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Nitric oxide, carbon monoxide and hydrogen sulfide are three endogenous gasotransmitters that serve a role in regulating normal and pathological cellular activities. They can stimulate or inhibit cancer cell proliferation and invasion, as well as interfere with cancer cell responses to drug treatments. Understanding the molecular pathways governing the interactions between these gases and the tumor microenvironment can be utilized for the identification of a novel technique to disrupt cancer cell interactions and may contribute to the conception of effective and safe cancer therapy strategies. The present review discusses the effects of these gases in modulating the action of chemotherapies, as well as prospective pharmacological and therapeutic interfering approaches. A deeper knowledge of the mechanisms that underpin the cellular and pharmacological effects, as well as interactions, of each of the three gases could pave the way for therapeutic treatments and translational research.

The spreadsheet file reported herein provides centroid data, descriptive of deuterium uptake, for the FabFragment of NISTmAb (PDB: 5K8A) reference material, as measured by the bottom-up hydrogen-deuterium exchange mass spectrometry (HDX-MS) method. The protein sample was incubated in deuterium-rich solutions under uniform pH and salt concentrations between 3.6 oC and 25.4 oC for seven intervals ranging over (0 to 14,400) s plus a ∞pseudo s control. The deuterium content of peptic peptide fragments were measured by mass spectrometry. These data were reported by fifteen laboratories, which conducted the measurements using orbitrap and Q-TOF mass spectrometers. The cohort reported ≈ 78,900 centroids for 430 proteolytic peptide sequences of the heavy and light chains of NISTmAb, providing nearly 100 % coverage. In addition, some groups reported ≈ 10,900 centroid measurements for 77 peptide sequences of the Fc fragment. The instrumentation and physical and chemical conditions under which these data were acquired are documented.

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.