Collision-induced Unfolding Research Articles

Gas-Phase Unfolding Reveals Stability Shifts Associated with Substrate Binding in Modular Polyketide Synthases.

Native mass spectrometry (MS), ion mobility (IM), and collision-induced unfolding (CIU) have all been widely used to study the binding of small molecules to proteins and their complexes. Despite many successes in detecting subtle gas-phase stability differences in smaller systems dominated by single-domain subunits, studies targeting complexes comprised of large, multidomain subunits still face many challenges. For example, polyketide synthases (PKSs) are multiprotein enzymes that use their modular architecture to produce polyketide natural products and form the basis for nearly one-third of pharmaceuticals. Here, we describe the development of CIU methods capable of extracting information from these multiprotein complexes and demonstrate the current limits of quantitative CIU technology by probing the stabilities ∼280 kDa PKS dimer protein complexes. Our approach detects the evidence of the stability shifts associated with substrate binding that accounts for <0.1% of the mass for the intact assembly.

Time-Resolved Ion Mobility-Mass Spectrometry Reveals Structural Transitions in the Disassembly of Modular Polyketide Syntheses.

The type 1 polyketide synthase (PKS) assembly line uses its modular structure to produce polyketide natural products that form the basis of many pharmaceuticals. Currently, several cryoelectron microscopy (cryo-EM) structures of a multidomain PKS module have been constructed, but much remains to be learned. Here we utilize ion-mobility mass spectrometry (IM-MS) to record size and shape information and detect different conformational states of a 207 kDa didomain dimer comprised of ketosynthase (KS) and acyl transferase (AT), excised from full-length module. Furthermore, gas-phase stability differences between these different conformations are captured by collision induced unfolding (CIU) technology. Additionally, through tracking these forms as a function of time, we elucidate a detailed disassembly pathway for KS-AT dimers for the first time.

CIUSuite 3: Next-Generation CCS Calibration and Automated Data Analysis Tools for Gas-Phase Protein Unfolding Data.

Ion mobility-mass spectrometry (IM-MS) has become a technology deployed across a wide range of structural biology applications despite the challenges in characterizing closely related protein structures. Collision-induced unfolding (CIU) has emerged as a valuable technique for distinguishing closely related, iso-cross-sectional protein and protein complex ions through their distinct unfolding pathways in the gas phase. With the speed and sensitivity of CIU analyses, there has been a rapid growth of CIU-based assays, especially regarding biomolecular targets that remain challenging to assess and characterize with other structural biology tools. With information-rich CIU data, many software tools have been developed to automate laborious data analysis. However, with the recent development of new IM-MS technologies, such as cyclic IM-MS, CIU continues to evolve, necessitating improved data analysis tools to keep pace with new technologies and facilitating the automation of various data processing tasks. Here, we present CIUSuite 3, a software package that contains updated algorithms that support various IM-MS platforms and supports the automation of various data analysis tasks such as peak detection, multidimensional classification, and collision cross section (CCS) calibration. CIUSuite 3 uses local maxima searches along with peak width and prominence filters to detect peaks to automate CIU data extraction. To support both the primary CIU (CIU1) and secondary CIU (CIU2) experiments enabled by cyclic IM-MS, two-dimensional data preprocessing is deployed, which allows multidimensional classification. Our data suggest that additional dimensions in classification improve the overall accuracy of class assignments. CIUSuite 3 also supports CCS calibration for both traveling wave and drift tube IM-MS, and we demonstrate the accuracy of a new single-field CCS calibration method designed for drift tube IM-MS leveraging calibrant CIU data. Overall, CIUSuite 3 is positioned to support current and next-generation IM-MS and CIU assay development deployed in an automated format.

