PUBLICATIONS: Before 2005

 
Fetal alcohol exposure alters neurosteroid levels in the developing rat brain.
Caldeira JC, Wu Y, Mameli M, Purdy RH, Li PK, Akwa Y, Savage DD, Engen JR, Valenzuela CF.
J Neurochem. 2004 Sep;90(6):1530-9.

ABSTRACT
Neurosteroids are modulators of neuronal function that may play important roles in brain maturation. We determined whether chronic prenatal ethanol exposure altered neurosteroid levels in the developing brain. Rat dams were exposed to: (i) a 5% ethanol-containing liquid diet that produces peak maternal blood alcohol levels near the legal intoxication limit (approximately 0.08 g/dL); (ii) an isocaloric liquid diet containing maltose-dextrin instead of ethanol with pair-feeding; (iii) rat chow ad libitum. Neurosteroid levels were assessed in offspring brains using radioimmunoassay or gas chromatography-mass spectrometry techniques. A prenatal ethanol exposure-induced increase in pregnenolone sulfate levels, but not dehydroepiandrosterone sulfate levels, was evident at the earliest time point studied (embryonic day 14). This effect lasted until post-natal day 5. Levels of other neurosteroids were assessed at embryonic day 20; pregnenolone levels, but not allopregnanolone levels, were elevated. Pregnenolone sulfate levels were not altered in the maternal brain. Neither pregnenolone nor pregnenolone sulfate levels were significantly altered in the fetal liver, placenta and maternal blood, indicating that the effect of ethanol is not secondary to accumulation of peripherally-produced steroids. Fetal ethanol exposure has been shown to decrease both cellular and behavioral responsiveness to neurosteroids, and our findings provide a plausible explanation for this effect.
 
Pubmed: 15341536

 
What mass spectrometry can reveal about protein function.

Wu Y, Engen JR. Analyst. 2004 Apr;129(4):290-6. Cover Feature

Pubmed: 15042158

Analysis of protein complexes with hydrogen exchange and mass spectrometry.
Engen JR.
Analyst. 2003 Jun;128(6):623-8.

ABSTRACT
Analysis of protein complexes using hydrogen exchange (HX) combined with high resolution electrospray mass spectrometry (MS) is demonstrated. HX MS offers the possibility to analyze the strength of binding in protein complexes, to identify regions that undergo binding induced structural changes, and to study the nature (hydrophobic, electrostatic, etc.) of binding between two or more proteins. In the current work, a heteromeric complex containing UBC9 (an E2 conjugating enzyme) and SUMO-1 (a ubiquitin-like modifier) was investigated by incubating the complex in D2O and measuring the amount of deuterium incorporation with MS. SUMO-1 had significant changes in deuterium levels when bound to UBC9. In contract, few or no changes in deuterium levels were detected in UBC9 when part of the complex, even at the binding interface. Titrations were used to estimate the binding constant for the complex. The nature of the interface was probed by creating a site-directed mutant form of UBC9. The mutant form showed no detectable binding to SUMO-1 and thereby suggested that binding between these two proteins is primarily electrostatically driven. This application of HX MS demonstrates its value in the study of protein complexes and protein machinery.
 
Pubmed: 12866878

 
Regulation of c-Fes tyrosine kinase activity by coiled-coil and SH2 domains: analysis with Saccharomyces cerevisiae.
Takashima Y, Delfino FJ, Engen JR, Superti-Furga G, Smithgall TE.
Biochemistry. 2003 Apr 1;42(12):3567-74.

ABSTRACT
The c-Fes protein-tyrosine kinase regulates the growth and differentiation of diverse cell types, including myeloid hematopoietic cells, vascular endothelial cells, and neurons. Structurally, Fes is composed of a unique N-terminal region with coiled-coil oligomerization motifs, followed by SH2 and kinase domains. Although Fes kinase activity is tightly regulated in cells, the structural basis for its negative regulation is not clear. In this report, c-Fes was expressed in Saccharomyces cerevisiae to determine whether regulation is kinase-intrinsic or dependent upon protein factors found in mammalian cells. Wild-type Fes kinase activity was completely repressed in yeast and did not affect cell growth. Mutation or deletion of the more N-terminal c-Fes coiled-coil domain reversed negative regulation, leading to strong kinase activation and suppression of yeast cell growth. Similarly, replacement of the wild-type SH2 domain with that of v-Src induced strong kinase activation and the growth-inhibitory phenotype. Immunoblotting with phosphospecific antibodies shows that activation of Fes by either mechanism induced autophosphorylation of the activation loop tyrosine residue (Tyr 713). These data support the idea that Fes naturally adopts an inactive conformation in vivo, and that maintenance of the inactive structure requires the coiled-coil and SH2 domains.
 
Pubmed: 12653561

 
Determining the site of spin trapping of the equine myoglobin radical by combined use of EPR, electrophoretic purification, and mass spectrometry.
Harris MN, Burchiel SW, Winyard PG, Engen JR, Mobarak CD, Timmins GS.
Chem Res Toxicol. 2002 Dec;15(12):1589-94.

