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1.  Activation Loop Dynamics Determine the Different Catalytic Efficiencies of B Cell- and T Cell-Specific Tec Kinases 
Science signaling  2013;6(290):ra76.
Itk and Btk are nonreceptor tyrosine kinases of the Tec family that signal downstream of the T cell receptor (TCR) and B cell receptor (BCR), respectively. Despite their high sequence similarity and related signaling roles, Btk is a substantially more active kinase than Itk. We showed that substitution of six of the 619 amino acid residues of Itk with those of Btk was sufficient to completely switch the activities of Itk and Btk. The substitutions responsible for the swap in activity are all localized to the activation segment of the kinase domain. Nuclear magnetic resonance and hydrogen-deuterium exchange mass spectrometry analyses revealed that Itk and Btk had distinct protein dynamics in this region, which could explain the observed differences in catalytic efficiency between these kinases. Introducing Itk with enhanced activity into T cells led to enhanced and prolonged TCR signaling compared to that in cells with wild-type Itk. These findings imply that evolutionary pressures have led to Tec kinases having distinct enzymatic properties depending on the cellular context. We suggest that the weaker catalytic activities observed for T cell–specific kinases is one mechanism to regulate cellular activation and prevent aberrant immune responses.
PMCID: PMC4089953  PMID: 23982207
2.  Accessing the Reproducibility and Specificity of Pepsin and other Aspartic Proteases 
Biochimica et biophysica acta  2012;1834(6):1222-1229.
The aspartic protease pepsin is less specific than other endoproteinases. Because aspartic proteases like pepsin are active at low pH, they are utilized in hydrogen deuterium exchange mass spectrometry (HDX MS) experiments for digestion under hydrogen exchange quench conditions. We investigated the reproducibility, both qualitatively and quantitatively, of online and offline pepsin digestion to understand the compliment of reproducible pepsin fragments that can be expected during a typical pepsin digestion. The collection of reproducible peptides was identified from >30 replicate digestions of the same protein and it was found that the number of reproducible peptides produced during pepsin digestion becomes constant above 5-6 replicate digestions. We also investigated a new aspartic protease from the stomach of the rice field eel (Monopterus albus Zuiew) and compared digestion efficiency and specificity to porcine pepsin and aspergillopepsin. Unique cleavage specificity was found for rice field eel pepsin at arginine, asparagine, and glycine. Different peptides produced by the various proteases can enhance protein sequence coverage and improve the spatial resolution of HDX MS data.
PMCID: PMC3553288  PMID: 23063535
Mass spectrometry; aspergillopepsin; factor XIII; hydrogen exchange; rice field eel; Monopterus albus Zuiew; online digestion
3.  Partial cooperative unfolding in proteins as observed by hydrogen exchange mass spectrometry 
International reviews in physical chemistry  2012;32(1):10.1080/0144235X.2012.751175.
Many proteins do not exist in a single rigid conformation. Protein motions, or dynamics, exist and in many cases are important for protein function. The analysis of protein dynamics relies on biophysical techniques that can distinguish simultaneously existing populations of molecules and their rates of interconversion. Hydrogen exchange (HX) detected by mass spectrometry (MS) is contributing to our understanding of protein motions by revealing unfolding and dynamics on a wide timescale, ranging from seconds to hours to days. In this review we discuss HX MS-based analyses of protein dynamics, using our studies of multi-domain kinases as examples. Using HX MS, we have successfully probed protein dynamics and unfolding in the isolated SH3, SH2 and kinase domains of the c-Src and Abl kinase families, as well as the role of inter- and intra-molecular interactions in the global control of kinase function. Coupled with high-resolution structural information, HX MS has proved to be a powerful and versatile tool for the analysis of the conformational dynamics in these kinase systems, and has provided fresh insight regarding the regulatory control of these important signaling proteins. HX MS studies of dynamics are applicable not only to the proteins we illustrate here, but to a very wide range of proteins and protein systems, and should play a role in both classification of and greater understanding of the prevalence of protein motion.
