Hydrogen/Deuterium Exchange Mass Spectrometry of Actin in Various Biochemical Contexts

Chik, John K.; Schriemer, David C.

doi:10.1016/j.jmb.2003.09.044

Cited by 27 publications

(30 citation statements)

References 54 publications

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“…The G to F transition of muscle actin was previously studied by HD exchange and mass spectrometry by Chik et al (16). Results of that study are in general agreement with our results.…”

Section: Discussionsupporting

confidence: 93%

“…The extent of these movements will directly translate into changes in filament dynamics and flexibility. We observed increased exchange of peptide 326 -340 in the filament compared with the monomer in yeast actin, whereas no such difference was observed with muscle actin in a previous study by Chik et al (16). Since, as discussed earlier, this peptide is in the pivot point region, this difference is consistent with greater twisting and perhaps greater flexibility of the monomer within the filament, in line with the apparently more dynamic behavior of yeast F-actin.…”

Section: Discussionsupporting

confidence: 84%

“…Since changes in conformation among different actin states will affect deuterium uptake, HD exchange coupled with mass spectrometry should provide us with a tool to study the states of actin present in solution. HD exchange detected with mass spectrometry was previously used successfully by Chik and co-workers (16) to assess changes in muscle actin structure due to the G-to F-actin transition and the binding of phallodin to F-actin and of DNase I to G-actin. More recently, the technique has been used to assess conformational changes in the Arp2/3 complex, which contains actin like subunits (17).…”

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confidence: 99%

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Actin Isoform-specific Conformational Differences Observed with Hydrogen/Deuterium Exchange and Mass Spectrometry

Stokasimov

Rubenstein

2009

Journal of Biological Chemistry

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Actin can exist in multiple conformations necessary for normal function. Actin isoforms, although highly conserved in sequence, exhibit different biochemical properties and cellular roles. We used amide proton hydrogen/deuterium (HD) exchange detected by mass spectrometry to analyze conformational differences between Saccharomyces cerevisiae and muscle actins in the G and F forms to gain insight into these differences. We also utilized HD exchange to study interdomain and allosteric communication in yeast-muscle hybrid actins to better understand the conformational dynamics of actin. Areas showing differences in HD exchange between G-and F-actins are areas of intermonomer contacts, consistent with the current filament models. Our results showed greater exchange for yeast G-actin compared with muscle actin in the barbed end pivot region and areas in subdomains 1 and 2 and for F-actin in monomer-monomer contact areas. These results suggest greater flexibility of the yeast actin monomer and filament compared with muscle actin. For hybrid G-actins, the muscle-like and yeastlike parts of the molecule generally showed exchange characteristics resembling their parent actins. A few exceptions were a peptide on top of subdomain 2 and the pivot region between subdomains 1 and 3 with muscle actin-like exchange characteristics although the areas were yeastlike. These results demonstrate that there is cross-talk between subdomains 1 and 2 and the large and small domains. Hybrid F-actin data showing greater exchange compared with both yeast and muscle actins are consistent with mismatched yeast-muscle interfaces resulting in decreased stability of the hybrid filament contacts.The ability of actin to engage in a wide range of physiological functions requires that it be subject to complex spatial and temporal control by a large array of actin-binding proteins (1-3). Such regulation demands that both the monomeric and filamentous forms of actin be able to exist in a number of different conformations. Some of these may be differentially recognized by different actin-binding proteins, and some might actually be induced by the interaction of actin with these proteins.Actin is highly conserved from yeast to humans. There is 87% sequence identity between yeast and muscle actin and 91% sequence identity between yeast and nonmuscle actins. Even with this high sequence similarity and minor differences in crystal structures, there are some major differences in yeast and muscle actin behavior. Yeast, compared with muscle actin, polymerizes faster and exchanges its bound nucleotide faster. In contrast to muscle actin, the yeast actin filament releases its P i almost immediately after ATP hydrolysis and the filament fragments more readily (4). Yeast cells cannot survive with muscle actin as the sole actin, and with ␤-nonmuscle actin as the only actin, yeast cells are very sick (5). A set of biochemical data indicates that the muscle filament is less flexible than yeast (6, 7). However, conformational and structural differences that could ex...