ABSTRACT
Although myoglobin protein radicals are thought important intermediates in peroxide-induced toxicity, the site of spin trapping of this radical in equine myoglobin using the trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) is unclear. We have combined EPR, electrophoretic adduct purification, and mass spectrometry approaches to unambiguously determine the site of trapping to be Tyr-103 and suggest that reports of trapping at Trp-7 or Trp-14 may be due to nonradical addition to proteolytically derived Trp-containing peptides with DBNBS. The technique developed here of combining electrophoretic separation of DBNBS adducts with MS of resultant peptides will also allow proteomic-like approaches to determining identities and sites of radical formation and translocation on complex mixtures of proteins.
 
Pubmed: 12482241

Using stable-isotope-labeled proteins for hydrogen exchange studies in complex mixtures.
Engen JR, Bradbury EM, Chen X.
Anal Chem. 2002 Apr 1;74(7):1680-6.

ABSTRACT
The use of mass spectrometry to measure hydrogen exchange rates for individual proteins in complex mixtures is described. Incorporation of stable-isotope-labeled (SIL) amino acids into a protein of interest during overexpression in bacteria produced distinctive isotope patterns in mass spectra of peptic peptides from the labeled protein. The isotope pattern was used as a signature for peptides originating from the SIL protein. In addition, stable-isotope labeling simplified identification of the peptic peptides by providing partial amino acid composition information. Despite the complex isotope patterns associated with SIL peptides, hydrogen exchange rates could still be measured for peptides from SIL protein and were found to be the same as exchange rates for unlabeled protein. Hydrogen exchange in a single protein of interest was measured in a complex mixture of proteins, a bacterial cell lysate. This methodology, which includes easy recognition of peptic peptides from the protein(s) of interest during hydrogen exchange studies in heterogeneous systems, will permit analysis of structural properties and dynamics of large protein complexes and complex protein systems.
 
Pubmed: 12033260

 
Intramolecular binding of a proximal PPII helix to an SH3 domain in the fusion protein SH3Hck : PPIIhGAP.
Gmeiner WH, Xu I, Horita DA, Smithgall TE, Engen JR, Smith DL, Byrd RA.
Cell Biochem Biophys. 2001;35(2):115-26.

ABSTRACT
SH3 domains are a conserved feature of many nonreceptor protein tyrosine kinases, such as Hck, and often function in substrate recruitment and regulation of kinase activity. SH3 domains modulate kinase activity by binding to polyproline helices (PPII helix) either intramolecularly or in target proteins. The preponderance of bimolecular and distal interactions between SH3 domains and PPII helices led us to investigate whether proximal placement of a PPII helix relative to an SH3 domain would result in tight, intramolecular binding. We have fused the PPII helix region of human GAP to the C-terminus of Hck SH3 and expressed the recombinant fusion protein in Escherichia coli. The fusion protein, SH3Hck : PPIIhGAP, folded spontaneously into a structure in which the PPII helix was bound intramolecularly to the hydrophobic crevice of the SH3 domain. The SH3Hck : PPIIhGAP fusion protein is useful for investigating SH3: PPII helix interactions, for studying concepts in protein folding and design, and may represent a protein structural motif that is widely distributed in nature.
 
Pubmed: 11892787

 
Phosphorylation and structure-based functional studies reveal a positive and a negative role for the activation loop of the c-Abl tyrosine kinase.
Dorey K, Engen JR, Kretzschmar J, Wilm M, Neubauer G, Schindler T, Superti-Furga G.
Oncogene. 2001 Dec 6;20(56):8075-84.

ABSTRACT
c-Abl is a nuclear and cytoplasmic tyrosine kinase involved in a variety of cellular growth and differentiation processes. In contrast to its oncogenic counterparts, like BCR-Abl, c-Abl is not constitutively tyrosine phosphorylated and its catalytic activity is very low. Here we report tyrosine phosphorylation of endogenous c-Abl and a concomitant increase in catalytic activity. Using Abl -/- cells reconstituted with mutated c-Abl forms, we show that phosphorylation and activity depend on Tyr412 in the activation loop. Tyr412 is also required for stimulation by PDGF or by cotransfection of active Src. Phosphorylation of Tyr412 can occur autocatalytically by a trans-mechanism and cause activation of otherwise inactive c-Abl, suggesting a positive feedback loop on c-Abl activity. In the recent structure of the Abl catalytic domain bound to the STI-571 inhibitor, unphosphorylated Tyr412 in the activation loop points inward and appears to interfere with catalysis. We mutated residues involved in stabilizing this inhibited form of the activation loop and in positioning Tyr412. These mutations resulted in tyrosine phosphorylation and activation of c-Abl, as if relieving c-Abl from inhibition. Tyr412 is therefore necessary both for activity and for regulation of c-Abl, by stabilizing the inactive or the active conformation of the enzyme in a phosphorylation-dependent manner.
 
Pubmed: 11781820

Investigating protein structure and dynamics by hydrogen exchange MS.