Modeling collisional kinetic energy damping, heating, and cooling of ions in mass spectrometers: A tutorial perspective

Many powerful methods in mass spectrometry rely on activation of ions by high-energy collisions with gas particles. For example, multiple Collision Induced Dissociation (CID) has been used for many years to determine structural information for ions ranging from small organics to large, native-like protein complexes. More recently, Collision Induced Unfolding (CIU) has proved to be a very powerful method for understanding high-order protein structure and detecting differences between similar proteins. Quantifying the thermochemistry underlying dissociation/unfolding in these experiments can be quite challenging without reliable models of ion heating and cooling. Established physical models of CID are valuable in predicting ion heating but do not explicitly include mechanisms for cooling, which may play a large part in CID/CIU in modern instruments. Ab initio and Molecular Dynamics methods are extremely computationally expensive for modeling CID/CIU of large analytes such as biomolecular ions. In this tutorial perspective, limiting behaviors of ion kinetic energy damping, heating, and cooling set by “extreme” cases are explored, and an Improved Impulsive Collision Theory and associated software (“Ion Simulations of the Physics of Activation”, IonSPA) are introduced that can model all of these for partially inelastic collisions. Finally, examples of modeled collisional activation of native-like protein ions under realistic experimental conditions are discussed, with an outlook toward the use of IonSPA in accessing the thermochemical information hidden in CID breakdown curves and CIU fingerprints.

Recent advances in gas phase unfolding: Instrumentation and applications.

Broader adoption of native mass spectrometry (MS) and ion mobility-mass spectrometry (IM-MS) has propelled the development of several techniques which take advantage of the selectivity, sensitivity, and speed of MS to make measurements of complex biological molecules in the gas phase. One such method, collision induced unfolding (CIU), has risen to prominence in recent years, due to its well documented capability to detect shifts in structural stability of biological molecules in response to external stimuli (e.g., mutations, stress, non-covalent interactions, sample conditions etc.). This increase in reported CIU measurements is enabled partly due to advances in IM-MS instrumentation by vendors, and alsoinnovative method development by researchers. This perspective highlights a few of these advances and concludes with a look forward toward the future of the gas phase unfolding field.

Ion Mobility-Mass Spectrometry and Collision-Induced Unfolding Rapidly Characterize the Structural Polydispersity and Stability of an Fc-Fusion Protein.

Fc-fusion proteins are an emerging class of protein therapeutics that combine the properties of biological ligands with the unique properties of the fragment crystallizable (Fc) domain of an immunoglobulin G (IgG). Due to their diverse higher-order structures (HOSs), Fc-fusion proteins remain challenging characterization targets within biopharmaceutical pipelines. While high-resolution biophysical tools are available for HOS characterization, they frequently demand extended time frames and substantial quantities of purified samples, rendering them impractical for swiftly screening candidate molecules. Herein, we describe the development of ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) workflows that aim to fill this technology gap, where we focus on probing the HOS of a model Fc-Interleukin-10 (Fc-IL-10) fusion protein engineered using flexible glycine-serine linkers. We evaluate the ability of these techniques to probe the flexibility of Fc-IL-10 in the absence of bulk solvent relative to other proteins of similar size, as well as localize structural changes of low charge state Fc-IL-10 ions to specific Fc and IL-10 unfolding events during CIU. We subsequently apply these tools to probe the local effects of glycine-serine linkers on the HOS and stability of IL-10 homodimer, which is the biologically active form of IL-10. Our data reveals that Fc-IL-10 produces significantly more structural transitions during CIU and broader IM profiles when compared to a wide range of model proteins, indicative of its exceptional structural dynamism. Furthermore, we use a combination of enzymatic approaches to annotate these intricate CIU data and localize specific transitions to the unfolding of domains within Fc-IL-10. Finally, we detect a strong positive, quadratic relationship between average linker mass and fusion protein stability, suggesting a cooperative influence between glycine-serine linkers and overall fusion protein stability. This is the first reported study on the use of IM-MS and CIU to characterize HOS of Fc-fusion proteins, illustrating the practical applicability of this approach.

