PUBLICATIONS: 2008

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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