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H/D exchange (HDX) mass spectrometry (MS) is a widely used technique for interrogating protein structure and dynamics. Backbone HDX is mediated by opening/closing (unfolding/refolding) fluctuations. In traditional HDX-MS, proteins are incubated in D2O as a function of time at constant temperature (T). There is an urgent need to complement this traditional approach with experiments that probe proteins in a T-dependent fashion, e.g., for assessing the stability of therapeutic antibodies. A key problem with such studies is the absence of strategies for interpreting HDX-MS data in the context of T-dependent protein dynamics. Specifically, it has not been possible thus far to separate T-induced changes of the chemical labeling step (kch) from thermally enhanced protein fluctuations. Focusing on myoglobin, the current work solves this problem by dissecting T-dependent HDX-MS profiles into contributions from kch(T), as well as local and global protein dynamics. Experimental profiles started off with surprisingly shallow slopes that seemed to defy the quasi-exponential kch(T) dependence. Just below the melting temperature (Tm) the profiles showed a sharp increase. Our analysis revealed that local dynamics dominate at low T, while global events become prevalent closer to Tm. About half of the backbone NH sites exhibited a canonical scenario, where local opening/closing was associated with positive ΔH and ΔS. Many of the remaining sites had negative ΔH and ΔS, thereby accounting for the shallowness of the experimental HDX-MS profiles at low T. In summary, this work provides practitioners with the tools to analyze proteins over a wide temperature range, paving the way toward T-dependent high-throughput screening applications by HDX-MS.

Proteins that are unfolded in solution produce higher charge states during electrospray ionization (ESI) than their natively folded counterparts. Protein charge states can be further increased by the addition of supercharging agents (SCAs) such as sulfolane. The mechanism whereby these supercharged [M + zH] z+ ions are formed under unfolded conditions remains unclear. Here we employed a combination of mass spectrometry (MS), ion mobility spectrometry (IMS), and molecular dynamics (MD) simulations for probing the ESI mechanism under denatured supercharging conditions. ESI of acid-unfolded apo-myoglobin (aMb) in the presence of sulfolane produced charge states around 27+, all the way to fully protonated (33+) aMb. MD simulations of aMb 27+ to 33+ in Rayleigh-charged water/sulfolane droplets culminated in electrostatically driven protein expulsion, consistent with the chain ejection model (CEM). The electrostatically stretched conformations predicted by these simulations were in agreement with IMS experiments. The CEM involves partitioning of mobile H+ between the droplet and the departing protein. Our results imply that supercharging of unfolded proteins is caused by residual sulfolane that stabilizes protonated sites on the protruding chains, thereby promoting H+ retention on the protein. The stabilization of charged sites is due to charge-dipole interactions mediated by the large dipole moment and the low volatility of sulfolane. Support for this mechanism comes from the experimental observation of sulfolane adducts on the most highly charged ions, a phenomenon previously noted by Venter ( J. Am. Soc. Mass Spectrom. 2012, 23, 489-497). The "CEM supercharging model" proposed here for unfolded proteins is distinct from the charge trapping mechanism believed to be operative during native ESI supercharging.

Previous structural studies of osteoprotegerin (OPG), a crucial negative regulator of bone remodeling and osteoclastogenesis, were mostly limited to the N-terminal ligand-binding domains. It is now known that the three C-terminal domains of OPG also play essential roles in its function by mediating OPG dimerization, OPG–heparan sulfate (HS) interactions, and formation of the OPG–HS–receptor activator of nuclear factor κB ligand (RANKL) ternary complex. Employing hydrogen–deuterium exchange MS methods, here we investigated the structure of full-length OPG in complex with HS or RANKL in solution. Our data revealed two noteworthy aspects of the OPG structure. First, we found that the interconnection between the N- and C-terminal domains is much more rigid than previously thought, possibly because of hydrophobic interactions between the fourth cysteine-rich domain and the first death domain. Second, we observed that two hydrophobic clusters located in two separate C-terminal domains directly contribute to OPG dimerization, likely by forming a hydrophobic dimerization interface. Aided by site-directed mutagenesis, we further demonstrated that an intact dimerization interface is essential for the biological activity of OPG. Our study represents an important step toward deciphering the structure–function relationship of the full-length OPG protein.

Various activation methods are available for the fragmentation of gaseous protein complexes produced by electrospray ionization (ESI). Such experiments can potentially yield insights into quaternary structure. Collision-induced dissociation (CID) is the most widely used fragmentation technique. Unfortunately, CID of protein complexes is dominated by the ejection of highly charged monomers, a process that does not yield any structural insights. Using hemoglobin (Hb) as a model system, this work examines under what conditions CID generates structurally informative subcomplexes. Native ESI mainly produced tetrameric Hb ions. In addition, "noncanonical" hexameric and octameric complexes were observed. CID of all these species [(αβ)2, (αβ)3, and (αβ)4] predominantly generated highly charged monomers. In addition, we observed hexamer → tetramer + dimer dissociation, implying that hexamers have a tetramer··dimer architecture. Similarly, the observation of octamer → two tetramer dissociation revealed that octamers have a tetramer··tetramer composition. Gas-phase candidate structures of Hb assemblies were produced by molecular dynamics (MD) simulations. Ion mobility spectrometry was used to identify the most likely candidates. Our data reveal that the capability of CID to produce structurally informative subcomplexes depends on the fate of protein-protein interfaces after transfer into the gas phase. Collapse of low affinity interfaces conjoins the corresponding subunits and favors CID via monomer ejection. Structurally informative subcomplexes are formed only if low affinity interfaces do not undergo a major collapse. However, even in these favorable cases CID is still dominated by monomer ejection, requiring careful analysis of the experimental data for the identification of structurally informative subcomplexes.

The mechanisms whereby protein ions are released into the gas phase from charged droplets during electrospray ionization (ESI) continue to be controversial. Several pathways have been proposed. For native ESI the charged residue model (CRM) is favored; it entails the liberation of proteins via solvent evaporation to dryness. Unfolded proteins likely follow the chain ejection model (CEM), which involves the gradual expulsion of stretched-out chains from the droplet. According to the ion evaporation model (IEM) ions undergo electrostatically driven desorption from the droplet surface. The IEM is well supported for small precharged species such as Na+. However, it is unclear whether proteins can show IEM behavior as well. We examined this question using molecular dynamics (MD) simulations, mass spectrometry (MS), and ion mobility spectrometry (IMS) in positive ion mode. Ubiquitin was chosen as the model protein because of its structural stability which allows the protein charge in solution to be controlled via pH adjustment without changing the protein conformation. MD simulations on small ESI droplets (3 nm radius) showed CRM behavior regardless of the protein charge in solution. Surprisingly, many MD runs on larger droplets (5.5 nm radius) culminated in IEM ejection of ubiquitin, as long as the protein carried a sufficiently large positive solution charge. MD simulations predicted that nonspecific salt adducts are less prevalent for IEM-generated protein ions than for CRM products. This prediction was confirmed experimentally. Also, collision cross sections of MD structures were in good agreement with IMS data. Overall, this work reveals that the CRM, CEM, and IEM all represent viable pathways for generating gaseous protein ions during ESI. The IEM is favored for proteins that are tightly folded and highly charged in solution and for droplets in a suitable size regime.