Distinct antithrombin activation modes for fondaparinux and natural heparins detected using millisecond hydrogen deuterium exchange and collision induced unfolding

Low molecular weight heparin and synthetic mimetics such as fondaparinux show different binding kinetics, protease specificity, and clinical effects. A combination of allosteric and template-mediated bridging mechanisms have been proposed to explain the differences in rate acceleration and specificity. The difficulty in working with heterogeneous heparin species has rendered a crystallographic interpretation of the differences in antithrombin activation between mimetics and natural heparin inaccessible. In this study, we examine the allosteric changes in antithrombin caused by binding fondaparinux, enoxaparin and depolymerized natural heparins using millisecond hydrogen deuterium exchange mass spectrometry (TRESI-HDX MS) and relate these conformational changes to complex stability in the gas phase using collision induced unfolding (CIU). This exploration reveals that in addition to the dynamic changes caused by fondaparinux, long chain heparins reduce structural flexibility proximal to Arg393, the cleavable residue in the reactive centre loop of the protein. These local changes in protein dynamics are associated with an increase in overall complex stability that increases with heparin chain length. Ultimately, these results shed light on the molecular mechanisms underlying differences in activity and specificity between heparin mimetics and natural heparins.

Native ESI-MS and Collision-Induced Unfolding (CIU) of the Complex between Bacterial Elongation Factor-Tu and the Antibiotic Enacyloxin IIa.

Collision-induced unfolding (CIU) of protein ions, monitored by ion mobility-mass spectrometry, can be used to assess the stability of their compact gas-phase fold and hence provide structural information. The bacterial elongation factor EF-Tu, a key protein for mRNA translation in prokaryotes and hence a promising antibiotic target, has been studied by CIU. The major [M + 12H]12+ ion of EF-Tu unfolded in collision with Ar atoms between 40 and 50 V, corresponding to an Elab energy of 480-500 eV. Binding of the cofactor analogue GDPNP and the antibiotic enacyloxin IIa stabilized the compact fold of EF-Tu, although dissociation of the latter from the complex diminished its stabilizing effect at higher collision energies. Molecular dynamics simulations of the [M + 12H]12+ EF-Tu ion showed similar qualitative behavior to the experimental results.

Native ion mobility-mass spectrometry reveals the binding mechanisms of anti-amyloid therapeutic antibodies.

One of the most important attributes of anti-amyloid antibodies is their selective binding to oligomeric and amyloid aggregates. However, current methods of examining the binding specificities of anti-amyloid β (Aβ) antibodies have limited ability to differentiate between complexes that form between antibodies and monomeric or oligomeric Aβ species during the dynamic Aβ aggregation process. Here, we present a high-resolution native ion-mobility mass spectrometry (nIM-MS) method to investigate complexes formed between a variety of Aβ oligomers and three Aβ-specific IgGs, namely two antibodies with relatively high conformational specificity (aducanumab and A34) and one antibody with low conformational specificity (crenezumab). We found that crenezumab primarily binds Aβ monomers, while aducanumab preferentially binds Aβ monomers and dimers and A34 preferentially binds Aβ dimers, trimers, and tetrameters. Through collision induced unfolding (CIU) analysis, our data indicate that antibody stability is increased upon Aβ binding and, surprisingly, this stabilization involves the Fc region. Together, we conclude that nIM-MS and CIU enable the identification of Aβ antibody binding stoichiometries and provide important details regarding antibody binding mechanisms.

Investigation of Structure-Stabilizing Elements in Proteins by Ion Mobility Mass Spectrometry and Collision-Induced Unfolding.

A recently developed proteolytic reactor, designed for protein structural investigation, was coupled to ion mobility mass spectrometry to monitor collisional cross section (CCS) evolution of model proteins undergoing trypsin-mediated mono enzymatic digestion. As peptides are released during digestion, the CCS of the remaining protein structure may deviate from the classical 2/3 power of the CCS-mass relationship for spherical structures. The classical relationship between CCS and mass (CCS = A × M2/3) for spherical structures, assuming a globular shape in the gas phase, may deviate as stabilizing elements are lost during digestion. In addition, collision-induced unfolding (CIU) experiments on partially digested proteins provided insights into the CCS resilience in the gas phase to ion activation, potentially due to the presence of stabilizing elements. The study initially investigated a model peptide ModBea (3 kDa), assessing the impact of disulfide bridges on CCS resilience in both reduced and oxidized forms. Subsequently, β-lactoglobulin (2 disulfide bridges), calmodulin (Ca2+ coordination cation), and cytochrome c (heme) were selected to investigate the influence of common structuring elements on CCS resilience. CIU experiments probed the unfolding process, evaluating the effect of losing specific peptides on the energy landscapes of partially digested proteins. Comparisons of the TWCCSN2→He to trend curves describing the CCS/mass relationship revealed that proteins with structure-stabilizing elements consistently exhibit TWCCSN2→He and greater resilience toward CIU compared to proteins lacking these elements. The integration of online digestion, ion mobility, and CIU provides a valuable tool for identifying structuring elements in biopolymers in the gas phase.

