Conformational Analysis of Intrinsically Disordered Proteins Using Mass Spectrometry-Based Approaches

Abstract

Intrinsically disordered proteins (IDPs) have regions that are highly flexible and lack stable secondary or tertiary structure. Recently, there has been a growing interest in IDPs due to their important roles in many biological processes and functions. Although many studies have shown that IDPs participate in functional interactions, less is known about the structural details of the interactions. Over the past two decades, mass spectrometry (MS) has become a powerful technique for biophysical characterization of proteins due to its high sensitivity and variety of choices for sample preparation and instrumentation. Here I present the application of two important mass spectrometry-based approaches: hydrogen exchange (HX) and fast photochemical oxidation of proteins (FPOP) to study disordered proteins. HX-MS was applied to better understand the mechanism of calcineurin activation. Calcineurin is a heterodimeric phosphatase that plays essential roles in cellular processes. Previous work has established that at high calcium concentration, calmodulin binds calcium ions, resulting in calmodulin binding to the intrinsically disordered regulatory domain of calcineurin. Calmodulin binding causes release of the autoinhibitory domain from the active site, activating calcineurin. My results with full-length calcineurin demonstrate that the regulatory domain is unstructured in the absence of calmodulin, while it folds upon binding to calmodulin. This result confirms previous work on the isolated regulatory-autoinhibitory domain construct. Additionally, I have observed calmodulin-induced changes in peptides located in other domains of calcineurin. Finally, and surprisingly, I found no changes in the structuring of the calcineurin autoinhibitory domain upon calmodulin binding. I present results from all regions of the calcineurin to describe the mechanism of calcineurin activation. I also propose a new model of calcineurin activation upon calmodulin binding. The degree of structure measurement in IDPs can provide important information about the mechanisms by which IDPs undergo coupled folding and binding. Different approaches to quantify the degree of structure in IDPs using millisecond HX were explored. A quench-flow device, built in-house, for HX labeling on the millisecond timescale was employed. It is essentially impossible to determine the degree of structure without having an accurate unprotected reference state. The interaction domains of the activator for thyroid and retinoid receptors (ACTR) and the CREB binding protein (CBP) were used as model IDPs to explore the best approach to produce an unprotected reference state for millisecond HX. ACTR is a near-random coil IDP that has some residual helicity while CBP is a molten globular IDP that transiently becomes unstructured as revealed by NMR and HX-MS. The approaches explored to obtain an unprotected reference state in HX experiments were chemical exchange calculations, addition of denaturing agents, and millisecond HX labeling of peptic peptides obtained from the IDP. It was found that peptic reference peptides can be used as an accurate unprotected reference state for determining the degree of the structure. Due to its fast labeling timeframe, FPOP might reveal states of IDPs that are undetectable by HX. The application of the FPOP technique for characterizing IDPs was also evaluated. To explore the applicability of this technique for studying IDPs, ACTR and CBP as model systems that co-fold upon binding were used. The FPOP technique was utilized to study ACTR and CBP in their free and bound forms. The data show that FPOP provided useful information to compare two states of IDPs. The usefulness and limitations of FPOP analysis to characterize and localize regions of protein-protein interactions involving IDPs are illustrated.