PUBLICATIONS: 2007-2011


Antibody mechanics on a membrane-bound HIV segment essential for gp41-targeted viral neutralization.
Kim M, Sun Z-YJ, Rand KD, Shi X, Song L, Cheng Y, Fahmy AF, Majumdar S, Ofek G, Yang Y, Kwong PD, Wang J-H, Engen JR, Wagner G, Reinherz EL.
Nat Struct Mol Biol. 2011. Nov 4;18(11):1235-1243.

ABSTRACT

Broadly neutralizing antibodies such as 2F5 are directed against the membrane-proximal external region (MPER) of HIV-1 GP41 and recognize well-defined linear core sequences. These epitopes can be engrafted onto protein scaffolds to serve as immunogens with high structural fidelity. Although antibodies that bind to this core GP41 epitope can be elicited, they lack neutralizing activity. To understand this paradox, we used biophysical methods to investigate the binding of human 2F5 to the MPER in a membrane environment, where it resides in vivo. Recognition is stepwise, through a paratope more extensive than core binding site contacts alone, and dynamic rearrangement through an apparent scoop-like movement of heavy chain comple-mentarity determining region 3 (CDRH3) is essential for MPER extraction from the viral membrane. Core-epitope recognition on the virus requires the induction of conformational changes in both the MPER and the paratope. Hence, target neutralization through this lipid-embedded viral segment places stringent requirements on the plasticity of the antibody combining site.

Pubmed: 22002224


Hydrogen Exchange Mass Spectrometry for Conformational Analysis of Proteins.
Engen JR, Wales TE, Shi X.
Encyclopedia of Analytical Chemistry, 2011. DOI: 10.1002/9780470027318.a9201.
Online ISBN: 9780470027318, Wiley, Robert A. Meyers, Editor-in-Chief. 

Wiley EAC   1. Hydrogen Exchange in Proteins
  1.1. Introduction
  1.2. Which hydrogens and how
    1.2.1. Exchangeable hydrogens
    1.2.2. The exchange reaction mechanism(s)
    1.2.3. Factors influencing exchange rates
       1.2.3.1. pH
       1.2.3.2. Temperature
  1.3. Exchange into proteins
    1.3.1. The two-process model
    1.3.2. Detecting HX in proteins
       1.3.2.1. Nuclear magnetic resonance (NMR)
       1.3.2.2. Mass spectrometry (MS)
  1.4. Pros and cons of HX
  1.5. Applications
2. HX MS experiments
  2.1. Labeling
  2.2. Digestion
  2.3. Separation
  2.4. Ionization
 
3. Data analysis
  3.1. Relative Deuterium Level
  3.2. Correcting for back exchange
  3.3. Visualization and automated data analysis
  3.4. Information from peak shape: EX1 vs.EX2 kinetics

ABSTRACT
Proteins contain labile hydrogen atoms at the backbone amide positions.  These hydrogens are in continuous exchange with hydrogen in surrounding solvent.  By incubating proteins with an isotope of hydrogen, usually deuterium, this exchange process can be monitored experimentally.  Because  hydrogen exchange is a function of protein conformation and dynamics, the rate of hydrogen exchange reports on aspects of protein structure.  One method for detection of exchange is mass spectrometry as proteins will increase in mass as deuteration proceeds.  This article provides background on how and why hydrogens exchange, how the exchange is controlled by several parameters, what measuring exchange is good for, and then gives a general overview of how to make these measurements with mass spectrometry.
 


Electron transfer dissociation in a travelling wave ion guide at tuned Z-spray ion source conditions allows for site-specific hydrogen/deuterium exchange measurements.
Rand KD, Pringle SD, Morris M, Engen JR, Brown J.
J Am Soc Mass Spectrom. 2011. Oct;22(10):1784-1793.

ABSTRACT
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 travelling wave ion guide of a quadrupole-time-of-flight (Q-TOF) mass spectrometer with a “Z-spray” type ion source, to measure the hydrogen exchange of individual residues in a peptide. 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 travelling wave ion guide without prior gas-phase hydrogen scrambling. We show that ETD and supplemental collisional activation in consecutive travelling wave ion guides allows for enhanced extraction of residue-specific deuterium contents of specific peptides during an HX-ETD experiment. Our results demonstrate the feasibility- and illustrate the advantages of performing HX-ETD experiments on a high-resolution Q-TOF instrument equipped with travelling wave ion guides. Determination of parameters of the Z-spray ion source that contribute to ion heating are also pertinent to a growing number of MS applications that similarly rely on an energetically gentle transfer of ions into the gas-phase, such as the analysis of biomolecular structure by native mass spectrometry combined with gas-phase ion-ion/ion-neutral reactions or ion mobility spectrometry.
 
Pubmed: 21952892


Conformational transitions in the membrane scaffold protein of phospholipid bilayer nanodiscs.
Morgan CR, Hebling CM, Rand KD, Stafford DW, Jorgenson JW, Engen JR.
Mol. Cell Proteomics 2011. Sept;10(9):M111.010876.

ABSTRACT

Phospholipid bilayer nanodiscs are model membrane systems that provide an environment where membrane proteins are highly stable and monodisperse without the use of detergents or liposomes.  Nanodiscs consist of a discoidal phospholipid bilayer encircled by two copies of an amphipathic alpha helical membrane scaffold protein which is modeled from apolipoprotein A-1.  Hydrogen exchange mass spectrometry (HX MS) was used to probe the structure and dynamics of the scaffold protein in the presence and absence of lipid.  Upon nanodisc self-assembly, the entire scaffold protein gained significant protection from exchange, consistent with a large, protein-wide, structural rearrangement.  This protection was short-lived and the scaffold protein was highly deuterated within two hours.  Several regions of the scaffold protein, in both the lipid-free and lipid-associated states, displayed EX1 unfolding kinetics.  The rapid deuteration of the scaffold protein and the presence of correlated unfolding events both indicate that nanodiscs are dynamic rather than rigid bodies in solution.  This work provides a catalog of the expected scaffold protein peptic peptides in a nanodisc-HX MS experiment and their deuterium uptake signatures, data which can be used as a benchmark to verify correct assembly and nanodisc structure.  Such reference data will be useful control data for all HX MS experiments involving nanodiscs in which transmembrane or lipid-associated proteins are the primary molecule(s) of interest. 

