PUBLICATIONS: 2015

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Subtle dynamic changes accompany Hck activation by HIV-1 Nef and are reversed by an antiretroviral kinase inhibitor.
Wales TE, Hochrein JM, Morgan CR, Emert-Sedlak LA, Smithgall TE, Engen JR.
Biochemistry 2015. Oct 20;54(41):6382-6391.


ABSTRACT

The HIV-1 virulence factor Nef interacts with the macrophage Src-family kinase Hck, resulting in constitutive kinase activation that contributes to viral replication and immune escape.  Previous chemical library screens identified the diphenylfuranopyrimdine kinase inhibitor DFP-4AB, which selectively inhibits Nef-dependent Hck activity in biochemical assays and potently blocks HIV replication in vitro.  In the present study, hydrogen exchange mass spectrometry (HX MS) was used to study conformational changes in downregulated Hck that result from Nef binding, as well as the impact of DFP-4AB on these changes.  Remarkably, interaction with Nef induced only subtle changes in deuterium uptake by Hck, with the most significant changes in the N-lobe of the kinase domain adjacent to the docking site for Nef on the SH3 domain.  No changes in hydrogen exchange were observed in the Hck SH2 domain or C-terminal tail, indicating that this regulatory interaction is unaffected by Nef binding.  When HX MS was performed in the presence of DFP-4AB, the effect of Nef on Hck N-lobe dynamics was completely reversed.  These results show that constitutive activation of Hck by HIV-1 Nef requires only modest changes to the conformational dynamics of the overall kinase structure.  DFP-4AB reverses these effects, consistent with its activity against this Nef-induced signaling event in HIV-infected cells. 

Pubmed: 26440750

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Hydrogen exchange mass spectrometry of proteins at Langmuir monolayers.
Pirrone GF, Vernon BC, Kent MS, Engen JR.
Anal. Chem. 2015. Jul 21;87(14):7022-7029.


ABSTRACT

Hydrogen exchange (HX) mass spectrometry (MS) is valuable for providing conformational information for proteins/peptides that are very difficult to analyze with other methods such as peripheral membrane proteins and peptides that interact with membranes. We developed a new type of HX MS measurement that integrates Langmuir monolayers. A lipid monolayer was generated, a peptide or protein associated with it, and then the monolayer-associated peptide or protein was exposed to deuterium. The deuterated species was recovered from the monolayer, digested, and deuterium incorporation monitored by MS. Test peptides showed that deuterium recovery in an optimized protocol was equivalent to deuterium recovery in conventional solution HX MS. The reproducibility of the measurements was high, despite the requirement of generating a new monolayer for each deuterium labeling time. We validated that known conformational changes in the presence of a monolayer/membrane could be observed with the peptide melittin and the myristoylated protein Arf-1. Results in an accompanying paper show that the method can reveal details of conformational changes in a protein (HIV-1 Nef), which adopts a different conformation, depending on whether or not it is able to insert into the lipid layer. Overall, the HX MS Langmuir monolayer method provided new and meaningful conformational information for proteins that associate with lipid layers. The combination of HX MS results with neutron or X-ray reflection of the same proteins in Langmuir monolayers can be more informative than the isolated use of either method.

Pubmed: 26134943

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The membrane-associated conformation of HIV-1 Nef investigated with hydrogen exchange mass spectrometry at a Langmuir monolayer.
Pirrone GF, Emert-Sedlak LA, Wales TE, Smithgall TE, Kent MS, Engen JR.
Anal. Chem. 2015. Jul 21;87(14):7030-7035.