"Native" electrospray ionization (ESI) mass spectrometry (MS) aims to transfer proteins from solution into the gas phase while maintaining solution-like structures and interactions. The ability to control the charge states of protein ions produced in these experiments is of considerable importance. Supercharging agents (SCAs) such as sulfolane greatly elevate charge states without significantly affecting the protein structure in bulk aqueous solution. The origin of native ESI supercharging remains contentious. According to one model, SCAs trigger unfolding within ESI droplets. In contrast, the "charge trapping model" envisions that SCAs impede the ejection of charge carriers (e.g., NH4+ or Na+) from the droplet. We addressed this controversy experimentally and computationally by employing 18C6 crown ether as a mechanistic probe in native ESI-MS experiments on holo-myoglobin. Remarkably, 18C6 suppressed the supercharging capability of sulfolane. Molecular dynamics (MD) simulations reproduced the experimental charge states. The MD data revealed that 18C6 altered the location of charge carriers in the ESI droplets. Without 18C6, sulfolane covered the droplets in an ionophobic layer that impeded charge carrier access to the surface. In contrast, 18C6 complexation caused charge carrier enrichment in this surface layer, thereby promoting charge ejection. For late droplets, all the water had left and the protein was encapsulated in sulfolane; charge ejection at this stage continued only in the presence of 18C6. As a result, evaporation to dryness of charge-depleted water/sulfolane/18C6 droplets produced low protein charge states, whereas charge-abundant water/sulfolane droplets generated high charge states. Our data support the view that native ESI supercharging is caused by charge trapping. Unfolding within the droplet may play an ancillary role under some conditions, but for the cases examined here, protein structural changes are not a causative factor for supercharging. Our conclusions are bolstered by dendrimer supercharging experiments.

Allosteric proteins possess dynamically coupled residues for the propagation of input signals to distant target binding sites. The input signals usually correspond to "effector is present" or "effector is not present". Many aspects of allosteric regulation remain incompletely understood. This work focused on S100A11, a dimeric EF-hand protein with two hydrophobic target binding sites. An annexin peptide (Ax) served as the target. Target binding is allosterically controlled by Ca2+ over a distance of ∼26 Å. Ca2+ promotes formation of a [Ca4 S100 Ax2] complex, where the Ax peptides are accommodated between helices III/IV and III'/IV'. Without Ca2+ these binding sites are closed, precluding interactions with Ax. The allosteric mechanism was probed by microsecond MD simulations in explicit water, complemented by hydrogen exchange mass spectrometry (HDX/MS). Consistent with experimental data, MD runs in the absence of Ca2+ and Ax culminated in target binding site closure. In simulations on [Ca4 S100] the target binding sites remained open. These results capture the essence of allosteric control, revealing how Ca2+ prevents binding site closure. Both HDX/MS and MD data showed that the metalation sites become more dynamic after Ca2+ loss. However, these enhanced dynamics do not represent the primary trigger of the allosteric cascade. Instead, a labile salt bridge acts as an incessantly active "agitator" that destabilizes the packing of adjacent residues, causing a domino chain of events that culminates in target binding site closure. This agitator represents the starting point of the allosteric signal propagation pathway. Ca2+ binding rigidifies elements along this pathway, thereby blocking signal transmission. This blocking mechanism does not conform to the commonly held view that allosteric communication pathways generally originate at the sites where effectors interact with the protein.

The E3 ligase parkin ubiquitinates outer mitochondrial membrane\ud proteins during oxidative stress and is linked to early-onset\ud Parkinson’s disease. Parkin is autoinhibited but is activated by the\ud kinase PINK1 that phosphorylates ubiquitin leading to parkin\ud recruitment, and stimulates phosphorylation of parkin’s N-terminal\ud ubiquitin-like (pUbl) domain. How these events alter the\ud structure of parkin to allow recruitment of an E2~Ub conjugate\ud and enhanced ubiquitination is an unresolved question. We\ud present a model of an E2~Ub conjugate bound to the phosphoubiquitin-loaded\ud C-terminus of parkin, derived from NMR chemical\ud shift perturbation experiments. We show the UbcH7~Ub conjugate\ud binds in the open state whereby conjugated ubiquitin binds to the\ud RING1/IBR interface. Further, NMR and mass spectrometry experiments\ud indicate the RING0/RING2 interface is re-modelled,\ud remote from the E2 binding site, and this alters the reactivity of\ud the RING2(Rcat) catalytic cysteine, needed for ubiquitin transfer.\ud Our experiments provide evidence that parkin phosphorylation\ud and E2~Ub recruitment act synergistically to enhance a weak\ud interaction of the pUbl domain with the RING0 domain and rearrange\ud the location of the RING2(Rcat) domain to drive parkin\ud activity.

It is believed that enzyme catalysis is facilitated by conformational dynamics of the protein scaffold that surrounds the active site, yet the exact nature of catalytically relevant protein motions remains largely unknown. Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) reports on backbone H-bond fluctuations. HDX/MS thus represents a promising avenue for probing the relationship between enzyme dynamics and catalysis. A seemingly straightforward strategy for such studies involves comparative measurements during substrate turnover and in the resting state. We examined the feasibility of this approach using rabbit muscle pyruvate kinase (rM1-PK) which catalyzes the conversion of phosphoenolpyruvate and Mg-ADP to pyruvate and Mg-ATP. HDX/MS revealed that catalytically active rM1-PK undergoes significant rigidification in the active site. This finding is counterintuitive, considering the purported correlation between dynamics and catalysis. Interestingly, virtually the same rigidification was seen upon exposing rM1-PK to substrates or products in the absence of turnover. These data imply that the active site dynamics during turnover are dominated by protein-ligand binding interactions. These interactions stabilize H-bonds in the vicinity of the active site, thereby masking subtle dynamic features that might be uniquely associated with catalysis. Our data uncover an inherent problem with side-by-side turnover/resting state measurements, i.e., the difficulty to design a suitable reference state against which the working enzyme can be compared. Comparative HDX/MS experiments on enzyme dynamics should therefore be interpreted with caution.

The 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. ᅟ

In addition to serving as respiratory electron shuttle, ferri-cytochrome c (cyt c) acts as a peroxidase; i.e., it catalyzes the oxidation of organic substrates by H

Royal jelly (RJ) triggers the development of female honeybee larvae into queens. This effect has been attributed to the presence of major royal jelly protein 1 (MRJP1) in RJ. MRJP1 isolated from royal jelly is tightly associated with apisimin, a 54-residue α-helical peptide that promotes the noncovalent assembly of MRJP1 into multimers. No high-resolution structural data are available for these complexes, and their binding stoichiometry remains uncertain. We examined MRJP1/apisimin using a range of biophysical techniques. We also investigated the behavior of deglycosylated samples, as well as samples with reduced apisimin content. Our mass spectrometry (MS) data demonstrate that the native complexes predominantly exist in a (MRJP14 apisimin4) stoichiometry. Hydrogen/deuterium exchange MS reveals that MRJP1 within these complexes is extensively disordered in the range of residues 20-265. Marginally stable secondary structure (likely antiparallel β-sheet) exists around residues 266-432. These weakly structured regions interchange with conformers that are extensively unfolded, giving rise to bimodal (EX1) isotope distributions. We propose that the native complexes have a "dimer of dimers" quaternary structure in which MRJP1 chains are bridged by apisimin. Specifically, our data suggest that apisimin acts as a linker that forms hydrophobic contacts involving the MRJP1 segment 316VLFFGLV322. Deglycosylation produces large soluble aggregates, highlighting the role of glycans as aggregation inhibitors. Samples with reduced apisimin content form dimeric complexes with a (MRJP12 apisimin1) stoichiometry. The information uncovered in this work will help pave the way toward a better understanding of the unique physiological role played by MRJP1 during queen differentiation.

The mechanisms whereby protein ions are liberated from charged droplets during electrospray ionization (ESI) remain under investigation. Compact conformers electrosprayed from aqueous solution in positive ion mode likely follow the charged residue model (CRM), which envisions analyte release after solvent evaporation to dryness. The concentration of nonvolatile salts such as NaCl increases sharply within vanishing CRM droplets, promoting nonspecific pairing of Cl(-) and Na(+) with charged groups on the protein surface. For unfolded proteins, it has been proposed that ion formation occurs via the chain ejection model (CEM). During the CEM proteins are expelled from the droplet long before complete solvent evaporation has taken place. Here we examine whether salt adduction levels support the view that folded and unfolded proteins follow different ESI mechanisms. Solvent evaporation during the CEM is expected to be less extensive and, hence, the salt concentration at the point of protein release should be substantially lower than for the CRM. CEM ions should therefore exhibit lower adduction levels than CRM species. We explore the adduction behavior of several proteins that were chosen to allow comparative studies on folded and unfolded structures in the same solution. In-source activation eliminates chloride adducts via HCl release, generating protein ions that are heterogeneously charged because of sodiation and protonation. Sodiation levels measured under such conditions provide estimates of the salt adduction behavior experienced by the "nascent" analyte ions. Sodiation levels are significantly reduced for unfolded proteins, supporting the view that these species are indeed formed via the CEM.