Pubmed: 21715319

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On the solution conformation and dynamics of the HIV-1 Viral Infectivity Factor.
Marcsisin SR, Narute PS, Emert-Sedlak L, Kloczewiak M, Smithgall TE, Engen JR.
J Mol Biol. 2011. Special Issue "Structural and Molecular Biology of HIV" Jul 29;410(5):1008-1022.

ABSTRACT
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.

Pubmed: 21763503


Escherichia coli processivity clamp β from DNA polymerase III is dynamic in solution.

Fang J, Engen JR, Beuning PJ.
Biochemistry. 2011. Jul 5;50(26):5958-5968. 
 

ABSTRACT
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”.

Pubmed: 21657794


The utility of Hydrogen/Deuterium exchange mass spectrometry in biopharmaceutical comparability studies.
Houde D, Berkowitz SA, Engen JR.
J. Pharm. Sci. 2011. Jun 1;100(6):2071-2086.


ABSTRACT

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.  

Pubmed: 21491437

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False EX1 signatures caused by sample carryover during HX MS analyses.

Fang J, Rand KD, Beuning PJ, Engen JR.
Int. J. Mass. Spectrom. 2011. Special Issue "Hydrogen Exchange Mass Spectrometry" Apr 30;302(1-3):19-25. 

ABSTRACT
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.

Pubmed: 21643454

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Special focus issue: Hydrogen Exchange Mass Spectrometry.

Engen JR, Jørgensen TJ.
Int. J. Mass. Spectrom. 2011. Apr 30;302(1-3):1-2. 

Special Issue Table of Contents


The effects of HIV-1 Nef on human N-myristoyl transferase 1.
Morgan CR, Miglionico BV, Engen JR.
Biochemistry. 2011. Apr 26;50(16):3394-3403. 

ABSTRACT

The HIV-1 accessory protein Nef is N-terminally myristoylated and this post-translational 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.

Pubmed: 21449607


Evidence for increased exposure of the Notch1 metalloprotease cleavage site upon conversion to an activated conformation.
Tiyanont K, Wales TE, Aste-Amezaga M, Aster JC, Engen JR, Blacklow SC.
Structure. 2011. Apr 13;19(4):546-554.

ABSTRACT

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.

Preview by K. Raphael

Pubmed: 21481777


Allosteric interactions between the myristate- and ATP-site of the Abl kinase.
Iacob RE, Zhang J, Gray NS, Engen JR.
PLoS One. 2011. Jan 10;6(1):e15929.

ABSTRACT

Abl kinase inhibitors targeting the ATP binding pocket are currently employed as potent anti-leukemogenic agents but drug resistance has become a significant clinical limitation.  Recently, a compound that binds to the myristate pocket of Abl (GNF-5) was shown to act cooperatively with nilotinib, an ATP-competitive inhibitor to target the recalcitrant ‘T315I’ gatekeeper mutant of Bcr-Abl.  To uncover an explanation for how drug binding at a distance from the kinase active site could lead to inhibition and how inhibitors could combine their effects, hydrogen exchange mass spectrometry (HX MS) was employed to monitor conformational effects in the presence of both dasatinib, a clinically approved ATP-site inhibitor, and GNF-5.  While dasatinib binding to wild type Abl clearly influenced Abl conformation, no binding was detected between dasatinib and T315I.  GNF-5, however, elicited the same conformational changes in both wild type and T315I, including changes to dynamics within the ATP site located approximately 25 Å from the site of GNF-5 interaction.  Simultaneous binding of dasatinib and GNF-5 to T315I caused conformational and/or dynamics changes in Abl such that effects of dasatinib on T315I were the same as when it bound to wild type Abl.  These results provide strong biophysical evidence that allosteric interactions play a role in Abl kinase downregulation and that targeting sites outside the ATP binding site can provide an important pharmacological tool to overcome mutations that cause resistance to ATP-competitive inhibitors.

Pubmed: 21264348

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Molecular insight into the conformational dynamics of the Elongin BC complex and its interaction with HIV-1 Vif.
Marcsisin SR, Engen JR.
J Mol Biol. 2010. Oct 8;402(5):892-904.

ABSTRACT
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.

Pubmed: 20728451


Neutron reflectometry study of the conformation of HIV Nef bound to lipid membranes.

Kent MS, Murton JK, Sasaki DY, Satija S, Akgun B, Nanda H, Curtis JE, Majewski J, Morgan CR, Engen JR.
Biophys J. 2010. Sep 22;99(6):1940-1948. 

ABSTRACT
Nef is an HIV-1 accessory protein that directly contributes to AIDS progression. Nef is myristoylated on the N-terminus, associates with membranes, and may undergo a transition from a solution conformation to a membrane-associated conformation. It has been hypothesized that conformational rearrangements enables membrane-associated Nef to interact with cellular proteins. Despite its medical relevance, there is no direct information about the conformation of membrane-bound Nef. In this work we used neutron reflection (NR) to reveal the first details of the conformation of membrane-bound Nef. The conformation of Nef was probed upon binding to Langmuir monolayers through the interaction of an N-terminal His tag with a synthetic metal-chelating lipid, which models one of the possible limiting cases for myr-Nef. The data indicate insertion of residues into the lipid headgroups during interaction, and that the core domain lies directly against the lipid headgroups with a thickness of ~40 Å. Binding of Nef through the N-terminal His tag apparently facilitates insertion of residues as no insertion occurred upon binding of Nef through weak electrostatic interactions in the absence of the specific interaction through the His tag.