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“…The G to F transition of muscle actin was previously studied by HD exchange and mass spectrometry by Chik et al (16). Results of that study are in general agreement with our results.…”

Section: Discussionsupporting

confidence: 93%

Section: Discussionsupporting

confidence: 84%

mentioning

confidence: 99%

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Actin Isoform-specific Conformational Differences Observed with Hydrogen/Deuterium Exchange and Mass Spectrometry

Stokasimov

Rubenstein

2009

Journal of Biological Chemistry

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“…The ␣:␤ intensity imbalance can also be eliminated by a strongly elevated declustering potential in the ion sampling interface. In conclusion, important factors that have to be considered for the ESI-MS analysis of protein mixtures are (1) conformational effects, resulting in differential surface activities, and (2) properties [4][5][6][7], and their charge state distributions [8 -11]. Unfolded conformations generally result in higher ESI charge states than tightly folded structures, an effect that mirrors the lower compactness and the larger solventaccessible surface area of the unfolded state [12,13].…”

mentioning

confidence: 99%

“…Different polypeptide chains in solution can be distinguished based on their mass, their ligand-binding behavior [2,3], their hydrogen-deuterium exchange (HDX) properties [4][5][6][7], and their charge state distributions [8 -11]. Unfolded conformations generally result in higher ESI charge states than tightly folded structures, an effect that mirrors the lower compactness and the larger solventaccessible surface area of the unfolded state [12,13].…”

mentioning

confidence: 99%

Analysis of protein mixtures by electrospray mass spectrometry: Effects of conformation and desolvation behavior on the signal intensities of hemoglobin subunits

Kuprowski

Boys

Konermann

2007

J. Am. Soc. Mass Spectrom.

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The determination of solution-phase protein concentration ratios based on ESI-MS intensity ratios is not always straightforward. For example, equimolar mixtures of hemoglobin ␣-and ␤-subunits consistently result in much higher peak intensities for the ␣-chain. The current work explores the origin of this effect. Under mildly acidic conditions (pH 3.4) ␣-globin is extensively unfolded, whereas ␤-globin retains residual structure. Because of its greater nonpolar character, the more unfolded ␣-subunit can more effectively compete for charge. This leads to suppression of ␤-globin signals under conditions where the protein ion yield is limited by the charge concentration on the initially formed ESI droplets. More balanced intensities are observed when operating under charge excess conditions and/or in a solvent environment where both proteins are unfolded to a similar degree (pH 2.2). However, even in these cases the overall ␣-globin peak intensity is still twice as high as that of the ␤-subunit. The persistent imbalance under these conditions originates from the different declustering behaviors of the two proteins. A considerable fraction of ␤-globin undergoes incomplete desolvation during ESI, thereby reducing the intensity of bare [␤ ϩ zH] zϩ ions. When including the contributions of incompletely desolvated species, the overall ␣:␤ ion intensity ratio is close to unity. The ␣:␤ intensity imbalance can also be eliminated by a strongly elevated declustering potential in the ion sampling interface. In conclusion, important factors that have to be considered for the ESI-MS analysis of protein mixtures are (1) conformational effects, resulting in differential surface activities, and (2) properties [4][5][6][7], and their charge state distributions [8 -11]. Unfolded conformations generally result in higher ESI charge states than tightly folded structures, an effect that mirrors the lower compactness and the larger solventaccessible surface area of the unfolded state [12,13]. The combination of these features results in an unsurpassed selectivity that greatly facilitates the detection of coexisting species. One problem, however, that can complicate the analysis of ESI-MS data is that the measured ion intensities do not necessarily reflect the relative concentrations of the corresponding proteins in solution [14]. The apparent ionization efficiencies of different biomacromolecules can vary by several orders of magnitude [15]. The situation is further complicated by ion-suppression effects that may occur in protein mixtures and in the presence of other solutes [16 -19]. An improved understanding of the relationship between ESI-MS signal response and solution-phase concentration would be beneficial for a wide range of applications.The upper limit of the ionization efficiency in ESI-MS is determined by the molar concentration of excess charge, C q , on the initially formed electrospray droplets [19,20]. C q can be estimated based on the relationshipwhere K is the conductivity of the solution, ␥ is the surface tension of t...

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Protein structure and dynamics studied by mass spectrometry: H/D exchange, hydroxyl radical labeling, and related approaches

2008

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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.

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Hydrogen/Deuterium Exchange Mass Spectrometry of Actin in Various Biochemical Contexts

Cited by 27 publications

References 54 publications

Actin Isoform-specific Conformational Differences Observed with Hydrogen/Deuterium Exchange and Mass Spectrometry

Actin Isoform-specific Conformational Differences Observed with Hydrogen/Deuterium Exchange and Mass Spectrometry

Analysis of protein mixtures by electrospray mass spectrometry: Effects of conformation and desolvation behavior on the signal intensities of hemoglobin subunits

Protein structure and dynamics studied by mass spectrometry: H/D exchange, hydroxyl radical labeling, and related approaches

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