PMCID: PMC3652491  PMID: 23682200
Src-family kinase; Hck; Lck; SH3 domain; SH2 domain; Abl; deuterium; HDX; protein dynamics, flexibility
4.  Conformational Analysis of Recombinant Monoclonal Antibodies with Hydrogen/Deuterium Exchange Mass Spectrometry 
Understanding the conformation of antibodies, especially those of therapeutic value, is of great interest. Many of the current analytical methods used to probe protein conformation face issues in the analysis of antibodies, either due to the nature of the antibody itself or the limitations of the method. One method that has recently been utilized for conformational analysis of antibodies is hydrogen/deuterium exchange mass spectrometry (H/DX MS). H/DX MS can be used to probe the conformation and dynamics of proteins in solution, requires small sample quantities, is compatible with many buffer systems, and provides peptide-level resolution. The application of H/DX MS to immunoglobulin gamma 1 (IgG1) recombinant monoclonal antibodies can provide information about IgG1 conformation, dynamics, and changes to conformation as a result of protein modification(s), changes in storage conditions, purification procedures, formulation, and many other parameters. In this article we provide a comprehensive HD/X MS protocol for the analysis of an antibody.
PMCID: PMC3713497  PMID: 23475726
immunoglobin; deuterium; conformation; protein structure; antibody; hydrogen exchange
5.  Considerations in the analysis of hydrogen exchange mass spectrometry data 
i. Summary
A major component of a hydrogen exchange mass spectrometry experiment is the analysis of protein and peptide mass spectra to yield information about deuterium incorporation. The processing of data that are produced includes the identification of each peptic peptide to create a master table/array of peptide sequence, retention time and retention time range, mass range and undeuterated mass. The amount of deuterium incorporated into each of the peptides in this array must then be determined. Various software platforms have been developed in order to perform this specific type of data analysis. We describe the fundamental parameters to be considered at each step along the way and how data processing, either by an individual or by software, must approach the analysis.
PMCID: PMC3713502  PMID: 23666730
Deuterium; software; algorithm; protein dynamics; isotope
6.  Sulfonyl Fluoride Inhibitors of Fatty Acid Amide Hydrolase 
Journal of medicinal chemistry  2012;55(22):10074-10089.
Sulfonyl fluorides are known to inhibit esterases. Early work from our laboratory has identified hexadecyl sulfonylfluoride (AM374) as a potent in vitro and in vivo inhibitor of fatty acid amide hydrolase (FAAH). We now report on later generation sulfonyl fluoride analogs that exhibit potent and selective inhibition of FAAH. Using recombinant rat and human FAAH we show that 5-(4-hydroxyphenyl)pentanesulfonyl fluoride (AM3506) has similar inhibitory activity for both the rat and the human enzyme, while rapid dilution assays and mass spectrometry analysis suggest that the compound is a covalent modifier for FAAH and inhibits its action in an irreversible manner. Our SAR results are highlighted by molecular docking of key analogs.
PMCID: PMC3678964  PMID: 23083016
7.  Hydrogen exchange mass spectrometry: Are we out of the quicksand? 
Although the use of hydrogen exchange (HX) mass spectrometry (MS) to study proteins and protein conformation is now over 20 years old, the perception lingers that it still has “issues”. Is this method, in fact, still in the quicksand with many remaining obstacles to overcome? We do not think so. This critical insight addresses the “issues” and explores several broad questions including: have the limitations of HX MS been surmounted and has HX MS achieved “indispensable” status in the pantheon of protein structural analysis tools.
PMCID: PMC3389995  PMID: 22476891
8.  The natural product cucurbitacin E inhibits depolymerization of actin filaments 
ACS chemical biology  2012;7(9):1502-1508.