Pubmed: 20858440


Post-translational modifications differentially affect IgG1 conformation & receptor binding.
Houde D, Peng Y, Berkowitz SA, Engen JR.
Mol. Cell Proteomics 2010. Aug;9(8):1716-1728.

ABSTRACT

Post-translational modifications (PTMs) can have profound effects on protein structure and protein dynamics, and thereby
can influence protein function.  To understand and connect PTM-induced functional differences with any resulting
conformational changes, the conformational changes must be detected and localized to specific parts of the protein.  We
illustrate these principles here with a study of the functional and conformational changes that accompany modifications to a monoclonal immunoglobulin gamma 1 (IgG1) antibody.  IgG1s are large and heterogeneous proteins capable of incorporating a multiplicity of PTMs, both in vivo and in vitro.  For many IgG1s, these PTMs can play a critical role in affecting conformation, biological function, and the ability of the antibody to initiate a potential adverse biological response.  We investigated the impact of differential galatosylation, methionine oxidation, and fucosylation on solution conformation using hydrogen/deuterium exchange mass spectrometry and probed the effects of IgG1 binding to the FcgRIIIa receptor.  The results showed that methionine oxidation and galactosylation both impact IgG1 conformation while fucosylation appears to have little or no impact to the conformation.  FcgRIIIa binding was strongly influenced by both the glycan structure/ composition (namely galatose and glucose) and conformational changes that were induced by some of the modifications. 

Pubmed: 20103567


Conformational analysis of membrane proteins in phospholipid bilayer nanodiscs by hydrogen exchange mass spectrometry.

Hebling CM, Morgan CR, Stafford DW, Jorgenson JW, Rand KD, Engen JR.
Anal. Chem. 2010. Jul 1;82(13):5415-5419.

ABSTRACT
The study of membrane protein structure and enzymology has traditionally been hampered by the inherent insolubility of membrane proteins in aqueous environments and experimental challenges in emulating an in vivo lipid environment.  Phospholipid bilayer nanodiscs have recently been shown to be of great use for the study of membrane proteins since they offer a controllable, stable, and monodisperse model membrane with a native-like lipid bilayer.  Here we report the integration of nanodiscs with hydrogen exchange (HX) mass spectrometry (MS) experiments, thereby allowing for analysis of the native conformation of membrane proteins.  Gamma-glutamyl carboxylase (GGCX), an ~94 kDa transmembrane protein, was inserted into nanodiscs and labeled with deuterium oxide under native conditions.  Analytical parameters including sample-handling and chromatographic separation were optimized to measure the incorporation of deuterium into GGCX.  Coupling nanodisc technology with HX MS offers an effective approach for investigating the conformation and dynamics of membrane proteins in their native environment and is therefore capable of providing much needed insight into the function of membrane proteins.

Pubmed: 20518534


Hydrogen Exchange Mass Spectrometry: What is it and what can it tell us?
Marcsisin SR, Engen JR.
Anal Bioanal Chem. 2010. Jun 1;397(3):967-972.

ABSTRACT

Proteins are undoubtedly some of the most essential molecules of life.  While much is known about many proteins, some aspects still remain mysterious. One particularly important aspect of understanding proteins is determining how structure helps dictate function. Continued development and implementation of biophysical techniques that provide information about protein conformation and dynamics is essential.  In this review, we discuss hydrogen exchange mass spectrometry and how this method can be used to learn about protein conformation and dynamics.  The basic concepts of the method are described, the workflow illustrated, and a few examples of its application are provided.

Pubmed: 20195578

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Conformational dynamics of the Escherichia coli DNA polymerase manager protein UmuD and UmuD′.

Fang J, Rand KD, Silva MC, Wales TE, Engen JR, Beuning PJ.
J Mol Biol. 2010. Apr 23;398(1):40-53.

ABSTRACT
The expression of Escherichia coli umuD gene products is upregulated as part of the SOS response to DNA damage. UmuD is initially produced as a 139-amino acid protein, which subsequently cleaves off its N-terminal 24-amino acids in a RecA/ssDNA-dependent reaction, giving UmuD′.  The two forms of the umuD gene products play different roles in the cell. UmuD is implicated in a primitive DNA damage checkpoint and prevents DNA pol IV-dependent -1 frameshift mutagenesis, while the cleaved form facilitates UmuC-dependent mutagenesis via formation of DNA pol V (UmuD′2C). Thus, the cleavage of UmuD is a crucial switch that regulates replication and mutagenesis via numerous protein-protein interactions. A UmuD variant, UmuD3A, has been identified that is noncleavable but is a partial biological mimic of the cleaved form, UmuD′. We used hydrogen-deuterium exchange mass spectrometry (HXMS) to probe the conformations of UmuD, UmuD′, and UmuD3A. In HXMS experiments, backbone amide hydrogens that are solvent-accessible or not involved in hydrogen bonding become labeled with deuterium over time. Our HXMS results reveal that the N-terminal arm of UmuD, which is truncated in the cleaved form UmuD′, is dynamic. Residues that are likely to contact the N-terminal arm show more deuterium exchange in UmuD′ and UmuD3A than in UmuD. These observations suggest that noncleavable UmuD3A mimics the cleaved form UmuD′ because in both cases the arms are relatively unbound from the globular domain. Gas phase hydrogen exchange experiments, which specifically probe the exchange of side-chain hydrogens and are carried out on shorter time scales than solution experiments, show that UmuD′ incorporates more deuterium than either UmuD or UmuD3A. This work indicates that these three forms of the UmuD gene products are highly flexible, which is likely of critical importance for their many protein interactions.