Many protein investigations by electrospray ionization (ESI) mass spectrometry (MS) strive to ensure a "native" solvent environment, i.e., nondenaturing conditions up to the point of gas-phase ion formation. Ideally, these studies would employ a volatile pH buffer to mitigate changes in H(+) concentration that can occur during ESI. Ammonium acetate is a commonly used additive, despite its low buffering capacity at pH 7. Ammonium bicarbonate provides greatly improved pH stabilization, thus offering an interesting alternative. Surprisingly, protein analyses in bicarbonate at pH 7 tend to result in the formation of very high charge states, similar to those obtained when electrospraying unfolded proteins in a denaturing solvent. This effect has been reported previously (Sterling, H. J.; Cassou, C. A.; Susa, A. C.; Williams, E. R. Anal. Chem. 2012, 84, 3795), but its exact mechanistic origin remains unclear. ESI-mediated unfolding does not take place in acetate under otherwise identical conditions. We demonstrate that heating of protein-containing bicarbonate solutions results in extensive foaming, caused by CO2 outgassing. In contrast, acetate solutions do not generate foam. Protein denaturation caused by gas bubbles is a well-known phenomenon. Adsorption to the gas/liquid interface is accompanied by major conformational changes that allow the protein to act as a surfactant. The foaming of beer is a manifestation of this effect. Bubble formation in bicarbonate during ESI is facilitated by collisional and blackbody droplet heating. Our data imply that heat and bubbles act synergistically to cause unfolding during the electrospray process, while proteins reside in ESI droplets. Because of this effect we advise against the use of ammonium bicarbonate for native ESI-MS. Ammonium acetate represents a gentler droplet environment, despite its low buffering capacity.

The 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.

Mass spectrometry (MS)-based protein conformational studies are a rapidly growing field. The characterization of partially disordered conformers is of particular interest because these species are not amenable to classical high-resolution techniques. Such equilibrium intermediates can often be populated by exposure to mildly acidic pH. Hydroxyl radical (·OH) introduces oxidative modifications at solvent-accessible side chains, while buried sites are protected. ·OH can be generated by laser photolysis of H(2)O(2) (fast photochemical oxidation of proteins-FPOP). The resulting labeling pattern can be analyzed by MS. The characterization of partially disordered intermediates usually involves comparative measurements under different solvent conditions. It can be challenging to separate structurally induced labeling changes from pH-mediated "secondary" effects. The issue of secondary effects in FPOP has received little prior attention. We demonstrate that with a proper choice of conditions (e.g., in the absence of pH-dependent ·OH scavengers) such undesired phenomena can be almost completely eliminated. Using apomyoglobin as a model system, we map the structure of an intermediate that is formed at pH 4. This species retains a highly protected helix G that is surrounded by partially protected helices A, B, and H. Our results demonstrate the utility of FPOP for the structural characterization of equilibrium intermediates. The near absence of an intrinsic pH dependence represents an advantage compared to hydrogen/deuterium exchange MS.

Electrospray ionization (ESI) allows the transfer of multi-protein complexes into the gas phase, thereby providing a simple approach for monitoring the stoichiometry of these noncovalent assemblies by mass spectrometry (MS). It remains unclear, however, whether the measured ion abundance ratios of free and bound species are suitable for determining solution-phase binding affinities (K d values). Many types of mass spectrometers employ rf-only quadrupoles as ion guides. This work demonstrates that the settings used for these devices are a key factor for ensuring uniform transmission behavior, which is a prerequisite for meaningful affinity measurements. Using bovine β-lactoglobulin and hemoglobin as model systems, it is demonstrated that under carefully adjusted conditions the “direct” ESI-MS approach is capable of providing K d values that are in good agreement with previously published solution-phase data. Of the several ion sources tested, a regular ESI emitter operated with pressure-driven flow at 1 μL min–1 provided the most favorable results. Potential problems in these experiments include conformationally-induced differences in ionization efficiencies, inadvertent collision-induced dissociation, and ESI-induced clustering artifacts. A number of simple tests can be conducted to assess whether or not these factors are prevalent under the conditions used. In addition, the fidelity of the method can be scrutinized by performing measurements over a wide concentration range. Overall, this work supports the viability of the direct ESI-MS approach for determining binding affinities of protein–protein complexes in solution.

Conformational dynamics are thought to be a prerequisite for the catalytic activity of enzymes. However, the exact relationship between structural fluctuations and function is not well understood. In this work hydrogen/deuterium exchange (HDX) and electrospray ionization mass spectrometry (ESI-MS) are used for exploring the conformational dynamics of thermolysin. Amide HDX reflects the internal mobility of proteins; regions that undergo frequent unfolding-refolding show faster exchange than segments that are highly stable. Thermolysin is a zinc protease with an active site that is located between two lobes. Substrate turnover is associated with hinge bending that leads to a closed conformation. Product release regenerates the open form, such that steady-state catalysis involves a continuous closing/opening cycle. HDX/ESI-MS with proteolytic peptide mapping in the absence of substrate shows that elements in the periphery of the two lobes are most mobile. A comparison with previous X-ray data suggests that these peripheral regions undergo quite pronounced structural changes during the catalytic cycle. In contrast, active site residues exhibit only a moderate degree of backbone flexibility, and the central zinc appears to be in a fairly rigid environment. The presence of both rigid and moderately flexible elements in the active site may reflect a carefully tuned balance that is required for function. Interestingly, the HDX behavior of catalytically active thermolysin is indistinguishable from that of the free enzyme. This result is consistent with the view that catalytically relevant motions preexist in the resting state and that enzyme function can only be performed within the limitations given by the intrinsic dynamics of the protein. The data presented in this work indicate the prevalence of stochastic elements in the function of thermolysin, rather than supporting a deterministic mechanism.

Exposure of proteins to hydroxyl radicals induces the incorporation of oxygen atoms into solvent-exposed side chains. Earlier studies have employed this approach for mapping protein-protein interactions in mass spectrometry-based footprinting experiments. This work explores whether the overall level of gamma-ray mediated oxidative labeling can be used for monitoring large-scale conformational changes. According to a recently developed kinetic model (Tong, X.; Wren, J. C.; Konermann, L. Anal. Chem. 2007, 79, 6376-6382), the apparent first-order rate constant for oxidative labeling can be approximated as k(app) = k(RAD)/([P](tot) + C/k(u)), where k(RAD) is the primary rate of *OH formation, [P](tot) is the protein concentration, C reflects the presence of competing radical deactivation channels, and ku is the rate constant at which hydroxyl radicals react with the protein. The current study introduces conformational effects into this model by proposing that k(u) = [see text for formula] , where N is the number of amino acids, alphai is a measure for the solvent exposure of residue i, and k(ch)(i) is the oxidation rate constant that would apply for a completely solvent-exposed side chain. Using myoglobin and cytochrome c as model systems, it is demonstrated that unfolding by addition of H(3)PO(4) increases k(app) by up to 30% and 70%, respectively. Unfolding by other commonly used denaturants such as organic acids or urea results in dramatically lower oxidation levels than for the native state, a behavior that is due to the radical scavenging activity of these substances (corresponding to an increased value of C). Control experiments on model peptides are suitable for identifying such "secondary" effects, i.e., factors that modify oxidation levels without being related to conformational changes. In conclusion, the overall *OH labeling level represents a viable probe of large-scale protein conformational changes only under conditions where secondary effects are known to be minimal and where [P](tot) is constant.