Not All Binding Sites Are Equal: Site Determination and Folding State Analysis of Gas-Phase Protein-Metallodrug Adducts.

Modern approaches in metallodrug research focus on compounds that bind protein targets rather than DNA. However, the identification of protein targets and binding sites is challenging. Using intact mass spectrometry and proteomics, we investigated the binding of the antimetastatic agent RAPTA-C to the model proteins ubiquitin, cytochrome c, lysozyme, and myoglobin. Binding to cytochrome c and lysozyme was negligible. However, ubiquitin bound up to three Ru moieties, two of which were localized at Met1 and His68 as [Ru(cym)], and [Ru(cym)] or [Ru(cym)(PTA)] adducts, respectively. Myoglobin bound up to four [Ru(cym)(PTA)] moieties and five sites were identified at His24, His36, His64, His81/82 and His113. Collision-induced unfolding (CIU) studies via ion-mobility mass spectrometry allowed measuring protein folding as a function of collisional activation. CIU of protein-RAPTA-C adducts showed binding of [Ru(cym)] to Met1 caused a significant compaction of ubiquitin, likely from N-terminal S-Ru-N chelation, while binding of [Ru(cym)(PTA)] to His residues of ubiquitin or myoglobin induced a smaller effect. Interestingly, the folded state of ubiquitin formed by His functionalization was more stable than Met1 metalation. The data suggests that selective metalation of amino acids at different positions on the protein impacts the conformation and potentially the biological activity of anticancer compounds.

Collision-Induced Unfolding Reveals Disease-Associated Stability Shifts in Mitochondrial Transfer Ribonucleic Acids.

Ribonucleic acids (RNAs) remain challenging targets for structural biology, creating barriers to understanding their vast functions in cellular biology and fully realizing their applications in biotechnology. The inherent dynamism of RNAs creates numerous obstacles in capturing their biologically relevant higher-order structures (HOSs), and as a result, many RNA functions remain unknown. In this study, we describe the development of native ion mobility-mass spectrometry and collision-induced unfolding (CIU) for the structural characterization of a variety of RNAs. We evaluate the ability of these techniques to preserve native structural features in the gas phase across a wide range of functional RNAs. Finally, we apply these tools to study the elusive mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes-associated A3243G mutation. Our data demonstrate that our experimentally determined conditions preserve some solution-state memory of RNAs via the correlated complexity of CIU fingerprints and RNA HOS, the observation of predicted stability shifts in the control RNA samples, and the retention of predicted magnesium binding events in gas-phase RNA ions. Significant differences in collision cross section and stability are observed as a function of the A3243G mutation across a subset of the mitochondrial tRNA maturation pathway. We conclude by discussing the potential application of CIU for the development of RNA-based biotherapeutics and, more broadly, transcriptomic characterization.

Collision induced unfolding and molecular dynamics simulations of norovirus capsid dimers reveal strain-specific stability profiles.

Collision induced unfolding (CIU) is a method used with ion mobility mass spectrometry to examine protein structures and their stability. Such experiments yield information about higher order protein structures, yet are unable to provide details about the underlying processes. That information can however be provided using molecular dynamics simulations. Here, we investigate the gas-phase unfolding of norovirus capsid dimers from the Norwalk and Kawasaki strains by employing molecular dynamics simulations over a range of temperatures, representing different levels of activation, together with CIU experiments. The dimers have highly similar structures, but their CIU reveals different stability that can be explained by the different dynamics that arises in response to the activation seen in the simulations, including a part of the sequence with previously observed strain-specific dynamics in solution. Our findings show how similar protein variants can be examined using mass spectrometric techniques in conjunction with atomistic molecular dynamics simulations to reveal differences in stability as well as differences in how and where unfolding takes place upon activation.