Pubmed: 20206636


Targeting Bcr-Abl by combining allosteric with ATP-binding-site inhibitors.
Zhang J, Adrián FJ, Jahnke W, Cowan-Jacob SW, Li AG, Iacob RE, Sim T, Powers J, Dierks C, Sun F, Guo GR, Ding Q, Okram B, Choi Y, Wojciechowski A, Deng X, Liu G, Fendrich G, Strauss A, Vajpai N, Grzesiek S, Tuntland T, Liu Y, Bursulaya B, Azam M, Manley PW, Engen JR, Daley GQ, Warmuth M, Gray NS.
Nature 2010. Jan 28;463(7280):501-506.

ABSTRACT

In an effort to find new pharmacological modalities to overcome resistance to ATP-binding-site inhibitors of Bcr–Abl, we recently reported the discovery of GNF-2, a selective allosteric Bcr–Abl inhibitor. Here, using solution NMR, X-ray crystallography, mutagenesis and hydrogen exchange mass spectrometry, we show that GNF-2 binds to the myristate-binding site of Abl, leading to changes in the structural dynamics of the ATP-binding site. GNF-5, an analogue of GNF-2 with improved pharmacokinetic properties, when used in combination with the ATP-competitive inhibitors imatinib or nilotinib, suppressed the emergence of resistance mutations in vitro, displayed additive inhibitory activity in biochemical and cellular assays against T315I mutant human Bcr–Abl and displayed in vivo efficacy against this recalcitrant mutant in a murine bone-marrow transplantation model. These results show that therapeutically relevant inhibition of Bcr–Abl activity can be achieved with inhibitors that bind to the myristate-binding site and that combining allosteric and ATP-competitive inhibitors can overcome resistance to either agent alone.
 
PDB code: 3K5V

Pubmed: 20072125


Novel mutant-selective EGFR kinase inhibitors against EGFR T790M.
Zhou W, Ercan D, Chen L, Yun CH, Li D, Capelletti M, Cortot AB, Chirieac L, Iacob RE, Padera R,
Engen JR, Wong KK, Eck MJ, Gray NS, Jänne PA
.
Nature 2009. Dec 24;462(7276):1070-1074.

ABSTRACT

The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors in EGFR-mutant non-small-cell lung cancer (NSCLC) is limited by the development of drug-resistance mutations, including the gatekeeper T790M mutation. Strategies targeting EGFR T790M with irreversible inhibitors have had limited success and are associated with toxicity due to concurrent inhibition of wild-type EGFR. All current EGFR inhibitors possess a structurally related quinazoline-based core scaffold and were identified as ATP-competitive inhibitors of wild-type EGFR. Here we identify a covalent pyrimidine EGFR inhibitor by screening an irreversible kinase inhibitor library specifically against EGFR T790M. These agents are 30- to 100-fold more potent against EGFR T790M, and up to 100-fold less potent against wild-type EGFR, than quinazoline-based EGFR inhibitors in vitro. They are also effective in murine models of lung cancer driven by EGFR T790M. Co-crystallization studies reveal a structural basis for the increased potency and mutant selectivity of these agents. These mutant-selective irreversible EGFR kinase inhibitors may be clinically more effective and better tolerated than quinazoline-based inhibitors. Our findings demonstrate that functional pharmacological screens against clinically important mutant kinases represent a powerful strategy to identify new classes of mutant-selective kinase inhibitors.
 
PDB code: 3IKA

Pubmed: 20033049


Gas-phase hydrogen/deuterium exchange in a travelling wave ion guide for the examination of protein conformations.
Rand KD, Pringle SD, Murphy JP 3rd, Fadgen KE, Brown J, Engen JR.
Anal. Chem. 2009. Dec 15;81(24):10019-10028.

ABSTRACT
Accumulating evidence suggests that solution-phase conformations of small globular proteins and large molecular protein assemblies can be preserved for milliseconds after electrospray ionization.  Thus, the study of proteins in the gas-phase on this time-scale is highly desirable. Here we demonstrate that a travelling wave ion guide (TWIG) of a Synapt mass spectrometer offers a highly suitable environment for rapid and efficient gas-phase hydrogen/deuterium exchange (HDX).  Gaseous ND3 was introduced into either the source TWIG or the TWIG located just after the ion mobility cell, such that ions underwent HDX as they passed through the ND3 on the way to the time-of-flight analyzer.  The extent of deuterium labeling could be controlled by varying the quantity of ND3 or the speed of the travelling wave.  The gas-phase HDX of model peptides corresponded to labeling of primarily fast exchanging sites due to the short labeling times (ranging from 0.1 to 10 ms).  In addition to peptides, gas-phase HDX of ubiquitin, cytochrome c, lysozyme and apomyoglobin were examined.  We conclude that HDX of protein ions in a TWIG is highly sensitive to protein conformation, enables the detection of conformers present on sub-milliseconds timescales and can readily be combined with ion mobility spectrometry.  
 
Pubmed: 19921790


Investigating Solution-Phase Protein Structure and Dynamics by Hydrogen Exchange Mass Spectrometry.
Morgan CR, Engen JR.
Curr Protoc Protein Sci. 2009. Nov;Chapter 17:Unit17.6.

ABSTRACT

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 this unit, hydrogen exchange theory is discussed as it applies to HX-MS protocols, the practice of HX-MS including data analysis and interpretation is explained in detail, and recent advancements in technology which greatly increase the depth of information gained from the technique are highlighted.