This work employs electrospray mass spectrometry (ESI-MS) and UV-vis spectroscopy for monitoring the mechanism of acid-induced hemoglobin (Hb) denaturation. The protein for these experiments has been freshly prepared from bovine blood. All three Hb derivatives studied (oxyHb, metHb, and cyanometHb) respond to gradual changes from pH 6.8 to 2.1 in a manner that can be described by a stepwise sequential unfolding mechanism: (alphahbetah)2 --> 2 alphahbetah --> 2 alphahfolded + 2 betahfolded --> 2 alphaaunfolded + 2 betaaunfolded + 4 heme (superscripts "h" and "a" refer to holo- and apo-forms, respectively). The results obtained on these freshly prepared samples are significantly different from those of similar experiments previously conducted on metHb obtained commercially as lyophilized powder. Those earlier experiments suggested a highly asymmetric behavior of the two globin chains, involving a heme-deficient dimer (alphahbetaa) as a mechanistically important intermediate on the (dis)assembly pathway. Importantly, heme-deficient dimers are virtually undetectable for the freshly prepared Hb derivatives studied herein at any pH. This apparent discrepancy is attributed to the occurrence of oxidative modifications in the commercial protein. Liquid chromatography and tandem mass spectrometry reveal significant levels of sulfoxide formation for all four methionine residues in commercially obtained metHb. The extent of these modifications for freshly prepared protein is lower by at least a factor of 10. It is concluded that the acid-induced denaturation of Hb follows a highly symmetric mechanism. The occurrence of other mechanisms (possibly involving asymmetric elements) under different solvent conditions cannot be ruled out.

Electrospray ionization mass spectrometry (ESI-MS) is a commonly used tool for characterizing conformational changes of proteins in solution. Different conformations can be distinguished on the basis of their ESI charge state distributions. ESI-MS studies carried out under semidenaturing conditions result in bi- or multimodal distributions that reflect the presence of coexisting conformers. This study explores whether the concentration ratios of these species in solution are reflected in the measured ion intensities. Experiments on two model proteins, lysozyme and myoglobin, reveal that non-native polypeptide chains tend to result in a much stronger signal response than natively folded species. The measured ion intensity ratios can differ from the actual concentration ratios by as much as 2 orders of magnitude. It is proposed that the higher ionization efficiency of unfolded proteins is due to their partially hydrophobic character, which results in a larger surface activity and facilitates protein transfer into ion-producing progeny droplets. Conversely, natively folded proteins have a lower affinity for the air/liquid interface, such that ionization of these conformers is suppressed. The extent of ion suppression is strongly dependent on the experimental conditions such as flow rate and protein concentration, which determine if ESI occurs in a charge deficient or a charge surplus regime. These aspects should be taken into account for the design of ESI-MS-based protein folding experiments and for studies that use ion intensity ratios for the determination of protein-ligand binding affinities.

The native structure of hemoglobin (Hb) comprises two alpha- and two beta-subunits, each of which carries a heme group. There appear to be no previous studies that report the in vitro folding and assembly of Hb from highly unfolded alpha- and beta-globin in a "one-pot" reaction. One difficulty that has to be overcome for studies of this kind is the tendency of Hb to aggregate during refolding. This work demonstrates that denaturation of Hb in 40% acetonitrile at pH 10.0 is reversible. A dialysis-mediated solvent change to a purely aqueous environment of pH 8.0 results in Hb refolding without any apparent aggregation. Fluorescence, Soret absorption, circular dichroism, and ESI mass spectra of the protein recorded before unfolding and after refolding are almost identical. By employing an externally pressurized dialysis cell that is coupled on-line to an ESI mass spectrometer, changes in heme binding behavior, protein conformation, and quaternary structure can be monitored as a function of time. The process occurs in a stepwise sequential manner, leading from monomeric alpha- and beta-globin to heterodimeric species, which then assemble into tetramers. Overall, this mechanism is consistent with previous studies employing the mixing of folded alpha- and beta-globin. However, some unexpected features are observed, e.g., a heme-deficient beta-globin dimer that represents an off-pathway intermediate. Monomeric beta-globin is capable of binding heme before forming a complex with an alpha-subunit. This observation suggests that holo-alpha-apo-beta globin does not represent an obligatory intermediate during Hb assembly, as had been proposed previously. The on-line dialysis/ESI-MS approach developed for this work represents a widely applicable tool for studying the folding and self-assembly of noncovalent biological complexes.

Electrospray ionization (ESI)-mass spectrometry (MS) can provide information on protein-ligand interactions via detection of the corresponding complexes as gaseous ions. Unfortunately, some systems are prone to dissociation upon transfer into the gas phase. The reversible oxygen binding to hemoglobin (Hb) has been extensively studied in solution using a wide range of biophysical techniques. In addition to O2, ferrous (Fe(II)) Hb can bind CO. High affinity interactions with CN(-) are limited to the ferric (Fe(III)) state. In analogous fashion, CN(-), CO, and O2 bind to myoglobin (Mb). It remains unclear whether any of these ligand-bound forms can be observed by ESI-MS. In this work we demonstrate the successful detection of MbCN, while MbCO and MbO2 do not survive under ESI-MS conditions. Control experiments suggest that an older report of "MbO2" detection by ESI-MS may involve the misassignment of oxidation artifacts formed under corona discharge conditions. The situation is more favorable for ESI-MS studies on Hb. The most intense signal in the HbCN mass distribution corresponds to the expected complex with four cyanide moieties bound. Ligand loss during ESI-MS is around 20%. HbCO is detectable as well, albeit with a more noticeable level of ligand dissociation (∼50%) which produces the 2CO-bound state as the highest intensity ion in the spectrum. In addition, our data suggest that low levels of HbO2 can survive the transition into the gas phase, evident from +64 Da and +128 Da signals that can be assigned to Hb carrying two and four oxygen molecules, respectively. The application of collisional activation induces neutral ligand loss for all three Hb derivatives. It appears that this is the first report on the detection of MbCN, HbCO, and HbO2 in the gas phase. We hope that this work will pave the way towards future spectroscopic investigations of desolvated Mb and Hb, complementing the extensive literature on CN(-), CO, and O2 bound globins in the condensed phase.

Many protein structural investigations involve the use of H/D exchange (HDX) techniques. It is commonly thought that amide backbone protection arises from intramolecular H-bonding and/or burial of NH sites. Recently, fundamental HDX-related tenets have been called into question. The current work focuses on ubiquitin for exploring the defining features that distinguish amides in "open" (exchange-competent) and "closed" (exchange-incompetent) environments. Instead of relying on static X-ray structures, we employ all-atom molecular dynamics (MD) simulations for obtaining a dynamic view of the protein ground state and its surrounding solvent. The HDX properties for 57 out of 72 NH sites can be readily explained on the basis of backbone and side chain H-bonding, as well as solvent accessibility considerations. Unexpectedly, the same criteria fail for predicting the HDX characteristics of the remaining 15 amides. Significant protection is seen for numerous exposed NH sites that are not engaged in intramolecular H-bonds, whereas other amides that seemingly share the same features are unprotected. We scrutinize the proposal that H-bonding to crystallographically defined water can cause the protection of surface amides. For ubiquitin, the positioning of crystal water is not compatible with this idea. To further explore possible solvation effects, we tested for the presence of partially immobilized water networks. Our MD data reveal no difference in the solvation properties of protected vs unprotected surface amides, making it unlikely that restricted water dynamics can cause anomalous amide protection. The findings reported here suggest that efforts to deduce protein structural features on the basis of HDX protection factors may yield misleading results. This conclusion is relevant for initiatives that rely on sparse structural data as constraints for elucidating protein conformations. It may be necessary to pursue detailed quantum mechanical studies of the protein, the solvent, and the hydroxide catalyst for obtaining a comprehensive understanding of the factors that govern HDX rates. The considerable size of the systems involved makes such endeavors a daunting task.