Collision-Induced Unfolding, Tandem MS, Bottom-up Proteomics, and Interactomics for Identification of Protein Complexes in Native Surface Mass Spectrometry.

Endogenously occurring salts and nonvolatile matrix components in untreated biological surfaces can suppress protein ionization and promote adduct formation, challenging protein identification. Characterization of labile proteins within biological specimens is particularly demanding because additional purification or sample treatment steps can be time-intensive and can disrupt noncovalent interactions. It is demonstrated that the combined use of collision-induced unfolding, tandem mass spectrometry, and bottom-up proteomics improves protein characterization in native surface mass spectrometry (NSMS). This multiprong analysis is achieved by acquiring NSMS, MS/MS, ion mobility (IM), and bottom-up proteomics data from a single surface extracted sample. The validity of this multiprong approach was confirmed by the successful characterization of nine surface-deposited proteins, with molecular weights ranging from 8 to 147 kDa, in two separate mixtures. Bottom-up proteomics provided a list of proteins to match against observed proteins in NSMS and their detected subunits in tandem MS. The method was applied to characterize endogenous proteins from untreated chicken liver samples. The subcapsular liver sampling for NSMS analysis allowed for the detection of endogenous proteins with molecular weights of up to ∼220 kDa. Moreover, using IM-MS, collision cross sections and collision-induced unfolding pathways of enzymatic proteins and protein complexes of up to 145 kDa were obtained.

Development of an Automated, High-Throughput Methodology for Native Mass Spectrometry and Collision-Induced Unfolding.

Native ion mobility mass spectrometry (nIM-MS) has emerged as a useful technology for the rapid evaluation of biomolecular structures. When combined with collisional activation in a collision-induced unfolding (CIU) experiment, nIM-MS experimentation can be leveraged to gain greater insight into biomolecular conformation and stability. However, nIM-MS and CIU remain throughput limited due to nonautomated sample preparation and introduction. Here, we explore the use of a RapidFire robotic sample handling system to develop an automated, high-throughput methodology for nMS and CIU. We describe native RapidFire-MS (nRapidFire-MS) capable of performing online desalting and sample introduction in as little as 10 s per sample. When combined with CIU, our nRapidFire-MS approach can be used to collect CIU fingerprints in 30 s following desalting by using size exclusion chromatography cartridges. When compared to nMS and CIU data collected using standard approaches, ion signals recorded by nRapidFire-MS exhibit identical ion collision cross sections, indicating that the same conformational populations are tracked by the two approaches. Our data further suggest that nRapidFire-MS can be extended to study a variety of biomolecular classes, including proteins and protein complexes ranging from 5 to 300 kDa and oligonucleotides. Furthermore, nRapidFire-MS data acquired for biotherapeutics suggest that nRapidFire-MS has the potential to enable high-throughput nMS analyses of biopharmaceutical samples. We conclude by discussing the potential of nRapidFire-MS for enabling the development of future CIU assays capable of catalyzing breakthroughs in protein engineering, inhibitor discovery, and formulation development for biotherapeutics.

Enhanced Stability Differentiation of Therapeutic Polyclonal Antibodies with All Ion Unfolding-Ion Mobility-Mass Spectrometry.