Although small molecule actin modulators have been widely used as research tools, only one cell permeable small molecule inhibitor of actin depolymerization (jasplakinolide) is commercially available. We report that the natural product cucurbitacin E inhibits actin depolymerization and show that its mechanism of action is different from jasplakinolide. In assays using pure fluorescently labeled actin, cucurbitacin E specifically affected depolymerization without affecting polymerization. It inhibited actin depolymerization at sub-stoichiometric concentrations up to 1:6 cucurbitacin:actin E. Cucurbitacin E specifically binds to filamentous actin (F-actin) forming a covalent bond at residue Cys257, but not to monomeric actin (G-actin). Based on its compatibility with phalloidin staining, we show that cucurbitacin E occupies a different binding site on actin filaments. Using loss of fluorescence after localized photoactivation, we found that cucurbitacin E inhibited actin depolymerization in live cells. Cucurbitacin E is a widely available plant-derived natural product, making it a useful tool to study actin dynamics in cells and actin-based processes such as cytokinesis.
PMCID: PMC3448819  PMID: 22724897
9.  Pepsin immobilized on high-strength hybrid particles for continuous flow online digestion at 10,000 psi 
Analytical chemistry  2012;84(16):7256-7262.
Pepsin was immobilized on ethyl-bridged hybrid (BEH) particles and digestion performance was evaluated in a completely online format, with the specific intent of using the particles for hydrogen deuterium exchange mass spectrometry (HDX MS) experiments. Because the BEH particles are mechanically strong, they could withstand prolonged, continuous high-pressure at 10,000 psi. Online digestion was performed under isobaric conditions with continuous solvent flow, in contrast to other approaches where the pressure or flow is cycled. As expected, digestion efficiency at 10,000 psi was increased and reproducibly produced more peptic peptides versus digestion at 1,000 psi. Prototype columns made with the BEH pepsin particles exhibited robust performance and deuterium back-exchange was similar to that of other immobilized pepsin particles. These particles can be easily incorporated in existing HDX MS workflows to provide more peptide coverage in experiments where fast, efficient, and reproducible online pepsin digestion is desired.
PMCID: PMC3424325  PMID: 22856522
Pressure; online proteolysis; enzyme; bioreactor; ultra performance liquid chromatography; UPLC; LC; ethyl-bridged hybrid; BEH; acid protease
10.  Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars 
Nature reviews. Drug discovery  2012;11(7):527-540.
Biologics such as monoclonal antibodies are much more complex than small-molecule drugs, which raises challenging questions for the development and regulatory evaluation of follow-on versions of such biopharmaceutical products (also known as biosimilars) and their clinical use once patent protection for the pioneering biologic has expired. With the recent introduction of regulatory pathways for follow-on versions of complex biologics, the role of analytical technologies in comparing biosimilars with the corresponding reference product is attracting substantial interest in establishing the development requirements for biosimilars. Here, we discuss the current state of the art in analytical technologies to assess three characteristics of protein biopharmaceuticals that regulatory authorities have identified as being important in development strategies for biosimilars: post-translational modifications, three-dimensional structures and protein aggregation.
PMCID: PMC3714370  PMID: 22743980
11.  Endocannabinoid Enzyme Engineering: Soluble Human Thio-Monoacylglycerol Lipase (sol-S-hMGL) 
ACS Chemical Neuroscience  2012;3(5):393-399.
In the mammalian central nervous system, monoacylglycerol lipase (MGL) is principally responsible for inactivating the endocannabinoid signaling lipid 2-arachidonoylglycerol (2-AG) and modulates cannabinoid-1 receptor (CB1R) desensitization and signal intensity. MGL is also a drug target for diseases in which CB1R stimulation may be therapeutic. To inform the design of human MGL (hMGL) inhibitors, we have engineered a Leu(Leu169;Leu176)-to-Ser(Ser169;Ser176) double hMGL mutant (sol-hMGL) which exhibited enhanced solubility properties, and we further mutated this variant by substituting its catalytic-triad Ser122 with Cys (sol-S-hMGL). The hMGL variants hydrolyzed both 2-AG and a fluorogenic reporter substrate with comparable affinities. Our results suggest that the hMGL cysteine mutant maintains the same overall architecture as wild-type hMGL. The results also underscore the superior nucleophilic nature of the reactive catalytic Ser122 residue as compared to that of Cys122 in the sol-S-hMGL mutant and suggest that the nucleophilic character of the Cys122 residue is not commensurately enhanced within the three dimensional architecture of hMGL. The interaction of the sol-hMGL variants with the irreversible inhibitors AM6580 and N-arachidonylmaleimide (NAM) and the reversible inhibitor AM10212 was profiled. LC/MS analysis of tryptic digests from sol-S-hMGL directly demonstrate covalent modification of this variant by NAM and AM6580, consistent with enzyme thiol alkylation and carbamoylation, respectively. These data provide insight into hMGL catalysis, the key role of the nucleophilic character of Ser122, and the mechanisms underlying hMGL inhibition by different classes of small molecules.