Protein analyses by electrospray ionization (ESI) mass spectrometry can suffer from interferences caused by nonvolatile salts. The mechanistic basis of this effect remains to be fully investigated. In the current work we explore the behavior of proteins under native and denaturing conditions in the presence of NaCl, CsCl, and tetrabutyl ammonium chloride (NBu4Cl). All three salts interfere with the formation of "clean" [M + zH](z+) protein ions by progressively deteriorating spectral S/N ratios. We propose that salt interferences can be dissected into two independent aspects, i.e., (i) peak splitting by adduct formation and (ii) protein ion suppression. NaCl degrades the spectral quality by forming heterogeneous [M + zH + n(Na - H) + m(Cl + H)](z+) ions, while the integrated protein ion intensity remains surprisingly robust. Conversely, NBu4Cl does not cause any adduction, while dramatically reducing the protein ion yield. These findings demonstrate that adduct formation and protein ion suppression are indeed unrelated effects that may occur independently of one another. Other salts, such as CsCl, can give rise to a combination of the two scenarios. Molecular dynamics simulations of water droplets charged with either Na(+) or NBu4(+) provide insights into the mechanism underlying the observed effects. Na(+) containing droplets evolve relatively close to the Rayleigh limit (z/z(R) ≈ 0.74), whereas the z/z(R) values of NBu4(+) charged droplets are considerably lower (∼0.59). This difference is due to the high surface affinity of NBu4(+), which facilitates charge ejection from the droplet. We propose that the low z/z(R) values encountered in the presence of NBu4(+) suppress the Rayleigh fission of parent droplets in the ESI plume, thereby reducing the yield of progeny droplets that represent the precursors of gaseous protein ions. In addition, the rate of solvent evaporation is reduced in the presence of NBu4(+). Both of these factors lower the protein signal intensity. NaCl does not interfere with droplet fission, such that protein ions continue to form with high yield—albeit in heavily adducted form. Our findings expand on earlier proposals of charge competition as a key factor during the ESI process for salt-contaminated solutions.

This study reports the application of electrospray ionization (ESI) mass spectrometry (MS) with on-line rapid mixing for millisecond time-resolved studies of the refolding and assembly of a dimeric protein complex. Acid denaturation of S100A11 disrupts the native homodimeric protein structure. Circular dichroism and HSQC nuclear magnetic resonance measurements reveal that the monomeric subunits unfold to a moderate degree but retain a significant helicity and some tertiary structural elements. Following a rapid change in solution conditions to a slightly basic pH, the native protein reassembles with an effective rate constant of 6 s(-)(1). The ESI charge state distributions measured during the reaction suggest the presence of three kinetic species, namely, a relatively unfolded monomer (M(U)), a more tightly folded monomeric reaction intermediate (M(F)), and dimeric S100A11. These three forms exhibit distinct calcium binding properties, with very low metal loading levels for M(U), up to two calcium ions for M(F), and up to four for the dimer. Surprisingly, on-line pulsed hydrogen-deuterium exchange (HDX) reveals that each of the monomeric forms of the protein comprises two subspecies that can be distinguished on the basis of their isotope exchange levels. As the reaction proceeds, the more extensively labeled species are depleted. The exponential nature of the measured intensity-time profiles implies that the rate-determining step of the overall process is a unimolecular event. The kinetics are consistent with a sequential folding and assembly mechanism involving two increasingly nativelike monomeric intermediates en route to the native S100A11 dimer.

The possible involvement of "hidden" kinetic intermediates in the apparent two-state folding of some proteins is currently a matter of debate. This study uses time-resolved electrospray ionization (ESI) mass spectrometry with on-line pulsed hydrogen-deuterium exchange (HDX) for monitoring the refolding of acid/methanol-denatured ubiquitin. It is demonstrated that the ESI charge-state distribution (CSD) and the extent of HDX represent nonredundant probes of the protein structure in solution. When considered in isolation, the data provided by both of these probes are consistent with a two-state behavior, involving only denatured ubiquitin D and refolded protein F. However, a careful comparison of the CSD and HDX kinetics reveals the presence of an additional species, exhibiting a CSD like the folded protein but showing non-native HDX characteristics. This kinetic intermediate, D*, is in rapid equilibrium with D, such that the overall reaction is consistent with the mechanism D--D* --F. The results of this work suggest that the occurrence of transient intermediates may be more widespread than commonly thought, especially in cases where a cursory analysis indicates two-state behavior.

The application of a novel method for the identification of low-molecular-weight noncovalent ligands to a macromolecular target is reported. This technique is based on the measurement of analyte diffusion coefficients by electrospray mass spectrometry (ESI-MS) (Clark et al., Rapid Commun. Mass Spectrom. 2002, 16, 1454-1462). Potential ligands have large diffusion coefficients as long as they are free in solution. Binding to a macromolecular target, however, drastically reduces the diffusional mobility of any ligand species. Mixtures containing six different saccharides [ribose, rhamnose, glucose, maltose, maltotriose, and N,N',N''-triacetylchitotriose (NAG(3))] were screened for noncovalent binding to lysozyme. Of these six compounds, only NAG(3) is known to bind to the protein. In "direct" binding tests, NAG(3) shows a significantly reduced diffusion coefficient in the presence of the protein. No changes were observed for any of the other saccharides. In a second set of experiments, the use of a "competition" screening method was explored in which mixtures of candidate saccharides were tested for their ability to displace a reference ligand from the target. The addition of NAG(3)-containing mixtures significantly increased the diffusion coefficient of the reference ligand NAG(4) (N,N',N'',N'''-tetraacetylchitotetrose), whereas mixtures that did not contain NAG(3) had no effect. These data clearly indicate the potential of ESI-MS-based diffusion measurements as a novel tool to screen compound libraries for binding to proteins and other macromolecular targets. In contrast to conventional ESI-MS-based ligand-receptor binding studies, this method does not rely on the preservation of noncovalent interactions in the gas phase.

The "protein-folding problem" refers to the question of how and why a denatured polypeptide chain can spontaneously fold into a compact and highly ordered conformation. The classical description of this process in terms of reaction pathways has been complemented by models that describe folding as a biased conformational diffusion on a multidimensional energy landscape. The identification and characterization of short-lived intermediates provide important insights into the mechanism of folding. Pulsed hydrogen/deuterium exchange (HDX) methods are among the most powerful tools for studying the properties of kinetic intermediates. Analysis of pulse-labeled proteins by mass spectrometry (MS) provides information that is complementary to that obtained in nuclear magnetic resonance (NMR) studies; NMR data represent an average of entire protein ensembles, whereas MS can detect co-existing protein species. MS-based pulse-labeling experiments can distinguish between folding scenarios that involve parallel pathways, and those where folding is channeled through obligatory intermediates. The proteolytic digestion/MS technique provides spatially resolved information on the HDX pattern of folding intermediates. This method is especially important for proteins that are too large to be studied by NMR. Although traditional pulsed HDX protocols are based on quench-flow techniques, it is also possible to use electrospray (ESI) MS to analyze the reaction mixture on-line and "quasi-instantaneously" after labeling. This approach allows short-lived protein conformations to be studied by their HDX level, their ESI charge-state distribution, and their ligand-binding state. Covalent labeling of free cysteinyl residues provides an alternative approach to pulsed HDX experiments. Another promising development is the use of synchrotron X-rays to induce oxidation at specific sites within a protein for studying their solvent accessibility during folding.

A novel technique for studying protein folding kinetics is presented. It is based on a continuous-flow setup that is coupled to an electrospray (ESI) mass spectrometer and allows initiation of a folding reaction, followed by isotopic pulse labeling. The protein is electrosprayed "quasi-instantaneously" after exposure to the deuterated solvent. This approach yields structural information from the ESI charge state distribution and from the H/D exchange levels of individual protein states, while at the same time noncovalent interactions can be monitored. This technique is used to study the reconstitution of holomyoglobin (hMb) from unfolded apomyoglobin (aMb) and free heme. MS/MS is used to establish that a short-lived folding intermediate with two heme groups attached represents a protein-bound heme dimer. This state appears to have a compactness close to that of native hMb; however, isotopic labeling indicates a significantly perturbed structure. Another intermediate is bound to a single heme group and shows a charge state distribution similar to that of unfolded aMb. Exchange levels exhibited by this state are lower than for unfolded aMb, indicating that fewer hydrogens are exposed to the solvent and/or that more of them are involved in hydrogen bonding. Native hMb leads to the formation of low charge state ions (hMb(9+), hMb(8+)) and shows low exchange levels. However, early during reconstitution, a slightly unfolded form of the heme-protein complex contributes to the observed hMb(9+) ions. A peak width analysis reveals that the structural heterogeneity of some of the observed protein species decreases as reconstitution proceeds.