Pubmed: 19937720


Analysis of protein conformation and dynamics by Hydrogen/Deuterium exchange MS.

  Engen JR.
Anal. Chem. 2009. Oct 1;81(19):7870-5.

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ABSTRACT

Understanding as much as possible about proteins in the shortest amount of time has long been a goal of hydrogen exchange (HX) MS.  Recent advances have led to improvements in the technique, but has this goal yet been achieved?

Pubmed: 19788312


Characterization of IgG1 Conformation and Conformational Dynamics by Hydrogen / Deuterium Exchange Mass Spectrometry.
Houde D, Arndt J, Domeier W, Berkowitz SA, Engen JR.
Anal. Chem. 2009. Apr 1;81(7):2644-51.  Errata: Anal. Chem., 2009, 81 (14), p 5966.

ABSTRACT
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.
 
PDB code: 3FZU


Pubmed: 19265386


Isotope Exchange and Covalent Modification Strategies for Studying Protein Structure and Function.
Chen S, Engen JR.
Curr. Anal. Chem. Special Issue "The exciting ionic life of a protein in the hands of a mass spectrometrist".
2009. Apr 1;5(2):205-212.


ABSTRACT
Mass spectrometry can be used to obtain information about all levels of protein structure. To gain access to the conformation information found in the tertiary and quaternary structure, various labeling methods have been developed. These methods convert structural information into mass differences that can be observed with high-resolution protein/peptide mass spectrometry. Three methods are reviewed here: hydrogen/deuterium exchange, covalent modification (also called chemical modification) and hydroxyl radical footprinting. The general implementation of these methods is described and comparisons are made between the methods.


Conformational Dynamics of Proteins by Mass Spectrometry: Hydrogen / Deuterium Exchange and Covalent Labeling Approaches.
Houde D, Engen JR.
Chimica Oggi / Chemistry Today. 2009. Mar/Apr;27(2):12-15. 

ABSTRACT
As the bioanalytical analysis of proteins becomes evermore important, attention must be paid to the three-dimensional structure of these analytes. Protein function, viability and efficacy in the case of protein-based therapeutics are intricately connected to protein structure. Here we highlight mass spectrometry based approaches for studying protein conformation. By labeling proteins with hydrogen/deuterium exchange or covalent labeling methodology, many challenges associated with biophysical characterisation of protein structure and dynamics can be addressed. There are multiple advantages to using MS, not the least of which is the small amounts of material that are required.

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Aspartic Proteinases in Antarctic Fish.
De Luca V, Maria G, De Mauro G, Catara G, Carginale V, Ruggiero G, Capasso A, Parisi E, Brier S,
Engen JR, Capasso C.

Marine Genomics. 2009. Mar;2:1-10.

ABSTRACT
The present review surveys several recent studies of the aspartic proteinases from Antarctic Notothenioidei, a dominating fish group that has developed a number of adjustments at the molecular level to maintain metabolic function at low temperatures. Given the unique peculiarities of the Antarctic environment, studying the features of Antarctic aspartic proteinases could provide new insights into the role of these proteins in fish physiology. We describe here: (1) the biochemical properties of a cathepsin D purified from the liver of the hemoglobinless icefish Chionodraco hamatus; (2) the biochemical characterization of Trematomus bernacchii pepsins variants A1 and A2 obtained by heterologous expression in bacteria; and (3) the identification of two closely related, novel aspartic proteinases from the liver of the two Antarctic fish species mentioned above. Overall, the results show that Notothenioidei aspartic proteinases display a number of characteristics that are remarkably different from those of mammalian aspartic proteinases, including high turnover number or high catalytic efficiency. We have named the newly identified aspartic proteinases “Nothepsins” and classified them relative to aspartic proteinases from other species.


Conformational Disturbance in Abl Kinase upon Mutation and Deregulation.
Iacob RE, Dumitrescu TP, Zhang J, Gray NS, Smithgall TE, Engen JR.
Proc Natl Acad Sci U.S.A. 2009. Feb 3;106(5):1386-1391.
 
ABSTRACT
Protein dynamics are inextricably linked to protein function but there are few techniques that allow protein dynamics to be conveniently interrogated. For example, mutations and translocations give rise to aberrant proteins such as Bcr-Abl where changes in protein conformation and dynamics are believed to result in deregulated kinase activity that provides the oncogenic signal in Chronic Myelogeous Leukemia. Although crystal structures of the downregulated c-Abl kinase core have been reported, the conformational impact of mutations that render Abl resistant to small-molecule kinase inhibitors are largely unknown as is the allosteric interplay of the various regulatory elements of the protein. Hydrogen exchange mass spectrometry (HX MS) was employed to compare the conformations of wild-type Abl with a non-myristoylated form and with three clinically relevant imatinib resistance mutants (T315I, Y253H and E255V). A HX-resistant core localized to the interface between the SH2 and kinase domains, a region known to be important for maintaining the downregulated state. Conformational differences upon demyristoylation were consistent with the SH2 domain moving to the top of the small lobe of the kinase domain as a function of activation. There were conformational changes in the T315I mutant but, surprisingly, no major changes in conformation were detected in either the Y253H or the E255V mutants. Taken together, these results provide evidence that allosteric interactions and conformational changes play a major role in Abl kinase regulation in solution. Similar analyses could be performed on any protein to provide mechanistic details about conformational changes and protein function.

Pubmed: 19164531


Protein Analysis with Hydrogen-Deuterium Exchange Mass Spectrometry.
Mitchell JL, Engen JR.
Chapter 4 of “Protein Mass Spectrometry", Comprehensive Analytical Chemistry Series, 2009, Vol. 52, 83-102.
ISBN: 978-0-444-53055-4, Elsevier, Julian Whitelegge, Editor. 