ABSTRACT

In the companion paper to this work, we described development of a new type of hydrogen exchange (HX) mass spectrometry (MS) measurement that integrates Langmuir monolayers. With Langmuir monolayers, the lipid packing density can be reproducibly controlled and changed as desired. Analysis of HX in proteins that may undergo conformational changes as a function of lipid packing (for example, conformational rearrangements after insertion into a lipid layer) are then possible. We previously used neutron reflection to characterize just such a conformational change in the myristoylated HIV-1 Nef protein (myrNef): at high lipid packing density, myrNef could not insert into the lipids and maintained a compact conformation adjacent to the monolayer, whereas at lower lipid packing density, myrNef was able to insert N-terminal arm residues, causing displacement of the core domain away from the monolayer. In order to locate where conformation may have been altered by lipid association, we applied the HX MS Langmuir monolayer method to myrNef associated with monolayers of packing densities identical to those used for the prior neutron reflection measurements. The results show that the N-terminal region and the C-terminal unstructured loop undergo conformational changes when associated with a low density lipid monolayer. The results are not consistent with the hypothesis of myrNef dimerization upon membrane association in the absence of other myrNef binding partners. The HX MS Langmuir monolayer method provides new and meaningful information for myrNef that helps explain necessary conformational changes required for function at the membrane. 

Pubmed: 26133569


Analytical aspects of hydrogen exchange mass spectrometry.
Engen JR, Wales TE.
Annu Rev Anal Chem. 2015. Jul 22;8(1):127-148.

ABSTRACT

The analytical aspects of measuring hydrogen exchange by mass spectrometry are reviewed. The nature of analytical selectivity in hydrogen exchange is described, followed by a review of the analytical tools required to accomplish fragmentation, separation, and the mass spectrometry measurements under restrictive exchange quench conditions. In contrast to analytical quantitation that relies on measurements of peak intensity or area, quantitation in hydrogen exchange mass spectrometry depends on measuring a mass change with respect to an undeuterated or deuterated control, resulting in a value between zero and the maximum amount of deuterium that could be incorporated. Reliable quantitation is a function of experimental fidelity and to achieve high measurement reproducibility, a large number of experimental variables must be controlled during sample preparation and analysis. The method also reports on important qualitative aspects of the sample, including conformational heterogeneity and population dynamics.

Pubmed: 26048552


Replication in bioanalytical studies with HDX MS: Aim as high as possible.
Moroco JA, Engen JR.
Bioanalysis. 2015. 7(9):1065-1067.

ABSTRACT

Questions of reproducibility arise for an HDX MS experiment as for any analytical measurement.  How many replicates are required before there is reasonable confidence in the measured value?  What difference(s) between measurements would be considered “significant” or, more importantly, meaningful?  If one analyst in Laboratory A makes a measurement, how well can that measurement be reproduced with the identical sample by another analyst in Laboratory B with similar equipment and expertise?  The answers to these questions depend on a number of factors and clearer understanding of what types of measurements are possible and which ones have what type(s) of error.

Pubmed: 26039804


Noncognate DNA damage prevents the formation of the active conformation of the Y-family DNA polymerases DinB and DNA polymerase kappa.
Nevin P, Lu X, Zhang K, Engen JR, Beuning PJ.
FEBS J. 2015. Jul;282(14):2646-2660.

ABSTRACT
Y-family DNA polymerases are specialized to copy damaged DNA and are associated with an increased risk of mutagenesis due to their low fidelity. It is believed that the mechanism of nucleotide selection by Y-family DNA polymerases involves conformational changes preceding nucleotidyl transfer but there is limited experimental evidence for such structural changes. In particular, nucleotide-induced conformational changes in bacterial or eukaryotic Y-family DNA polymerases have to date not been characterized.  Using hydrogen/deuterium exchange mass spectrometry, we demonstrate here that the Escherichia coli Y-family DNA polymerase DinB and its human ortholog DNA polymerase κ undergo a conserved nucleotide-induced conformational change in the presence of undamaged DNA and the correct incoming nucleotide. Notably, this holds true for damaged DNA containing N2-furfuryl-deoxyguanosine that is efficiently copied by these two polymerases, but not for damaged DNA containing the major groove modification O6-methyl-deoxyguanosine that is a poor substrate. Our observations suggest that DinB and pol κ utilize a common mechanism for nucleotide selection involving a conserved prechemical conformational transition promoted by the correct nucleotide and only preferred DNA substrates.

Pubmed: 25899385

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Tag and capture flow hydrogen exchange mass spectrometry with a fluorous-immobilized probe.
Marcsisin SR, Liptak C, Marineau J, Bradner JE, Engen JR.
Anal. Chem. 2015. Jun 16;87(12):6349-6356.