PMCID: PMC3400385  PMID: 22860208
Active site; catalytic mechanism; drug design; enzyme inhibition; serine hydrolase
12.  On the solution conformation and dynamics of the HIV-1 Viral Infectivity Factor 
Journal of molecular biology  2011;410(5):1008-1022.
HIV-1 has evolved a cunning mechanism to circumvent the antiviral activity of the APOBEC3 family of host-cell enzymes. The HIV-1 virion infectivity factor, one of several HIV accessory proteins, targets APOBEC3 proteins for proteasomal degradation and down-regulates their expression at the mRNA level. Despite the importance of Vif for HIV-1 infection, there is little conformational data on Vif alone or in complex with other cellular factors due to incompatibilities with many structural techniques and difficulties in producing suitable quantities of protein for biophysical analysis. As an alternative, we have turned to hydrogen exchange mass spectrometry (HX MS), a conformational analysis method well suited for proteins that are difficult to study using X-ray crystallography and/or NMR. HX MS was used to probe the solution conformation of recombinant full-length HIV-1 Vif. Vif specifically interacted with the previously identified binding partner Hck and was able to cause kinase activation suggesting that the Vif studied by HX MS retained a biochemically competent conformation relevant to Hck interaction. HX MS analysis of Vif alone revealed low deuteration levels in the N-terminal portion indicating that this region contained structured or otherwise protected elements. In contrast, high deuteration levels in the C-terminal portion of Vif indicated that this region was likely unstructured in the absence of cellular interacting proteins. Several regions within Vif displayed conformational heterogeneity in solution including the APOBEC3G/F binding site and HCCH zinc finger. Taken together, these HX MS results provide new insights into the solution conformation of Vif.
PMCID: PMC3139145  PMID: 21763503
Accessory protein; APOBEC3F/G; E3 Ligase; hydrogen exchange; mass spectrometry; deuterium; Vif
13.  The Utility of Hydrogen/Deuterium Exchange Mass Spectrometry in Biopharmaceutical Comparability Studies 
Journal of pharmaceutical sciences  2010;100(6):2071-2086.
The function, efficacy, and safety of protein biopharmaceuticals are tied to their three-dimensional structure. The analysis and verification of this higher-order structure are critical in demonstrating manufacturing consistency and in establishing the absence of structural changes in response to changes in production. It is, therefore, essential to have reliable, high-resolution and high sensitivity biophysical tools capable of interrogating protein structure and conformation. Here, we demonstrate the use of hydrogen/deuterium exchange mass spectrometry (H/DX-MS) in biopharmaceutical comparability studies. H/DX-MS measurements can be conducted with good precision, consume only picomoles of protein, interrogate nearly the entire molecule with peptide level resolution, and can be completed in a few days. Structural comparability or lack of comparability was monitored for different preparations of interferon-β-1a. We present specific graphical formats for the display of H/DX-MS data that aid in rapidly making both the qualitative (visual) and quantitative assessment of comparability. H/DX-MS is capable of making significant contributions in biopharmaceutical characterization by providing more informative and confident comparability assessments of protein higher order structure than are currently available within the biopharmaceutical industry.