Hydrogen/deuterium exchange (HDX) methods are widely used for monitoring protein-ligand interactions. This approach relies on the fact that ligand binding can modulate the extent of protein structural fluctuations that transiently disrupt hydrogen bonds and expose backbone amides to the solvent. It is commonly observed that ligand binding causes a reduction of HDX rates. This reduction can be restricted to elements adjacent to the binding site, but other regions can be affected as well. Qualitatively, ligand-induced HDX protection can be rationalized on the basis of two-state models that equate structural dynamics with global unfolding/refolding. Unfortunately, such models tend to be unrealistic because the dynamics of native proteins are dominated by subglobal transitions and local fluctuations. Ligand binding lowers the ground-state free energy. It is not obvious why this should necessarily be accompanied by a depletion of excited-state occupancies, which would be required for a reduction of HDX rates. Here, we propose a framework that implies that ligand binding can either slow or accelerate amide deuteration throughout the protein. These scenarios are referred to as "type 1" and "type 2", respectively. Evidence for type 1 binding is abundant in the literature, whereas the viability of type 2 interactions is less clear. Using HDX mass spectrometry (MS), we demonstrate that the oxygenation of hemoglobin (Hb) provides a dramatic example of a type 2 scenario. The observed behavior is consistent with cooperative T → R switching, where part of the intrinsic O2 binding energy is reinvested for destabilization of the ground state. This destabilization increases the Boltzmann occupancy of unfolded conformers, thereby enhancing HDX rates. Surprisingly, O2 binding to myoglobin (Mb) also induces elevated HDX rates. These Mb data reveal that type 2 behavior is not limited to cooperative multisubunit systems. Although enhanced protection from deuteration is widely considered to be a hallmark of protein-ligand interactions, this work establishes that an overall deuteration increase also represents a viable outcome. HDX-based ligand screening assays, therefore, have to allow for canonical as well as noncanonical effects.

The operation of an electrospray ion source in the positive ion mode involves charge-balancing oxidation reactions at the liquid/metal interface of the sprayer capillary. One of these reactions is the electrolytic oxidation of water. The protons generated in this process acidify the analyte solution within the electrospray capillary. This work explores the effects of this acidification on the electrospray ionization (ESI) mass spectrum of the protein cytochrome c (cyt c). In aqueous solution containing 40% propanol, cyt c unfolds around pH 5.6. Mass spectra recorded under these conditions, using a simple ESI series circuit, display a bimodal charge-state distribution that reflects an equilibrium mixture of folded and unfolded protein in solution. These spectra are not strongly affected by electrochemical acidification. An "external loop" is added to the ESI circuit when the metal needle of the sample injection syringe is connected to ground. The resulting circuit represents two coupled electrolytic cells that share the ESI capillary as a common anode. Under these conditions, the rate of charge-balancing oxidation reactions is dramatically increased because the ion source has to supply electrons for both, the external circuit and the ESI circuit. The analytical implications of this effect are briefly discussed. Mass spectra of cyt c recorded with the syringe needle grounded are shifted to higher charge states, indicating that electrochemical acidification has caused the protein to unfold in the ion source. The acidification can be suppressed by increasing the flow rate and lowering the electrolyte concentration of the solution and by using an electrolyte that acts as redox buffer. The observed acidification is similar for sprayer capillaries made of platinum and stainless steel. Removal of the protective oxide layer on the stainless steel surface results in effective redox buffering for a few minutes.

The acid-induced denaturation of ferricytochrome c (cyt c) was examined in aqueous solutions containing different concentrations of methanol by electrospray ionization mass spectrometry (ESI MS) and optical spectroscopy. Circular dichroism, fluorescence, and absorption spectroscopy show that at a low concentration of methanol (3%) a decrease in pH induces a cooperative unfolding transition at around pH 2.6 that is accompanied by a breakdown of the native secondary and tertiary structure of the protein. In 50% methanol the breakdown of the tertiary structure occurs at around pH 4.0, whereas the alpha-helical content remains largely intact over the whole pH range studied. In ESI MS different protein conformations in solution are monitored by the different charge state distributions they generate during ESI. The ESI mass spectra recorded at near-neutral pH for both methanol concentrations are very similar and show a maximum at (cyt c + 8H+)8+. Despite the different conformations of the protein in solution, the acid-denatured states for the two methanol concentrations also show very similar mass spectra with a maximum at (cyt c + 17H+)17+. This indicates that the charge state distribution generated during ESI is not sensitive to the differences in the secondary structure of the denatured protein. The observed transition from low to high charge states is due to the breakdown of the tertiary structure in both cases. These findings suggest that ESI MS might be a general method to selectively monitor changes in the tertiary structure of proteins.

A new method for studying the folding kinetics of proteins is described. The method combines a continuous flow mixing technique with an electrospray mass spectrometer. Different protein conformations in solution are detected by the different charge states they produce during electrospray ionization. Unfolded proteins generally have more accessible protonation sites and give higher charge states than native proteins. The method is applied to study the refolding of acid-denatured cytochrome c. Global data analysis is used to obtain the exponential lifetimes which are associated with the refolding process. The kinetics can be described by two lifetimes of 0.17 +/- 0.02 and 8.1 +/- 0.9 s which are in accordance with the results of stopped flow experiments previously described in the literature. These lifetimes are associated with roughly 90 and 10% of the total intensity changes in the mass spectrum, respectively, and most likely reflect fast and slow refolding subpopulations of cytochrome c in solution.

Collision-induced dissociation (CID) of electrosprayed protein complexes usually involves asymmetric charge partitioning, where a single unfolded chain gets ejected that carries a disproportionately large fraction of charge. Using hemoglobin (Hb) tetramers as model system, we confirm earlier reports that bound metal ions can stabilize protein complexes under CID conditions. We examine the mechanism underlying this effect. Nonvolatile salts cause extensive adduct formation. Significant stabilization was observed for Mg(2+) and Ca(2+), whereas K(+), Rb(+), and Cs(+) had no effect. Precursor ion selection was used to examine Hb subpopulations with well-defined metal binding levels. K(+), Rb(+), and Cs(+)-adducted tetramers eject monomers that carry roughly one-quarter of the metal ions that were bound to the precursor. This demonstrates that charge migration during CID is exclusively due to proton transfer, not metal ion transfer. Also, replacement of highly mobile charge carriers (protons) with less mobile species (metal ions) does not exert a stabilizing influence under the conditions used here. Interestingly, Hb carrying stabilizing ions (Mg(2+) and Ca(2+)) generates monomeric CID products that are metal depleted. This effect is attributed to a combination of two factors: (1) Me(2+) binding stabilizes Hb via formation of chelation bridges (e.g., R-COO(-) Me(2+) (-)OOC-R); the more Me(2+) a subunit contains the more stable it is. (2) More than ~90% of the tetramers contain at least one subunit with a below-average number of Me(2+). The prevalence of monomeric CID products with depleted Me(2+) levels is caused by the tendency of these low metal-containing subunits to undergo preferential unfolding/ejection.

The coupling of electrospray ionization (ESI) with ion mobility-mass spectrometry (IM-MS) allows structural studies on biological macromolecules in a solvent-free environment. Collision cross sections (CCSs) measured by IM-MS provide a measure of analyte size. For native proteins and their complexes, many structural features can be preserved in the gas phase, making IM-MS a powerful approach for a range of bioanalytical applications. In addition to tightly folded conformers, a large number of partially disordered proteins participate in biological processes and disease mechanisms. It remains unclear to what extent IM-MS is suitable for exploring structural properties of these semifolded species. The current work addresses this question, using myoglobin as model system. This protein follows a sequential unfolding pathway that comprises two partially disordered states, i.e., apo-myoglobin (aMb) at pH 7 and pH 4. IM-MS data acquired for these two conformers were compared to those of native holo-myoglobin (hMb) at pH 7 and extensively unfolded aMb at pH 2. When examining individual aMb charge states, the degree of gas phase unfolding is not strongly correlated with the corresponding solution behavior. A key problem is that non-native conformers generate high ESI charge states, resulting in conformational transitions caused by intramolecular electrostatic repulsion. It is possible to establish a link between solution phase and gas phase structure when normalizing CCS distributions according to their respective ESI-MS signal intensities. This approach yields CCS averages that follow the expected progression hMbpH 7aMbpH 7aMbpH 4aMbpH 2. However, this trend mainly reflects the protonation behavior of the conformers during the ESI process, rather than a genuine memory of solution structure. Overall, our data reveal that electrostatically driven expansion as well as collapse events can lead to disparities between gaseous and solution structures for partially unfolded proteins. IM-MS data on non-native conformers should therefore be interpreted with caution.