ABSTRACT

Analysis of complex mixtures of proteins by hydrogen exchange (HX) mass spectrometry (MS) is limited by one’s ability to resolve the protein(s) of interest from the proteins that are not of interest.  One strategy to overcome this problem is to tag the target protein(s) to allow for rapid removal from the mixture for subsequent analysis.  Here we illustrate a new solution involving fluorous conjugation of a retrievable probe.  The appended fluorous tag allows for facile immobilization on a fluorous surface.  When a target protein is passed over the immobilized probe molecule it can be efficiently captured and then exposed to a flowing stream of deuterated buffer for hydrogen exchange.  The utility of this method is illustrated for a model system of the Elongin BC protein complex bound to a peptide from HIV Vif.  Efficient capture is demonstrated and deuteration when immobilized was identical to deuteration in conventional solution-phase hydrogen exchange MS.  Protein captured from a crude bacterial cell lysate could also be deuterated without need for separate purification steps before HX MS.  The advantages and disadvantages of the method are discussed in light of miniaturization and automation.

Pubmed: 26023704

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Utilizing Microchip Capillary Electrophoresis Electrospray Ionization for Hydrogen Exchange Mass Spectrometry.
Black WA, Stocks BB, Mellors JS, Engen JR, Ramsey JM.
Anal. Chem. 2015. Jun 16;87(12):6280-6287.


ABSTRACT

Hydrogen exchange (HX) mass spectrometry (MS) of complex mixtures requires a fast, reproducible, and high peak capacity separation prior to MS detection. The current paradigm relies on liquid chromatography (LC) with fast gradients performed at low temperatures to minimize back exchange. Unfortunately, under these conditions, the efficiency of LC is limited due to resistance to mass transfer, reducing the capability to analyze complex samples. Capillary electrophoresis (CE), on the other hand, is not limited by resistance to mass transfer, enabling very rapid separations that are not adversely affected by low temperature. Previously, we have demonstrated an integrated microfluidic device coupling CE with electrospray ionization (ESI) capable of very rapid and high efficiency separations. In this work, we demonstrate the utility of this microchip CE-ESI device for HX MS. High speed CE-ESI of a bovine hemoglobin pepsin digestion was performed in 1 minute with a peak capacity of 62 versus a similar LC separation performed in 7 minutes with peak capacity of 31. A room temperature CE method performed in 1.25 minutes provided similar deuterium retention as an 8.5 minute LC method conducted at 0 °C. Separation of a complex mixture with CE was done with considerably better speed and nearly triple the peak capacity than the equivalent separation by LC. Overall the results indicate the potential utility of microchip CE-ESI for HX MS.

Pubmed: 25992468

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Steric gate residues of Y-family DNA polymerases DinB and pol kappa are crucial for dNTP-induced conformational change.
Nevin P, Engen JR, Beuning PJ.
DNA Repair. 2015. May;29:65-73. 

ABSTRACT
Discrimination against ribonucleotides by DNA polymerases is critical to preserve DNA integrity. For many DNA polymerases, including those of the Y family, rNTP discrimination has been attributed to steric clashes between a residue near the active site, the steric gate, and the 2′-hydroxyl of the incoming rNTP. Here we used hydrogen/deuterium exchange (HDX) mass spectrometry (MS) to probe the effects of the steric gate in the Y-family DNA polymerases Escherichia coli DinB and human DNA pol κ. Formation of a ternary complex with a G:dCTP base pair in the active site resulted in slower hydrogen exchange relative to a ternary complex with G:rCTP in the active site. The protection from exchange was localized to regions both distal and proximal to the active site, suggesting that DinB and DNA pol κ adopt different conformations depending on the sugar of the incoming nucleotide. In contrast, when the respective steric gate residues were mutated to alanine, the differences in HDX between the dNTP- and rNTP-bound ternary complexes were attenuated such that for DinB(F13A) and pol κ(Y112A), ternary complexes with either G:dCTP or G:rCTP base pairs had similar HDX profiles. Furthermore, the HDX in these ternary complexes resembled that of the rCTP-bound state rather than the dCTP-bound state of the wild-type enzymes. Primer extension assays confirmed that DinB(F13A) and pol κ(Y112A) do not discriminate against rNTPs to the same extent as the wild-type enzymes. Our observations indicate that the steric gate is crucial for rNTP discrimination because of its role in specifically promoting a dNTP-induced conformational change and that rNTP discrimination occurs in a relatively closed state of the polymerases.