PMCID: PMC3164548  PMID: 21491437
biotherapeutic; interferon; protein conformation; biosimilar; protein drug; structure
14.  False EX1 signatures caused by sample carryover during HX MS analyses 
Discovery of EX1 kinetics in hydrogen exchange (HX) mass spectrometry (MS) experiments is rare. Proteins follow the EX1 kinetic regime when cooperative unfolding events simultaneously expose multiple residues to solvent such that they all become deuterated together before the region is able to refold. A number of factors can contribute to what we call “false EX1” in which it appears as though EX1 occurs in a protein when it probably does not. One of the contributors to false EX1 is peptide carryover between chromatographic runs. In this work, we explore the origins of peptide carryover in HX MS, describe how carryover causes mass spectra to indicate false EX1 kinetics and then describe an optimized washing protocol that can be used to eliminate peptide carryover. A series of solvent injections was developed and found to efficiently eliminate carryover signatures such that analysis of deuterium incorporation could be reliably followed for two proteins prone to high carryover.
PMCID: PMC3106990  PMID: 21643454
Peptide carryover; EX1 kinetics; hydrogen exchange; mass spectrometry; deuterium
15.  The effects of HIV-1 Nef on human N-myristoyl transferase 1† 
Biochemistry  2011;50(16):3394-3403.
The HIV-1 accessory protein Nef is N-terminally myristoylated and this posttranslational modification is essential for Nef function in AIDS progression. Transfer of a myristate group from myristoyl coenzyme A to Nef occurs cotranslationally and is catalyzed by human N-myristoyl transferase-1 (NMT). To investigate the conformational effects of myristoylation on Nef structure as well as to probe the nature of the Nef: NMT complex, we investigated various forms of Nef with hydrogen exchange mass spectrometry. Conformational changes in Nef were not detected as a result of myristoylation and NMT had no effect on deuterium uptake by Nef in a myrNef:NMT complex. However, myrNef binding did have an effect on NMT deuterium uptake. Major HX differences in NMT were primarily located around the active site, with more subtle differences, at the longer timepoints, across the structure. At the shortest timepoint, significant differences between the two states were observed in two regions which interact strongly with the phosphate groups of coenzyme A. Based on our results, we propose a model of the Nef:NMT complex in which only the myristoyl moiety holds the two proteins together in complex and speculate that perhaps NMT chaperones Nef to the membrane and thereby protect the myristic acid group from the cytosol rather than Nef operating through a myristic acid switch mechanism.
PMCID: PMC3086299  PMID: 21449607
Hydrogen exchange; mass spectrometry; protein dynamics; myristoyl coenzyme A; myristoylation
16.  Using Hydrogen/Deuterium Exchange Mass Spectrometry to Study Conformational Changes in Granulocyte Colony Stimulating Factor upon PEGylation 
PEGylation is the covalent attachment of polyethylene glycol to proteins, and it can be used to alter immunogenicity, circulating half life and other properties of therapeutic proteins. To determine the impact of PEGylation on protein conformation, we applied hydrogen/deuterium exchange mass spectrometry (HDX MS) to analyze Granulocyte Colony Stimulating Factor (G-CSF) upon PEGylation as a model system. The combined use of HDX automation technology and data analysis software allowed reproducible and robust measurements of the deuterium incorporation levels for peptic peptides of both PEGylated and non-PEGylated G-CSF. The results indicated that significant differences in deuterium incorporation were induced by PEGylation of G-CSF, although the overall changes observed were quite small. PEGylation did not result in gross conformational rearrangement of G-CSF. The data complexity often encountered in HDX MS measurements was greatly reduced though a data processing and presentation format designed to facilitate the comparison process. This study demonstrates the practical utility of HDX MS for comparability studies, process monitoring and protein therapeutic characterization in the biopharmaceutical industry.
PMCID: PMC3438904  PMID: 22227798
Protein conformation; polyethylene glycol; comparability; biopharmaceutical; post-translational modification; therapeutic protein
17.  Conformational locking upon cooperative assembly of Notch transcription complexes 
Structure(London, England:1993)  2012;20(2):340-349.