Kinetic measurements can provide insights into protein folding mechanisms. However, the initial (submillisecond) stages of folding still represent a formidable analytical challenge. A number of ultrarapid triggering techniques have been available for some time, but coupling of these techniques with detection methods that are capable of providing detailed structural information has proven to be difficult. The current work addresses this issue by combining submillisecond mixing with laser-induced oxidative labeling. Apomyoglobin (aMb) serves as a model system for our measurements. Exposure of the protein to a brief pulse of hydroxyl radical (·OH) at different time points during folding introduces covalent modifications at solvent accessible side chains. The extent of labeling is monitored using mass spectrometry-based peptide mapping, providing spatially resolved measurements of changes in solvent accessibility. The submillisecond mixer used here improves the time resolution by a factor of 50 compared to earlier ·OH labeling experiments from our laboratory. Data obtained in this way indicate that early aMb folding events are driven by both local and sequence-remote docking of hydrophobic side chains. Assembly of a partially formed A(E)G(H) scaffold after 0.2 ms is followed by stepwise consolidation that ultimately yields the native state. Major conformational changes go to completion within 0.1 s. The technique introduced here is capable of providing in-depth structural information on very short time scales that have thus far been dominated by low resolution (global) spectroscopic probes. By employing submillisecond mixing in conjunction with slower mixing techniques, it is possible to observe complete folding pathways, from fractions of a millisecond all the way to minutes.

The oxygen binding properties of vertebrate hemoglobins (Hb) have been explored in great detail. In contrast, folding and assembly of these heterotetrameric protein complexes remain poorly understood. Similar to investigations of other multisubunit systems, in vitro Hb refolding experiments are often plagued by aggregation. Here we monitor the refolding of bovine Hb by electrospray mass spectrometry (ESI-MS). This technique allows the observation of coexisting subunit combinations, heme binding states, and protein conformers. Exposure to 40% acetonitrile at pH 10 causes Hb disassembly and extensive subunit unfolding. Hb reassembly is triggered by solvent exchange. Experiments conducted at room temperature provide a low metHb refolding yield. A significantly improved yield is achieved by lowering the temperature to 4 °C and by supplementing the protein solution with KCN prior to denaturation. Comparative studies under "low-yield" and "high-yield" conditions report on the interplay between folding and misfolding. The tendency of β-globin to undergo aggregation is found to be the key impediment to the formation of native Hb. The α/β imbalance generated in this way favors the formation of non-native α-globin assemblies. Our data imply that hemin dicyanide formed in the presence of KCN remains weakly bound to denatured β-globin, thereby counteracting aggregation, such that the refolding yield is enhanced. In the absence of aggregation-related interference, Hb assembly follows a symmetric pathway. Monomeric α- and β-globin adopt a compact conformation upon heme binding. Heme-bound monomers then form heterodimers, and ultimately heterodimer association results in native Hb. This work highlights the utility of time-resolved ESI-MS investigations for interrogating the kinetic competition between on-pathway events and aberrant side reactions during the self-assembly of biomolecular complexes.

Electrosprayed multi-protein complexes can be dissociated by collisional activation in the gas phase. Typically, these processes follow a mechanism whereby a single subunit gets ejected with a disproportionately high amount of charge relative to its mass. This asymmetric behavior suggests that the departing subunit undergoes some degree of unfolding prior to being separated from the residual complex. These structural changes occur concomitantly with charge (proton) transfer towards the subunit that is being unraveled. Charge accumulation takes place up to the point where the subunit loses physical contact with the residual complex. This work develops a simple electrostatic model for studying the relationship between conformational changes and charge enrichment during collisional activation. Folded subunits are described as spheres that carry continuum surface charge. The unfolded chain is envisioned as random coil bead string. Simulations are guided by the principle that the system will adopt the charge configuration with the lowest potential energy for any backbone conformation. A finite-difference gradient algorithm is used to determine the charge on each subunit throughout the dissociation process. Both dimeric and tetrameric protein complexes are investigated. The model reproduces the occurrence of asymmetric charge partitioning for dissociation events that are preceded by subunit unfolding. Quantitative comparisons of experimental MS/MS data with model predictions yield estimates of the structural changes that occur during collisional activation. Our findings suggest that subunit separation can occur over a wide range of scission point structures that correspond to different degrees of unfolding.

Considerable progress in deciphering the mechanisms of protein folding has been made. However, most work in this area has focused on single-chain systems, whereas the majority of proteins are oligomers. The spontaneous assembly of intact multi-subunit systems from disordered building blocks encompasses the formation of intramolecular as well as intermolecular contacts. Both types of interaction affect the solvent accessibility of individual protein segments. This work employs pulsed hydroxyl radical (·OH) labeling for tracking time-dependent accessibility changes during folding and assembly of the S100A11 homodimer. ·OH induces covalent modifications at exposed residues. Structural snapshots are obtained by combining ·OH labeling with rapid mixing and mass spectrometry. The free subunits are found to possess a partially non-native hydrophobic core that prevents subunit association during the initial stages of the reaction. Instead, the protein forms an early (10 ms) monomeric intermediate that exhibits reduced solvent accessibility in regions distant from helices I and IV, which constitute the dimerization interface. Subunit association is complete after 800 ms, although the protein retains significant disorder in helices II and III at this point. Subsequent consolidation of these elements leads to the native state. The experimental strategy used here could become a general tool for deciphering kinetic mechanisms of biomolecular self-assembly processes.

This work combines pulsed hydrogen/deuterium exchange (HDX) and top-down mass spectrometry for the structural characterization of short-lived protein folding intermediates. A custom-built flow device with three sequential mixing steps is used for (i) triggering protein folding, (ii) pulsed D(2)O labeling, and (iii) acid quenching. The earliest folding time point that can be studied with this system is 10 ms. The mixing device was coupled online to the electrospray source of a Fourier transform mass spectrometer, where intact protein ions are fragmented by electron capture dissociation (ECD). The viability of this experimental strategy is demonstrated by applying it to the refolding of horse apo-myoglobin (aMb), a reaction known to involve a transient intermediate. Cooling of the mixing device to 0 °C reduces the reaction rate such that the folding process occurs within the experimentally accessible time window. Top-down ECD provides an average spatial resolution of ca. 2 residues, surpassing the resolution typically achieved in traditional proteolytic digestion/HDX studies. Amide back exchange is virtually eliminated by the short (∼1 s) duration of the acid quenching step. The aMb folding intermediate exhibits HDX protection in helices G and H, whereas the remainder of the protein is largely unfolded. Marginal protection is seen for helix A. Overall, the top-down ECD approach used here offers insights into the sequence of events leading from the unfolded state to the native conformation, with envisioned future applications in the areas of protein misfolding and aggregation. The time-resolved experiments reported herein represent an extension of our previous work, where HDX/MS with top-down ECD was employed for monitoring "static" protein structures under equilibrium conditions (Pan et al. J. Am. Chem. Soc. 2009, 131, 12801).