  1. Introduction
   1.1. Review of Protein Structure
   1.2. Obtaining information about conformation and dynamics
2. Experimental protocol
   2.1. Deuterium introduction
   2.2. Global versus local information
   2.3. HPLC and MS
   2.4. Data interpretation
3. Illustrative examples
   3.1. Protein conformation and the effects of mutation
   3.2. Binding interactions
   3.3. Investigating proteins lacking structural data
4. Conclusions

ABSTRACT
There are some properties of proteins that remain hidden to a mass spectrometer during simple molecular weight analyses. Some of these hidden protein properties include protein conformation, protein dynamics and protein interactions. How can these properties be revealed when mass spectrometers measure molecular weight not protein conformation? One way to uncover these properties with a mass spectrometer is to use a labeling method that “captures” the structural information before mass analysis occurs. The following chapter will describe one of these labeling methods: hydrogen-deuterium exchange.

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Tyrosine Phosphorylation in the SH3 Domain Disrupts Negative Regulatory Interactions within the c-Abl Kinase Core.

Chen S, O'Reilly LP, Smithgall TE, Engen JR.
J Mol Biol. 2008. Nov 7;383(2):414-423.

ABSTRACT
Recent studies have shown that trans-phosphorylation of the Abl SH3 domain at Tyr89 by Src-family kinases is required for the full transforming activity of Bcr-Abl. Tyr89 localizes to a binding surface of the SH3 domain that engages the SH2-kinase linker in the crystal structure of the c-Abl core. Displacement of SH3 from the linker is an event likely to influence efficient downregulation of c-Abl. Hydrogen-deuterium exchange (HX) and mass spectrometry (MS) were used to investigate whether Tyr89 phosphorylation affects the ability of the SH3 domain to interact intramolecularly with the SH2-kinase linker in cis as well as other peptide ligands in trans. HX MS analysis of SH3 binding showed that when various Abl constructs were phosphorylated at Tyr89 by the Src-family kinase Hck, SH3 was unable to engage a high-affinity ligand in trans and that cis interaction with the linker was dramatically reduced in a construct containing the SH3 and SH2 domains plus the linker. Phosphorylation of the Abl SH3 domain on Tyr89 also interfered with binding to the negative regulatory protein Abi-1 in trans. Site-directed mutagenesis of Tyr89 and Tyr245, another tyrosine phosphorylation site located in the linker that may also influence SH3 binding, implicated Tyr89 as the key residue necessary for disrupting regulation after phosphorylation. These results imply that phosphorylation at Tyr89 by Src-family kinases prevents engagement of the Abl SH3 domain with its intramolecular binding partner leading to enhanced Abl kinase activity and cellular signaling.

Pubmed: 18775435


Ion Mobility adds an Additional Dimension to Mass Spectrometry Analysis of Solution-Phase Hydrogen/Deuterium Exchange.
Iacob RE, Murphy JP 3rd, Engen JR.
Rapid Commun. Mass Spectrom. 2008. 22(18):2898-2904.

ABSTRACT
The goal of this study was to determine the utility of adding ion mobility spectrometry (IMS) to studies probing the solution-phase hydrogen/deuterium exchange (HX) of proteins. The HX profile of the Hck SH3 domain was measured at both the intact protein and the peptic peptide levels in the Waters Synapt HDMS system which uses a traveling wave to accomplish ion mobility separation prior to Tof m/z analysis. The results indicated a similar loss of deuterium with or without use of mobility in the Synapt and a level of deuterium loss comparable with a non-mobility Q-Tof instrument. The drift time of this small protein and its peptic peptides did not noticeably change due to solution-based deuterium incorporation. Importantly, ion mobility separations provided an orthogonal dimension of separation in addition to the RP-HPLC. The additional dimension of separation allowed for the deconvolution of overlapping isotopic patterns for co-eluting peptides and extraction of valuable deuterium incorporation data for those peptides. Taken together, these results indicate that including ion mobility separation in HX MS analyses further improves the mass spectrometry portion of such experiments.
 
Pubmed: 18727141


High-speed and High-resolution UPLC Separation at Zero Degrees Celsius.
Wales TE, Fadgen KE, Gerhardt GC, Engen JR.
Anal. Chem. 2008. 80(17):6815-6820.

ABSTRACT
The conformational properties of proteins can be probed with hydrogen/deuterium exchange mass spectrometry (HXMS).  In order to maintain the deuterium label during LC/MS analyses, chromatographic separation must be done rapidly (usually in under 8-10 minutes) and at zero degrees Celsius.  Traditional RP-HPLC with ~3 micron particles has shown generally poor chromatographic performance under these conditions and thereby has been prohibitive for HXMS analyses of larger proteins and many protein complexes.  Ultra performance liquid chromatography (UPLC) employs particles smaller than 2 microns in diameter to achieve superior resolution, speed, and sensitivity as compared to HPLC. UPLC has previously been shown to be compatible with the fast separation and low temperature requirements of HXMS.  Here we present construction and validation of a custom UPLC system for HXMS.  The system is based on the Waters nanoACQUITY platform and contains a Peltier-cooled module that houses the injection and switching valves, online pepsin digestion column, and C-18 analytical separation column.  Single proteins in excess of 95 kDa and a four-protein mixture in excess of 250 kDa have been used to validate the performance of this new system.  Near baseline resolution was achieved in 6 minute separations at 0 °C and displayed a median chromatographic peak width of ~2.7 sec at half height.  Deuterium recovery was similar to that obtained using a conventional HPLC and icebath.  This new system represents a significant advancement in HXMS technology that is expected to make the technique more accessible and mainstream in the near future. 
 