The Notch intracellular domain (NICD) forms a transcriptional activation complex with the DNA-binding factor CSL and a transcriptional co-activator of the Mastermind family (MAML). The "RAM" region of NICD recruits Notch to CSL, facilitating the binding of MAML at the interface between the ankyrin (ANK) repeat domain of NICD and CSL. Here, we report the X-ray structure of a human MAML1/RAM/ANK/CSL/DNA complex, and probe changes in component dynamics upon stepwise assembly of a MAML1/NICD/CSL complex using HX-MS. Association of CSL with NICD exerts remarkably little effect on the exchange kinetics of the ANK domain, whereas MAML1 binding greatly retards the exchange kinetics of ANK repeats 2–3. These exchange patterns identify critical features contributing to the cooperative assembly of Notch transcription complexes (NTCs), highlight the importance of MAML recruitment in rigidifying the ANK domain and stabilizing its interface with CSL, and rationalize the requirement for MAML1 in driving cooperative dimerization of NTCs on paired site DNA.
PMCID: PMC3285698  PMID: 22325781
18.  Molecular insight into the conformational dynamics of the Elongin BC complex and its interaction with HIV-1 Vif 
Journal of molecular biology  2010;402(5):892-904.
The HIV-1 Virion infectivity factor (Vif) inhibits the innate viral immunity afforded by the APOBEC3 family of cytidine deaminases. Vif targets the APOBEC3 family for poly-ubiquitination and subsequent proteasomal degradation by linking the Elongin BC dependent ubiquitin ligase complex with the APOBEC3 proteins. The interaction between Vif and the heterodimeric Elongin BC complex, which is mediated by Vif’s viral SOCS (suppressor of cytokine signaling) box, is essential for Vif function. The biophysical consequences of the full length Vif:Elongin BC interaction have not been extensively reported. In this study hydrogen exchange mass spectrometry (HX MS) was used to dissect the Vif:Elongin BC interaction. Elongin C was found to be highly dynamic in the Elongin BC complex while Elongin B was much more stable. Recombinant full length Vif interacted with the Elongin BC complex in vitro with a Kd of 1.9 μM and resulted in observable changes in deuterium uptake in both Elongin C and B. Upon binding to Elongin BC, no significant global conformational changes were detected in Vif by HX MS, but a short fragment of Vif that consisted of the viral SOCS box showed decreased deuterium incorporation upon Elongin BC incubation, suggesting that this region folds upon binding.
PMCID: PMC2949506  PMID: 20728451
Hydrogen exchange mass spectrometry; protein conformation; Viral SOCS box; APOBEC3; E3 ubiquitin ligase
Nature structural & molecular biology  2011;18(11):1235-1243.
Broadly neutralizing antibodies (BNAbs) such as 2F5 are directed against the HIV-1 GP41 membrane proximal external region (MPER) and recognize well-defined linear core sequences. These epitopes can be engrafted onto protein scaffolds, serving as immunogens with high structural fidelity. Although antibodies that bind to the core gp41 epitope can be elicited, they lack neutralizing activity. To understand this paradox, we used biophysical methods to investigate 2F5 binding to the MPER in a membrane environment where it resides. Recognition is stepwise, through a paratope more extensive than core binding site contacts alone, and dynamically rearranging via an apparent CDRH3 scoop-like movement essential for MPER extraction from the viral membrane. Core epitope recognition on the virus requires induction of conformational changes in both the MPER and paratope. Hence, target neutralization through this lipid-embedded viral segment places stringent requirements on antibody combining-site plasticity.
