Modupeola A. Sowole

Mass Spectrometry Methods for Studying Structure and Dynamics of Biological Macromolecules

■ CONTENTS Hydrogen/Deuterium Exchange 214 Fundamentals 214 Proteolytic Digestion-LC/MS 215 Characterization of Binding Interactions 216 HDX/MS of Intrinsically Disordered Proteins 216 Membrane Protein HDX/MS 217 Pulsed HDX/MS 217 Cytotoxic Protein Aggregates Studied by HDX/ MS 218 Application of HDX/MS to Protein Therapeutics 218 Single Amide Resolution 218 HDX/MS with Electron-Based Fragmentation 218 Covalent Labeling 220 General Considerations 220 Hydroxyl Radical Labeling 220 Covalent Cross-Linking 222 ESI Charge State Distributions 222 ESI Mechanism for Folded Proteins 222 CID of Multiprotein Complexes 223 ESI Mechanism for Unfolded Proteins 223 “Supercharging” and Related Phenomena 224 Native Mass Spectrometry and Ion Mobility Spectrometry 224 Preservation of Native-Like Structures in the Gas Phase 224 Ion Mobility Spectrometry and Other Techniques for Probing Gas Phase Structures 224 Protein−Protein Complexes 225 Other Types of Noncovalent Assemblies 225 Concluding Remarks 227 Author Information 227 Corresponding Author 227 Notes 227 Biographies 227 Acknowledgments 227 References 227

Protein Structural Studies by Traveling Wave Ion Mobility Spectrometry: A Critical Look at Electrospray Sources and Calibration Issues.

AbstractThe question whether electrosprayed protein ions retain solution-like conformations continues to be a matter of debate. One way to address this issue involves comparisons of collision cross sections (Ω) measured by ion mobility spectrometry (IMS) with Ω values calculated for candidate structures. Many investigations in this area employ traveling wave IMS (TWIMS). It is often implied that nanoESI is more conducive for the retention of solution structure than regular ESI. Focusing on ubiquitin, cytochrome c, myoglobin, and hemoglobin, we demonstrate that Ω values and collisional unfolding profiles are virtually indistinguishable under both conditions. These findings suggest that gas-phase structures and ion internal energies are independent of the type of electrospray source. We also note that TWIMS calibration can be challenging because differences in the extent of collisional activation relative to drift tube reference data may lead to ambiguous peak assignments. It is demonstrated that this problem can be circumvented by employing collisionally heated calibrant ions. Overall, our data are consistent with the view that exposure of native proteins to electrospray conditions can generate kinetically trapped ions that retain solution-like structures on the millisecond time scale of TWIMS experiments. Graphical Abstractᅟ

Electrochemical detection of hepatitis C viral NS3-4A protease

Here we lay the ground work for the detection of hepatitis C viral NS3-4A protease exploiting peptide-protein interaction. The NS3-4A protease is inhibited by N-terminal cleavage products. Our approach is based on the formation of a self-assembled monolayer (SAM) of a ferrocene amino acid derivative on an electrode surface. A short NS3-4A specific inhibitory peptide (Asp-Glu-Ile-Val-Pro-Nva) was then covalently attached to the electrode surface. The interaction of the peptide, through the C-terminal, with the protein was quantified using electrochemical techniques. The systems exhibit a linear relationship between the measured signal and NS3-4A concentration in the range of 10-100 pM with a detection limit of 5 pM.

Evidence of Allosteric Enzyme Regulation via Changes in Conformational Dynamics: A Hydrogen/Deuterium Exchange Investigation of Dihydrodipicolinate Synthase.

Dihydrodipicolinate synthase is a tetrameric enzyme of the diaminopimelate pathway in bacteria and plants. The protein catalyzes the condensation of pyruvate (Pyr) and aspartate semialdehyde en route to the end product lysine (Lys). Dihydrodipicolinate synthase from Campylobacter jejuni (CjDHDPS) is allosterically inhibited by Lys. CjDHDPS is a promising antibiotic target, as highlighted by the recent development of a potent bis-lysine (bisLys) inhibitor. The mechanism whereby Lys and bisLys allosterically inhibit CjDHDPS remains poorly understood. In contrast to the case for other allosteric enzymes, crystallographically detectable conformational changes in CjDHDPS upon inhibitor binding are very minor. Also, it is difficult to envision how Pyr can access the active site; the available X-ray data seemingly imply that each turnover step requires diffusion-based mass transfer through a narrow access channel. This study employs hydrogen/deuterium exchange mass spectrometry for probing the structure and dynamics of CjDHDPS in a native solution environment. The deuteration kinetics reveal that the most dynamic protein regions are in the direct vicinity of the substrate access channel. This finding is consistent with the view that transient opening/closing fluctuations facilitate access of the substrate to the active site. Under saturating conditions, both Lys and bisLys cause dramatically reduced dynamics in the inhibitor binding region. In addition, rigidification extends to regions close to the substrate access channel. This finding strongly suggests that allosteric inhibitors interfere with conformational fluctuations that are required for CjDHDPS substrate turnover. In particular, our data imply that Lys and bisLys suppress opening/closing events of the access channel, thereby impeding diffusion of the substrate into the active site. Overall, this work illustrates why allosteric control does not have to be associated with crystallographically detectable large-scale transitions. Our experiments provide evidence that in CjDHDPS allostery is mediated by changes in the extent of thermally activated conformational fluctuations.