Pubmed: 18672890


Structure and Dynamic Regulation of Src-Family Kinases.
Engen JR, Wales TE, Hochrein JM, Meyn MA 3rd, Ozkan SB, Bahar I, Smithgall TE.
Cell Mol Life Sci. 2008. Oct;65(19):3058-3073.

ABSTRACT
Src-family kinases are modular signaling proteins involved in a diverse array of cellular processes. All members of the Src family share the same domain organization, with modular SH3, SH2 and kinase domains followed by a C-terminal negative regulatory tail. X-ray crystallographic analyses of several Src family members have revealed critical roles for the SH3 and SH2 domains in the downregulation of the kinase domain. This review focuses on biological, biophysical, and computational studies that reveal conformationally distinct active states within this unique kinase family.
 
Pubmed: 18563293


Abl N-terminal Cap stabilization of SH3 domain dynamics.
Chen S, Dumitrescu TP, Smithgall TE, Engen JR.
Biochemistry. 2008. 47(21):5795-5803.

ABSTRACT
Crystal structures and other biochemical data indicate that the N-terminal cap (NCap) region of the Abelson tyrosine kinase (c-Abl) is important for maintaining the downregulated conformation of the kinase domain. The exact contributions that NCap makes in stabilizing the various intramolecular interactions within c-Abl are less clear. While the NCap appears important for locking the SH3/SH2 domains to the back of the kinase domain, there may be other more subtle elements of regulation. Hydrogen exchange (HX) and mass spectrometry (MS) were used to determine if the NCap contributes to intramolecular interactions involving the Abl SH3 domain. Under physiological conditions, the Abl SH3 domain underwent partial unfolding and its unfolding half-life was slowed during binding to the SH2-kinase linker, providing a unique assay to test NCap-induced stabilization of the SH3 domain in various constructs. The results showed that NCap stabilizes the dynamics of the SH3 domain in certain constructs but does not increase the relative affinity of the SH3 domain for the native SH2-kinase linker. The stabilization effect was absent in constructs of just NCap + SH3 but was obvious when the SH2 domain and the SH2-kinase linker were present. These results suggest that interactions between NCap and the SH3 domain can contribute to c-Abl stabilization in constructs that contain at least the SH2 domain, an effect that may partially compensate for the absence of the negative regulatory C-terminal tail found in the related Src family of kinases.
 
Pubmed: 18452309


Hydrogen Exchange Mass Spectrometry: Principles and Capabilities.
Brier S, Engen JR.
In “Mass Spectrometry Analysis for Protein-Protein Interactions and Dynamics", 2008. pp 11-43.
ISBN: 978-0-470-25886-6, Blackwell Publishing, Mark R. Chance, Editor.

  ABSTRACT
Hydrogen exchange (HX) detected by mass spectrometry (MS) is an extremely valuable method for understanding proteins. The hydrogen exchange reaction itself, which has been understood by examining the exchange behavior of small amide models and peptide analogues, imposes specific limits on the overall HX MS method. In this chapter, the fundamental concepts that govern the hydrogen exchange reaction will be described. These concepts build a foundation for a discussion of basic HX MS methodology and its application to various biological problems. Examples of applying the method to specific problems will be provided in subsequent chapters of this book.
 
CHAPTER 2
 
1. The chemistry of hydrogen exchange (HX)
   1.1. Principles of proton transfer
   1.2. Mechanisms of backbone amide hydrogen exchange
   1.3. Factors affecting hydrogen exchange
      1.3.1. pH effects
      1.3.2. Temperature effects
      1.3.3. Solvent and pressure effects
      1.3.4. Side-chain and ionic strength effects
2. HX mechanisms in proteins
3. Deuterium incorporation into proteins
   3.1. Continuous labeling
   3.2. Pulse labeling
   3.3. Other labeling strategies
4. Measuring HX with mass spectrometry (MS)
   4.1. Global versus local exchange
   4.2. Back-exchange
   4.3. Proteolysis before MS
   4.4. Mass measurements and data processing
5. Capabilities of HX MS in structural biology
   5.1. Protein folding studies
   5.2. Quality control
   5.3. Aid in structure elucidation
   5.4. Interactions & dynamics
6. Acknowledgements
7. References
8. Figure legends

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Mass Spectrometry Applications in Redox Biology.
Raza A, Engen JR.
In “Redox Biochemistry”, 2008.  Section 6.1, pp 228-237.
ISBN: 978-0-471-78625-5, John Wiley & Sons. Ruma Banerjee, Editor.

 
ABSTRACT
Mass spectrometry is a technique that is used to identify and differentiate molecules on the basis of their mass.  Mass spectrometry has been called the universal detector because almost all types of molecules are amenable to analysis.  A few examples include determining adulteration of honey, detecting steroids in athletes, identifying unknown proteins, determining the post-translational modifications of protein, confirming mutations in proteins, quantitating drugs in biological matrices and measuring the concentration of pollutants in the air.  Depending on the application, mass spectrometers are usually coupled to a separation method, i.e. gas chromatography & mass spectrometry (GC-MS) or liquid chromatography & mass spectrometry (LC-MS).  In this section, the focus will be on LC-MS, which is the most useful technique for biological applications, and on the applications of mass spectrometry in redox biology.

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Purification and characterization of pepsins A1 and A2 from the Antarctic rock cod Trematomus bernacchi.
Brier S, Maria G, Carginale V, Capasso A, Wu Y, Taylor RM, Borotto NB, Capasso C, Engen JR.
FEBS J. 2007, Dec;274(23):6152-6166.