PMCID: PMC3253551  PMID: 22002224
20.  ETD in a Traveling Wave Ion Guide at Tuned Z-Spray Ion Source Conditions Allows for Site-Specific Hydrogen/Deuterium Exchange Measurements 
The recent application of electron transfer dissociation (ETD) to measure the hydrogen exchange of proteins in solution at single-residue resolution (HX-ETD) paves the way for mass spectrometry-based analyses of biomolecular structure at an unprecedented level of detail. The approach requires that activation of polypeptide ions prior to ETD is minimal so as to prevent undesirable gas-phase randomization of the deuterium label from solution (i.e., hydrogen scrambling). Here we explore the use of ETD in a traveling wave ion guide of a quadrupole-time-of-flight (Q-TOF) mass spectrometer with a “Z-spray” type ion source, to measure the deuterium content of individual residues in peptides. We systematically identify key parameters of the Z-spray ion source that contribute to collisional activation and define conditions that allow ETD experiments to be performed in the traveling wave ion guide without gas-phase hydrogen scrambling. We show that ETD and supplemental collisional activation in a subsequent traveling wave ion guide allows for improved extraction of residue-specific deuterium contents in peptides with low charge. Our results demonstrate the feasibility, and illustrate the advantages of performing HX-ETD experiments on a high-resolution Q-TOF instrument equipped with traveling wave ion guides. Determination of parameters of the Z-spray ion source that contribute to ion heating are similarly pertinent to a growing number of MS applications that also rely on an energetically gentle transfer of ions into the gas-phase, such as the analysis of biomolecular structure by native mass spectrometry in combination with gas-phase ion-ion/ion-neutral reactions or ion mobility spectrometry.
PMCID: PMC3438897  PMID: 21952892
Hydrogen/deuterium exchange; Protein conformation; Electron transfer dissociation; Supplemental activation; Traveling wave ion guide
21.  Escherichia coli processivity clamp β from DNA polymerase III is dynamic in solution† 
Biochemistry  2011;50(26):5958-5968.
Escherichia coli DNA polymerase III is a highly processive replicase due to the presence of the β clamp protein that tethers DNA polymerases to DNA. The β clamp is a head-to-tail ring-shaped homodimer, in which each protomer contains three structurally similar domains. Although multiple studies have probed the functions of the β clamp, a detailed understanding of the conformational dynamics of the β clamp in solution is lacking. Here we used hydrogen exchange mass spectrometry to characterize the conformation and dynamics of the intact dimer β clamp and a variant form (I272A/L273A) with diminished ability to dimerize in solution. Our data indicate that the β clamp is not a static closed ring but rather is dynamic in solution. The three domains showed different dynamics though they share a highly similar tertiary structure. Domain I, which controls the opening of the clamp by dissociating from Domain III, contained several highly flexible peptides that underwent partial cooperative unfolding (EX1 kinetics) with a half-life ~4 h. The comparison between the β monomer variant and the wild-type β clamp showed that the β monomer was more dynamic. In the monomer, partial unfolding was much faster and additional regions of Domain III also underwent partial unfolding with a half-life ~1 h. Our results suggest that the δ subunit of the clamp loader may function as a “ring holder” to stabilize the transient opening of the β clamp, rather than as a “ring opener”.
PMCID: PMC3130017  PMID: 21657794
processivity clamp; hydrogen exchange; mass spectrometry; EX1 kinetics; PCNA; clamp loader
22.  Investigating solution-phase protein structure and dynamics by hydrogen exchange mass spectrometry 
Until recently, mass spectrometry (MS) was not often associated with the analysis of protein conformation and dynamics but rather as a method to measure molecular weight and quantify molecules. However, by taking advantage of labeling methods such as hydrogen exchange (HX), many details about protein conformation, dynamics and interactions can be revealed by mass spectrometry. In the current work we provide an update that covers hydrogen exchange theory as it applies to HX MS protocols, explain in detail the practice of HX MS including data analysis and interpretation, and highlight recent advancements in technology which greatly increase the depth of information gained from the technique.