Compared with monoclonal antibodies, polyclonal antibodies (pAbs) have rather significant characteristics, including lower cost, shorter production cycle, and higher affinity. Therefore, to facilitate their applications in clinic, it is equally critical to comprehensively characterize the conformational stabilities of pAb at the molecular weight-resolved scale, which is technically challenging due to the lack of an effective analytical tool capable of simultaneously providing both stability and molecular weight information within an acceptable error range. Ion mobility-mass spectrometry (IM-MS) has grown as an alternative to rapidly assess protein conformational stability with accurate molecular weight information maintained, especially when equipped with a collision-induced unfolding (CIU) regime. Dynamic and transient conformational intermediates can be captured with the CIU-IM-MS technique, adding to traditional static structural measurements with collisional cross section. Most CIU-IM-MS-centered protocols are focusing on the application to isolated, targeted protein ions, namely, analyzing one single charge state at one time, limiting its analytical throughput and speed. In this study, we employed an enhanced unfolding regime, all ion unfolding (AIU), capable of the simultaneous operation of numerous ions at a time during stepped unfolding processes to analyze pAb. Results show that AIU can quantitatively characterize the subtle differences in conformational stability among four structurally similar pAbs with improved resolving capability by around a 2-4-fold increment in both stability and structure differentiating parameters. Besides, AIU also benefits from considerably saved time cost and improved spectrum quality with an elevated signal-to-noise ratio.

Collision Induced Unfolding Enables the Quantitation of Isomass Biotherapeutics in Complex Biological Matrices.

Quantitative mass spectrometry has been widely used to evaluate the concentrations of molecules within a variety of biological matrices. Typically, such quantitative mass spectrometry analyses are predicated upon the production of mass-resolved precursor or fragment ions, leading to challenges surrounding the quantification of isomeric or conformationally distinct analytes. As such, new approaches are required for the label-free quantification of isomass proteins. Native ion-mobility MS (nIM-MS) in combination with collision induced unfolding (CIU) is a potentially enabling approach for such quantitative mass spectrometry methods as the technique can rapidly separate and detect many biomacromolecule isoforms. CIU uses collisional activation to capture the unfolding trajectory of ions in the gas phase, producing different intermediate structures that can be leveraged to distinguish protein structures that exhibit identical sizes at lower energies. Here we describe the deployment of quantitative CIU methodology to measure the concentrations of isomass pairs of biotherapeutics and sequence homologues in both standard and biological matrices. Our results cover three antibody pairs and include examples of mixed therapies where multiple biologics are commonly provided to patients. In all cases, CIU enables the production of resolved features for each antibody mixture probed, producing calibration curves with correlation coefficients ranging from 0.92 to 0.99, limits of detection ranging from 300 to 5000 nM and sensitivities ranging from 8.7 × 10-5 nM-1 to 6 × 10-3 μM-1. We conclude our report by projecting the future utility of CIU-enabled quantitative MS methods.

Gas-phase stability and thermodynamics of ligand-bound, binary complexes of chloramphenicol acetyltransferase reveal negative cooperativity.

The biological role of the bacterial chloramphenicol (Chl)-resistance enzyme, chloramphenicol acetyltransferase (CAT), has seen renewed interest due to the resurgent use of Chl against multi-drug-resistant microbes. This looming threat calls for more rationally designed antibiotic derivatives that have improved antimicrobial properties and reduced toxicity in humans. Herein, we utilize native ion mobility spectrometry-mass spectrometry (IMS-MS) to investigate the gas-phase structure and thermodynamic stability of the type I variant of CAT from Escherichia coli (EcCATI) and several EcCATI:ligand-bound complexes. EcCATI readily binds multiple Chl without incurring significant changes to its gas-phase structure or stability. A non-hydrolyzable acetyl-CoA derivative (S-ethyl-CoA, S-Et-CoA) was used to kinetically trap EcCATI and Chl in a ternary, ligand-bound state (EcCATI:S-Et-CoA:Chl). Using collision-induced unfolding (CIU)-IMS-MS, we find that Chl dissociates from EcCATI:S-Et-CoA:Chl complexes at low collision energies, while S-Et-CoA remains bound to EcCATI even as protein unfolding occurs. Gas-phase binding constants further suggest that EcCATI binds S-Et-CoA more tightly than Chl. Both ligands exhibit negative cooperativity of subsequent ligand binding in their respective binary complexes. While we observe no significant change in structure or stability to EcCATI when bound to either or both ligands, we have elucidated novel gas-phase unfolding and dissociation behavior and provided a foundation for further characterization of alternative substrates and/or inhibitors of EcCATI.