ABSTRACT
The Antarctic notothenioid Trematomus bernacchii (rock cod) lives at a constant mean temperature of -1.9 °C. Gastric digestion under these conditions relies on the proteolytic activity of aspartic proteases such as pepsin. To understand the molecular mechanisms of Antarctic fish pepsins, T. bernacchii pepsins A1 and A2 were cloned, overexpressed in E. coli, purified and characterized with a number of biochemical and biophysical methods. The properties of these two Antarctic isoenzymes were compared to porcine pepsin and found to be unique in a number of ways. Fish pepsins were found to be more temperature sensitive, generally less active at lower pH and more sensitive to inhibition by pepstatin than the mesophilic counterpart. The specificity of Antarctic fish pepsins was similar but not identical to pig pepsin, likely owing to changes in the sequence of fish enzymes near the active site. Gene duplication of Antarctic rock cod pepsins is the likely mechanism for adaptation to the harsh temperature environment in which these enzymes must function.
 
Pubmed: 17976195

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Allosteric Loss-of-Function Mutations in HIV-1 Nef from a Long-term Non-progressor.
Trible RP, Emert-Sedlak L, Wales TE, Ayyavoo V, Engen JR, Smithgall TE.
J Mol Biol. 2007, Nov 16;374(1):121-129.
 
ABSTRACT
Activation of Src family kinases by HIV-1 Nef may play an important role in the pathogenesis of HIV/AIDS. Here we investigated whether diverse Nef sequences universally activate Hck, a Src family member expressed in macrophages and other HIV-1 target cells. In general, we observed that Hck activation is a highly conserved Nef function. However, we identified an unusual Nef variant from an HIV-positive individual that did not develop AIDS which failed to activate Hck despite the presence of conserved residues linked to Hck SH3 domain binding and kinase activation. Amino acid sequence alignment with active Nef proteins revealed differences in regions not previously implicated in Hck activation, including a large internal flexible loop absent from available Nef structures. Substitution of these residues in active Nef compromised Hck activation without affecting SH3 domain binding. These findings show that residues at a distance from the SH3 domain binding site allosterically influence Nef interactions with a key effector protein linked to AIDS progression.
 
Pubmed: 17920628


Probing Protein Interactions using Hydrogen-Deuterium Exchange Mass Spectrometry.
Weis DD, Kaveti S, Wu Y, Engen JR.
In “Mass Spectrometry of Protein Interactions”, 2007
ISBN: 978-0-471-79373-1, John Wiley & Sons. Kevin Downard, Editor.

 

Cover art:
Inspired by Figure 3.3
CHAPTER 3
I. Introduction
II. Hydrogen exchange background
III. General HX MS method
   a. Location information provided by HX MS
   b. Revealing interactions by comparison
IV. Interactions of proteins
V. Examples
   a. Conformational changes of proteins during binding
   b. Protein:protein interactions
   c. Protein:peptide interactions
   d. Protein:small molecule interactions
VI. Conclusions
VII. References
VIII. Acknowledgements
IX. Figure legends
X. Figures

ABSTRACT
Hydrogen-deuterium exchange in combination with mass spectrometry has become a powerful analytical tool for the investigation of protein interactions and protein dynamics. The technique is based on the exchange of protein amide hydrogens for deuterium when a protein or protein complex is placed in D2O solution. The isotopic exchange can be localized and quantified by measuring the mass increase with high resolution mass spectrometry. The fundamentals of H/D exchange MS are described along with its applications in various areas of protein chemistry. The method can provide valuable information about protein folding, about the conformational changes in proteins during binding or activation and inactivation, and can also be used to determine the dissociation constants of proteins in complexes. The application of hydrogen exchange is illustrated with recent experiments involving protein:protein interactions and protein interactions with small molecules.

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The Abl SH2-kinase linker naturally adopts a conformation competent for SH3 domain binding.
Chen S, Brier S, Smithgall TE, Engen JR.
Protein Sci. 2007 Apr;16(4):572-581.

ABSTRACT
The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the downregulated conformation.  Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity.  Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain.  Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution.  Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain.  Truncation of the linker before W254 completely prevented cis-interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions.  The observation that the Abl linker sequence exhibits SH3 binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases.  These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation that in Src kinases, where the downregulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain.
 
Pubmed: 17327393

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Functional characterization and conformational analysis of the Herpesvirus saimiri Tip-C484 protein.
Mitchell JL, Trible RP, Emert-Sedlak LA, Weis DD, Lerner EC, Applen JJ, Sefton BM, Smithgall TE,
Engen JR
.
J Mol Biol. 2007 Mar 2;366(4):1282-1293.

ABSTRACT
Tyrosine kinase interacting protein (Tip) of Herpesvirus saimiri (HVS) activates the lymphoid-specific Src-family kinase Lck. The Tip:Lck interaction is essential for transformation and oncogenesis in HVS-infected cells. As there are no structural data for Tip, hydrogen exchange mass spectrometry was used to investigate the conformation of a nearly full-length form (residues 1-187) of Tip from HVS strain C484. Disorder predictions suggested that Tip would be mostly unstructured so great care was taken to ascertain whether recombinant Tip was functional. Circular dichroism and gel filtration analysis indicated an extended, unstructured protein. In vitro and in vivo binding and kinase assays confirmed that purified, recombinant Tip interacted with Lck, was capable of strongly activating Lck kinase activity and was multiply phosphorylated by Lck. Hydrogen exchange mass spectrometry of Tip then showed that the majority of backbone amide hydrogens became deuterated after only 10 seconds of labeling. Such a result suggested that Tip was almost totally unstructured in solution. Digestion of deuterium labeled Tip revealed some regions with minor protection from exchange. Overall, it was found that although recombinant Tip is still functional and capable of binding and activating its target Lck, it is largely unstructured.
 
Pubmed: 17207813