigations into the structure and function of IDPs reveal that they mediate a large number of crucial cellular processes and are associated with various disease processes. Despite the growing interest in IDPs, much remains unclear about the relationships between IDP structures, their interactions with other proteins and their mechanisms of action. Another important consideration is whether IDPs remain disordered in crowded conditions, such as the native cellular environments where IDPs function. Mass spectrometry (MS)-based approaches have been developed to probe IDP secondary structure and to complement other biophysical techniques in elucidating IDP functions. In addition, liquid-chromatography (LC) provides avenues for the removal of interference from complex protein samples containing high concentrations of polymeric crowding agents. Described herein, are studies based on hydrogen exchange (HX) measured by mass spectrometry (HX-MS) to: (1) propose a mechanism for calcineurin activation based on structural changes in its disordered regulatory domain, occurring upon binding calmodulin, (2) develop a method to probe the effects of polymer crowders on IDPs, and (3) investigate the effects of different crowder concentrations on transiently helical regions of random coil IDPs. Calcineurin is a heterodimeric phosphatase, whose activity is regulated by calcium, calmodulin, and a regulatory subunit. The mechanism of calcineurin activation is directly associated with binding of calmodulin to a disordered regulatory domain (RD) domain, located in the catalytic subunit. A calcineurin construct, comprising the RD, an autoinhibitory domain (AID), and a C terminal tail (referred to as the RD-AID-CT), was exposed to D2O to compare HX in its free and calmodulin-bound forms. Free RD-AID-CT was fully exchanged at every residue after 5 s of exposure to D2O. In the calmodulin-bound form HX was slowed in the calmodulin-binding domain and in an adjacent region. Slowed HX suggests that binding induces structure in the calmodulin-binding domain and an adjacent domain, in agreement with results from other biophysical techniques, such as CD and fluorescence anisotropy. The HX-MS data provided the spatial resolution required to localize calmodulin-induced structure to particular regions of RD-AID-CT. That localization provided enough information to propose a mechanism for calcineurin activation whereby the adoption of structure in the RD displaces the AID, exposing the active site cleft in the catalytic subunit. Following the discussion of calcineurin’s mechanism of activation, the development of a method for HX-MS analysis of CREB binding protein (CBP) in crowded buffers containing 300 g L−1 Ficoll, is outlined. CBP is a molten globular IDP consisting of three stable α-helices but a highly flexible tertiary structure. Ficoll is a synthetic polysaccharide used as an artificial crowder, but the high concentrations used to simulate cellular crowding present overwhelming interference to HX-MS analysis. To overcome this challenge, an LC method employing strong cation exchange (SCX) and reversed-phase extractions was developed to remove Ficoll before MS detection of CBP peptide segments. To validate the dual extraction approach, control experiments were performed using myoglobin in 300 g L−1 Ficoll samples, subjected to conventional HX-MS and SCX-based HX-MS workflows. Myoglobin peptide signals were lost in conventional HX-MS but were recovered after the SCX-based HX-MS workflows. Unstructured myoglobin peptides were used to ensure that Ficoll neither alters the amounts of deuterium in HX labeling buffers nor affects the rates of deuterium uptake. Afterwards, HX-MS measurements showed that some CBP regions are stabilized while α-helices are destabilized in the presence of Ficoll. The SCX-based HX-MS cleanup was applied to study crowding effects on a disordered domain of the activator of thyroid hormone and retinoid receptor (ACTR) in samples containing 300 g L−1 and 400 g L−1 of Ficoll. ACTR is a random coil IDP with transiently helical segments. Interestingly, all transiently helical regions of ACTR were destabilized in 300 g L−1 Ficoll, but one transiently helical region was stabilized in 400 g L−1 Ficoll. These results suggest that ACTR engages in non-specific interaction with Ficoll, disrupting intramolecular hydrogen bonds, in lower Ficoll concentrations. In contrast, ACTR is compacted by steric repulsions which can stabilize intramolecular hydrogen bonds in higher Ficoll concentrations. This information gives insights into the possible alterations of IDP structures, which could affect their functions, in different cellular compartments. | |