Structural Characterization of Cu(I)/Zn(II)-metallothionein-3 by Ion Mobility Mass Spectrometry and Top-Down Mass Spectrometry.

Mammalian zinc metallothionein-3 (Zn7MT3) plays an important role in protecting against copper toxicity by scavenging free Cu(II) ions or removing Cu(II) bound to β-amyloid and α-synuclein. While previous studies reported that Zn7MT3 reacts with Cu(II) ions to form Cu(I)4Zn(II)4MT3ox containing two disulfides (ox), the precise localization of the metal ions and disulfides remained unclear. Here, we undertook comprehensive structural characterization of the metal-protein complexes formed by the reaction between Zn7MT3 and Cu(II) ions using native ion mobility mass spectrometry (IM-MS). The complex formation mechanism was found to involve the disassembly of Zn3S9 and Zn4S11 clusters from Zn7MT3 and reassembly into Cu(I)xZn(II)yMT3ox complexes rather than simply Zn(II)-to-Cu(I) exchange. At neutral pH, the β-domain was shown to be capable of binding up to six Cu(I) ions to form Cu(I)6Zn(II)4MT3ox, although the most predominant species was the Cu(I)4Zn(II)4MT3ox complex. Under acidic conditions, four Zn(II) ions dissociate, but the Cu(I)4-thiolate cluster remains stable, highlighting the MT3 role as a Cu(II) scavenger even at lower than the cytosolic pH. IM-derived collision cross sections (CCS) reveal that Cu(I)-to-Zn(II) swap in Zn7MT3 with concomitant disulfide formation induces structural compaction and a decrease in conformational heterogeneity. Collision-induced unfolding (CIU) experiments estimated that the native-like folded Cu(I)4Zn(II)4MT3ox conformation is more stable than Zn7MT3. Native top-down MS demonstrated that the Cu(I) ions are exclusively bound to the β-domain in the Cu(I)4Zn(II)4MT3ox complex as well as the two disulfides, serving as a steric constraint for the Cu(I)4-thiolate cluster. In conclusion, this study enhances our comprehension of the structure, stability, and dynamics of Cu(I)xZn(II)yMT3ox complexes.

Effects of Hydrogen/Deuterium Exchange on Protein Stability in Solution and in the Gas Phase.

Mass spectrometry (MS)-based techniques are widely used for probing protein structure and dynamics in solution. H/D exchange (HDX)-MS is one of the most common approaches in this context. HDX is often considered to be a "benign" labeling method, in that it does not perturb protein behavior in solution. However, several studies have reported that D2O pushes unfolding equilibria toward the native state. The origin, and even the existence of this protein stabilization remain controversial. Here we conducted thermal unfolding assays in solution to confirm that deuterated proteins in D2O are more stable, with 2-4 K higher melting temperatures than unlabeled proteins in H2O. Previous studies tentatively attributed this phenomenon to strengthened H-bonds after deuteration, an effect that may arise from the lower zero-point vibrational energy of the deuterated species. Specifically, it was proposed that strengthened water-water bonds (W···W) in D2O lower the solubility of nonpolar side chains. The current work takes a broader view by noting that protein stability in solution also depends on water-protein (W···P) and protein-protein (P···P) H-bonds. To help unravel these contributions, we performed collision-induced unfolding (CIU) experiments on gaseous proteins generated by native electrospray ionization. CIU profiles of deuterated and unlabeled proteins were indistinguishable, implying that P···P contacts are insensitive to deuteration. Thus, protein stabilization in D2O is attributable to solvent effects, rather than alterations of intraprotein H-bonds. Strengthening of W···W contacts represents one possible explanation, but the stabilizing effect of D2O can also originate from weakened W···P bonds. Future work will be required to elucidate which of these two scenarios is correct, or if both contribute to protein stabilization in D2O. In any case, the often-repeated adage that "D-bonds are more stable than H-bonds" does not apply to intramolecular contacts in native proteins.