The S100 proteins comprise a group of EF-hand proteins that undergo a calcium-induced conformational change allowing them to interact with other proteins and produce a biological response. A unique feature of these proteins is the fact that they can form both homo- and heterodimers independent of calcium binding. The reported dissociation constants for several S100 proteins span a very large range, from 1-4 microM to

The structural characterization of short-lived intermediates provides insights into the mechanisms of protein folding and unfolding. Using holo-myoglobin as a model system, this work reports the application of oxidative pulse labeling for experiments of this kind. Protein unfolding is triggered by a pH jump from 6.5 to 3.2 in 150 mM NaCl. Subsequent (.-)OH exposure at various time points using laser photolysis of H2O2 leads to covalent modifications of solvent-exposed side chains within approximately 1 mus (Hambly, D. M.; Gross, M. L. J. Am. Soc. Mass Spectrom. 2005, 16, 2057-2063). Most of these modifications appear as 16 Da adducts in the mass spectrum of the intact protein. The overall extent of labeling increases with time, reflecting the exposure of reactive side chains that had previously been buried. Unfolding and disruption of heme-protein interactions go to completion within approximately 10 s. Spatially resolved information is obtained by monitoring the signal intensities of unmodified tryptic peptides. After 50 ms, many regions have lost most of their protection, whereas structure is retained in the B, E, F, and G helices. The BEF core remains partially folded, even after 500 ms, at which point helix G is fully unprotected. The observation of an "early" (BEFG) and a "late" (BEF) intermediate is in accord with optical stopped-flow measurements. Formation of these transient species is attributed to the persistence of heme-protein interactions during the early stages of the reaction. Overall, this work demonstrates the feasibility of laser-induced oxidative labeling as a tool for characterizing the structure of short-lived protein conformers. The combination of this approach with ultrarapid mixing or photochemical triggering should allow folding experiments in the submillisecond range.

This work uses electrospray ionization mass spectrometry (ESI-MS) in conjunction with hydrogen/deuterium exchange (HDX) and optical spectroscopy for characterizing the solution-phase properties of cytochrome c (cyt c) after heat exposure. Previous work demonstrated that heating results in irreversible denaturation for a subpopulation of proteins in the sample. However, that study did not investigate the physical reasons underlying this interesting effect. Here we report that the formation of oxidative modifications at elevated temperature plays a key role for the observed behavior. Tryptic digestion followed by tandem mass spectrometry is used to identify individual oxidation sites. Trp59 and Met80 are among the modified amino acids. In native cyt c both of these residues are buried deep within the protein structure, such that covalent modifications would be expected to be particularly disruptive. ESI-MS analysis after heat exposure results in a bimodal charge-state distribution. Oxidized protein appears predominantly in charge states around 11+, whereas a considerably lower degree of oxidation is observed for the 7+ and 8+ peaks. This finding confirms that different oxidation levels are associated with different solution-phase conformations. HDX measurements for different charge states are complicated by peak distortions arising from oxygen adduction. Nonetheless, comparison with simulated peak shapes clearly shows that the HDX properties are different for high- and low-charge states, confirming that interconversion between unfolded and folded conformers is blocked in solution. In addition to oxidation, partial aggregation upon heat exposure likely contributes to the formation of irreversibly denatured protein.

Mass spectrometry (MS) plays a central role in studies on protein structure and dynamics. This review highlights some of the recent developments in this area, with focus on applications involving the use of electrospray ionization (ESI) MS. Although this technique involves the transformation of analytes into highly nonphysiological species (desolvated gas-phase ions in the vacuum), ESI-MS can provide detailed insights into the solution-phase behavior of proteins. Notably, the ionization process itself occurs in a structurally sensitive manner. An increased degree of solution-phase unfolding is correlated with a higher level of protonation. Also, ESI allows the transfer of intact noncovalent complexes into the gas phase, thereby yielding information on binding partners, stoichiometries, and even affinities. A particular focus of this article is the use of hydrogen/deuterium exchange (HDX) methods and hydroxyl radical (.OH) labeling for monitoring dynamic and structural aspect of solution-phase proteins. Conceptual similarities and differences between the two methods are discussed. We describe a simple method for the computational simulation of protein HDX patterns, a tool that can be helpful for the interpretation of isotope exchange data recorded under mixed EX1/EX2 conditions. Important aspects of .OH labeling include a striking dependence on protein concentration, and the tendency of commonly used solvent additives to act as highly effective radical scavengers. If not properly controlled, both of these factors may lead to experimental artifacts.

There is an ongoing debate whether collision-induced dissociation (CID) of electrosprayed proteins after solution-phase hydrogen/deuterium exchange (HDX) is a viable approach for determining spatially resolved deuteration patterns. This work explores the use of two methods, source-CID and hexapole tandem mass spectrometry (MS/MS) on a quadrupole time-of-flight (Q-TOF) mass spectrometer, for measuring the fragment deuteration levels of regioselectively labeled ubiquitin. Both methods reveal that b-ions exhibit HDX levels significantly below that of the intact protein, whereas several y'' fragments are labeled to a much greater extent. These results are consistent with earlier source-CID data (Akashi, S.; Naito, Y.; Takio, K. Anal. Chem. 1999, 71, 4974-4980). However, the measured b-ion deuteration levels are in disagreement with the known solution-phase behavior of ubiquitin. Partial agreement is observed for y''-ions. Control experiments on homogeneously labeled ubiquitin (having the same average deuteration level at every exchangeable site) result in highly nonuniform fragment HDX levels. In particular, b-ions exhibit deuteration levels significantly below that of intact ubiquitin, thereby mimicking the behavior seen for the regioselectively labeled protein. This effect is likely caused by isotope fractionation during collisional activation, facilitated by the high mobility of charge carriers (scrambling) in the gas phase. The observation that the b-ion labeling behavior is largely independent of the spatial isotope distribution within solution-phase ubiquitin invalidates these ions as reporters of the protein deuteration pattern. This work questions the common practice of interpreting any nonuniformities in fragment deuteration as being indicative of regioselective solution-phase labeling. Artifactual deuterium enrichment or depletion during collisional activation may have contributed to the current lack of consensus as to whether HDX/CID represents a potentially viable tool for measuring solution-phase deuteration patterns.

This work reports the use of electrospray mass spectrometry for studying the conformational dynamics of enzymes by amide hydrogen/deuterium exchange (HDX) measurements. A rapid-mixing quench-flow approach allows comparisons to be made between the HDX kinetics of free enzymes with those under steady-state conditions. Experiments carried out on carboxypeptidase B in the absence of substrate and in the presence of saturating concentrations of hippuryl-Arg result in HDX kinetics that are indistinguishable. This finding implies that the conformational dynamics that mediate HDX are not significantly different in the resting state of the enzyme and during substrate turnover.

One of the greatest current challenges in proteomics is to develop an understanding of cellular communication and regulation processes, most of which involve noncovalent interactions of proteins with various binding partners. Mass spectrometry plays an important role in all aspects of these research efforts. This article provides a survey of mass spectrometry-based approaches for exploring protein-ligand interactions. A wide array of techniques is available, and the choice of method depends on the specific problem at hand. For example, the high-throughput screening of compound libraries for binding to a specific receptor requires different approaches than structural studies on multiprotein complexes. This review is directed to readers wishing to obtain a concise yet comprehensive overview of existing experimental techniques. Specific emphasis is placed on emerging methods that have been developed within the last few years.

The inducible nitric oxide synthase core oxygen domain (iNOS(COD)) is a homodimeric protein complex of ca. 100 kDa. In this work, the subunit disassembly and unfolding of the protein following a pH jump from 7.5 to 2.8 were monitored by on-line rapid mixing in conjunction with electrospray (ESI) time-of-flight mass spectrometry. Various protein species become populated during the denaturation process. These can be distinguished by their ligand binding behavior, and by the different charge states that they produce during ESI. Detailed intensity-time profiles were obtained for all of these species, and the kinetics were subjected to a global analysis which allows a model of the denaturation process to be developed. The data are described well by three relaxation times (tau(1) = 0.36 s, tau(2) = 0.62 s, and tau(3) = 3.3 s), each of which has a characteristic amplitude spectrum. The initial step of the reaction is the disruption of the iNOS(COD) dimer, to generate heme-bound monomeric species in various degrees of unfolding. This first step is accompanied by the loss of two tetrahydrobiopterin cofactors. Subsequent heme loss generates monomeric apoproteins exhibiting various degrees of unfolding. In addition, the formation of proteins that are bound to two heme groups is observed. A subpopulation of holo monomers undergoes substantial unfolding while retaining contact with the heme cofactor. Together with previous studies, the results of this work suggest that the occurrence of complex reaction mechanisms involving several short-lived intermediates is a common feature for the denaturation of large multiprotein complexes.