Peroxide-mediated oxidation and inhibition of the peptidyl-prolyl isomerase Pin1

Pin1 is a phosphorylation-dependent peptidyl-prolyl isomerase that plays a critical role in mediating protein conformational changes involved in signaling processes related to cell cycle control. Pin1 has also been implicated as being neuroprotective in aging-related neurodegenerative disorders including Alzheimers disease where Pin1 activity is diminished. Notably, recent proteomic analysis of brain samples from patients with mild cognitive impairment revealed that Pin1 is oxidized and also displays reduced activity. Since the Pin1 active site contains a functionally critical cysteine residue (Cys113) with a low predicted pK(a), we hypothesized that Cys113 is sensitive to oxidation. Consistent with this hypothesis, we observed that treatment of Pin1 with hydrogen peroxide results in a 32Da mass increase, likely resulting from the oxidation of Cys113 to sulfinic acid (Cys-SO(2)H). This modification results in loss of peptidyl-prolyl isomerase activity. Notably, Pin1 with Cys113 substituted by aspartic acid retains activity and is no longer sensitive to oxidation. Structural studies by X-ray crystallography revealed increased electron density surrounding Cys113 following hydrogen peroxide treatment. At lower concentrations of hydrogen peroxide, oxidative inhibition of Pin1 can be partially reversed by treatment with dithiothreitol, suggesting that oxidation could be a reversible modification with a regulatory role. We conclude that the loss of Pin1 activity upon oxidation results from oxidative modification of the Cys113 sulfhydryl to sulfenic (Cys-SOH) or sulfinic acid (Cys-SO(2)H). Given the involvement of Pin1 in pathological processes related to neurodegenerative diseases and to cancer, these findings could have implications for the prevention or treatment of disease.

Comparative Analysis of Oxy-Hemoglobin and Aquomet-Hemoglobin by Hydrogen/Deuterium Exchange Mass Spectrometry

AbstractThe function of hemoglobin (Hb) as oxygen transporter is mediated by reversible O2 binding to Fe(2+) heme in each of the α and β subunits. X-ray crystallography revealed different subunit arrangements in oxy-Hb and deoxy-Hb. The deoxy state is stabilized by additional contacts, causing a rigidification that results in strong protection against hydrogen/deuterium exchange (HDX). Aquomet-Hb is a dysfunctional degradation product with four water-bound Fe(3+) centers. Heme release from aquomet-Hb is relatively facile, triggering oxidative damage of membrane lipids. Aquomet-Hb crystallizes in virtually the same conformation as oxy-Hb. Hence, it is commonly implied that the solution-phase properties of aquomet-Hb should resemble those of the oxy state. This work compares the structural dynamics of oxy-Hb and aquomet-Hb by HDX mass spectrometry (MS). It is found that the aquomet state exhibits a solution-phase structure that is significantly more dynamic, as manifested by elevated HDX levels. These enhanced dynamics affect the aquomet α and β subunits in a different fashion. The latter undergoes global destabilization, whereas the former shows elevated HDX levels only in the heme binding region. It is proposed that these enhanced dynamics play a role in facilitating heme release from aquomet-Hb. Our findings should be of particular interest to the MS community because oxy-Hb and aquomet-Hb serve as widely used test analytes for probing the relationship between biomolecular structure in solution and in the gas phase. We are not aware of any prior comparative HDX/MS experiments on oxy-Hb and aquomet-Hb. Figureᅟ

Conformational characterization of the intrinsically disordered protein Chibby: Interplay between structural elements in target recognition.

The protein Chibby (Cby) is an antagonist of the Wnt signaling pathway, where it inhibits the binding between the transcriptional coactivator β‐catenin and the Tcf/Lef transcription factors. The 126 residue Cby is partially disordered; its N‐terminal half is unstructured while its C‐terminal half comprises a coiled‐coil domain. Previous structural analyses of Cby using NMR spectroscopy suffered from severe line broadening for residues within the proteins C‐terminal half, hindering detailed characterization of the coiled‐coil domain. Here, we use hydrogen/deuterium exchange‐mass spectrometry (HDX‐MS) to examine Cbys C‐terminal half. Results reveal that Cby is divided into three structural elements: a disordered N‐terminal half, a coiled‐coil domain, and a C‐terminal unstructured extension consisting of the last ∼ 25 residues (which we term C‐terminal extension). A series of truncation constructs were designed to assess the roles of individual structural elements in protein stability and Cby binding to TC‐1, a positive regulator of the Wnt signaling pathway. CD and NMR data show that Cby maintains coiled‐coil structure upon deletion of either disordered region. NMR and ITC binding experiments between Cby and TC‐1 illustrate that the interaction is retained upon deletion of either Cbys N‐terminal half or its C‐terminal extension. Intriguingly, Cbys C‐terminal half alone binds to TC‐1 with significantly greater affinity compared to full‐length Cby, implying that target binding of the coiled‐coil domain is affected by the flanking disordered regions.