PMCID: PMC2844867  PMID: 19937720
proteins; mass spectrometry; hydrogen exchange
23.  Evidence for Increased Exposure of the Notch1 Metalloprotease Cleavage Site upon Conversion to an Activated Conformation 
Notch proteins are transmembrane receptors that normally adopt a resting state poised to undergo activating proteolysis upon ligand engagement. Receptor quiescence is maintained by three LIN12/Notch repeats (LNRs), which wrap around a heterodimerization domain (HD) divided by furin cleavage at site S1 during maturation. Ligand binding initiates signaling by inducing sensitivity of the HD to proteolysis at the regulated S2 cleavage site. Here, we used hydrogen exchange mass spectrometry to examine the solution dynamics of the Notch1 negative regulatory region in autoinhibited states before and after S1 cleavage, in a proteolytically sensitive “on” state, and in a complex with an inhibitory antibody. Conversion to the “on” state leads to accelerated deuteration in the S2 region and in nearby secondary structural elements within the HD. In contrast, complexation with the inhibitory antibody retards deuteration around the S2 site. Together, these studies reveal how S2 site exposure is promoted by receptor activation and suppressed by inhibitory antibodies.
PMCID: PMC3075624  PMID: 21481777
24.  HIV-1 Nef interaction influences the ATP-binding site of the Src-family kinase, Hck 
BMC Chemical Biology  2012;12:1.
Nef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src kinase family. Nef preferentially activates Hck, a Src-family kinase (SFK) strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain. Recently, we identified a series of kinase inhibitors that preferentially inhibit Hck in the presence of Nef. These compounds also block Nef-dependent HIV replication, validating the Nef-SFK signaling pathway as an antiretroviral drug target. Our findings also suggested that by binding to the Hck SH3 domain, Nef indirectly affects the conformation of the kinase active site to favor inhibitor association.
To test this hypothesis, we engineered a "gatekeeper" mutant of Hck with enhanced sensitivity to the pyrazolopyrimidine tyrosine kinase inhibitor, NaPP1. We also modified the RT loop of the Hck SH3 domain to enhance interaction of the kinase with Nef. This modification stabilized Nef:Hck interaction in solution-based kinase assays, as a way to mimic the more stable association that likely occurs at cellular membranes. Introduction of the modified RT loop rendered Hck remarkably more sensitive to activation by Nef, and led to a significant decrease in the Km for ATP as well as enhanced inhibitor potency.
These observations suggest that stable interaction with Nef may induce Src-family kinase active site conformations amenable to selective inhibitor targeting.
PMCID: PMC3328272  PMID: 22420777
25.  Rapid characterization of IgG1 conformation and conformational dynamics by hydrogen/deuterium exchange mass spectrometry 
Analytical chemistry  2009;81(7):2644-2651.
Protein function is dictated by protein conformation. For the protein biopharmaceutical industry, therefore, it is important to have analytical tools that can detect changes in protein conformation rapidly, accurately and with high sensitivity. In this paper we show that hydrogen/deuterium exchange mass spectrometry (H/DX-MS) can play an important role in fulfilling this need within the industry. H/DX-MS was used to assess both global and local conformational behavior of a recombinant monoclonal IgG1 antibody, a major class of biopharmaceuticals. Analysis of exchange into the intact, glycosylated IgG1 (and the Fab and Fc regions thereof) showed that the molecule was folded, highly stable and highly amenable to analysis by this method using less than a nanomole of material. With improved chromatographic methods, peptide identification algorithms and data-processing steps, the analysis of deuterium levels in peptic peptides produced after labeling was accomplished in 1–2 days. Based on peptic peptide data, exchange was localized to specific regions of the antibody. Changes to IgG1 conformation as a result of deglycosylation were determined by comparing exchange into the glycosylated and deglycosylated forms of the antibody. Two regions of the IgG1 (residues 236-253 and 292-308) were found to have altered exchange properties upon deglycosylation. These results are consistent with previous findings concerning the role of glycosylation in the interaction of IgG1 with Fc receptors. Moreover, the data clearly illustrate how H/DX-MS can provide important characterization information on the higher order structure of antibodies and conformational changes that these molecules may experience upon modification.
PMCID: PMC2779575  PMID: 19265386
antibody; glycosylation; biopharmaceutical; crystallography

Results 1-25 (39)