Pubmed: 25684709

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Hydrogen/Deuterium exchange mass spectrometry applied to IL-23 interaction characteristics: potential impact for therapeutics.
Iacob RE, Krystek SR, Huang RY-C, Wei H, Tao L, Lin Z, Morin PE, Doyle ML, Tymiak AA, Engen JR, Chen G

Expert Rev Proteomics. 2015. Apr;12(2):159-169.

ABSTRACT
IL-23 is an important therapeutic target for the treatment of inflammatory diseases. Adnectins are targeted protein therapeutics that are derived from domain III of human fibronectin and have a similar protein scaffold to antibodies. Adnectin 2 was found to bind to IL-23 and compete with the IL-23/IL-23R interaction, posing a potential protein therapeutic. Hydrogen/deuterium exchange mass spectrometry and computational methods were applied to probe the binding interactions between IL-23 and Adnectin 2 and to determine the correlation between the two orthogonal methods. This review summarizes the current structural knowledge about IL-23 and focuses on the applicability of hydrogen/deuterium exchange mass spectrometry to investigate the higher order structure of proteins, which plays an important role in the discovery of new and improved biotherapeutics.
 
Pubmed: 25711416


Inhibition of pro-apoptotic BAX by a noncanonical interaction mechanism.
Barclay LA, Wales TE, Garner TP, Wachter F, Lee S, Guerra RM, Stewart ML, Braun CR, Bird GH,
Gavathiotis E, Engen JR, Walensky LD.

Mol. Cell. 2015. Mar 5;57(5):873-886.


ABSTRACT

BCL-2 is a negative regulator of apoptosis implicated in homeostatic and pathologic cell survival. The canonical anti-apoptotic mechanism involves entrapment of activated BAX by a groove on BCL-2, preventing BAX homo-oligomerization and mitochondrial membrane poration. The BCL-2 BH4 domain also confers anti-apoptotic functionality, but the mechanism is unknown. We find that a synthetic α-helical BH4 domain binds to BAX with nanomolar affinity and independently inhibits the conformational activation of BAX. Hydrogen-deuterium exchange mass spectrometry demonstrated that the N-terminal conformational changes in BAX induced by a triggering BIM BH3 helix were suppressed by the BCL-2 BH4 helix. Structural analyses localized the BH4 interaction site to a groove formed by residues of α1, α1–α2 loop, and α2–α3 and α5–α6 hairpins on the BAX surface. These data reveal a previously unappreciated binding site for targeted inhibition of BAX and suggest that the BCL-2 BH4 domain may participate in apoptosis blockade by a noncanonical interaction mechanism.   

Pubmed: 25684204

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Applications of Hydrogen/Deuterium exchange MS from 2012 to 2014.
Pirrone GF, Iacob RE, Engen JR.
Anal. Chem. 2015. Jan 6;87(1):99-118.
Special Issue: Fundamental and Applied reviews in Analytical Chemistry 2015


ABSTRACT

Hydrogen/Deuterium exchange (HDX) detected by mass spectrometry (MS) is extraordinarily useful in the study of many aspects of proteins, especially the analysis of protein conformation and dynamics.  While once a challenging and therefore sparingly used method, modern HDX MS is more straightforward, rapid and routine than in the past.  As a result, the breadth of applications of the method has expanded.  This Review catalogs applications of HDX MS that have appeared in the literature during the 30 months from January 2012 to June 2014.  As penetration of the method into nonacademic sectors where confidentiality is necessary is also at an all-time high, many more applications of this method likely exist that have not been reported in the literature.

Pubmed: 25398026