Engen JR, Smith DL. Anal Chem. 2001 May 1;73(9):256A-265A Cover Feature

Pubmed: 11354508

 
Investigating the higher order structure of proteins. Hydrogen exchange, proteolytic fragmentation, and mass spectrometry.
Engen JR, Smith DL.
Methods Mol Biol. 2000;146:95-112.

abstract
It has been apparent for some years that the structures of proteins are dynamic rather than static. For some proteins, dynamics is essential to function (e.g., refs. 1–7). These structural changes have been detected for more than 30 yr by observing hydrogen exchange between peptide amide hydrogens and solvent containing the hydrogen isotopes tritium or deuterium (Refs. 8-10). Although tritium is no longer used extensively for this purpose, deuterium is widely used in hydrogen exchange studies, especially in multidimensional nuclear magnetic resonance (NMR), in which amide hydrogen signals disappear on deuteration. Since deuterium weighs 1 Dalton more than protium, hydrogen exchange in proteins can also be detected by mass spectrometry. This approach is complementary to NMR in some respects and clearly advantageous in others.
 Pubmed: 10948498

 
Hydrogen exchange shows peptide binding stabilizes motions in Hck SH2.
Engen JR, Gmeiner WH, Smithgall TE, Smith DL.
Biochemistry. 1999 Jul 13;38(28):8926-35.

ABSTRACT
Src-homology-2 domains are small, 100 amino acid protein modules that are present in a number of signal transduction proteins. Previous NMR studies of SH2 domain dynamics indicate that peptide binding decreases protein motions in the pico- to nanosecond, and perhaps slower, time range. We suggest that amide hydrogen exchange and mass spectrometry may be useful for detecting changes in protein dynamics because hydrogen exchange rates are relatively insensitive to the time domains of the dynamics. In the present study, hydrogen exchange and mass spectrometry were used to probe hematopoietic cell kinase SH2 that was either free or bound to a 12-residue high-affinity peptide. Hydrogen exchange rates were determined by exposing free and bound SH2 to D(2)O, fragmenting the SH2 with pepsin, and determining the deuterium level in the peptic fragments. Binding generally decreased hydrogen exchange along much of the SH2 backbone, indicating a widespread reduction in dynamics. Alterations in the exchange of the most rapidly exchanging amide hydrogens, which was detected following acid quench and analysis by mass spectrometry, were used to locate differences in low-amplitude motion when SH2 was bound to the peptide. In addition, the results indicate that hydrogen exchange from the folded form of SH2 is an important process along the entire SH2 backbone.
 
Pubmed: 10413466

 
Comparison of SH3 and SH2 domain dynamics when expressed alone or in an SH(3+2) construct: the role of protein dynamics in functional regulation.
Engen JR, Smithgall TE, Gmeiner WH, Smith DL.
J Mol Biol. 1999 Apr 2;287(3):645-56.

ABSTRACT
Protein dynamics play an important role in protein function and regulation of enzymatic activity. To determine how additional interactions with surrounding structure affects local protein dynamics, we have used hydrogen exchange and mass spectrometry to investigate the SH2 and SH3 domains of the protein tyrosine kinase Hck. Exchange rates of isolated Hck SH3 and SH2 domains were compared with rates for the same domains when part of a larger SH(3+2) construct. Increased deuterium incorporation was observed for the SH3 domain in the joint construct, particularly near the SH2 interface and the short sequence that connects SH3 to SH2, implying greater flexibility of SH3 when it is part of SH(3+2). Slow cooperative unfolding of the SH3 domain occurred at the same rate in isolated SH3 as in the SH(3+2) construct, suggesting a functional significance for this unfolding. The SH2 domain displayed relatively smaller changes in flexibility when part of the SH(3+2) construct. These results suggest that the domains influence each other. Further, our results imply a link between functional regulation and structural dynamics of SH3 and SH2 domains.
 
Pubmed: 10092465

 
Identification and localization of slow, natural, cooperative unfolding in the hematopoietic cell kinase SH3 domain by amide hydrogen exchange and mass spectrometry.
Engen JR, Smithgall TE, Gmeiner WH, Smith DL.
Biochemistry. 1997 Nov 25;36(47):14384-91.

ABSTRACT
Protein unfolding on a fast time scale (milliseconds-minutes) has been widely reported, but slower unfolding events (10 min-hours) have received less attention. Amide hydrogen exchange (HX) and mass spectrometry (MS) were used to investigate the unfolding dynamics of the hematopoietic cell kinase (Hck) SH3 domain. Analysis of mass spectra after deuterium exchange into intact Hck SH3 indicates a cooperative unfolding event involving 24-61% of the domain and occurring with a half-life of approximately 20 min under physiological conditions. To identify the unfolding region, SH3 was incubated in D2O and proteolytically fragmented into peptides that were analyzed by mass spectrometry. Correlation of HX rates and isotope patterns reveals cooperative unfolding in several regions, including the C-terminal half of the RT-loop and a beta-sheet flanking the binding site. Binding of a prolyl-rich segment from the HIV Nef protein slows unfolding by a factor of 3. Further analysis yields a KD of 25 microM for the Nef peptide. These results demonstrate that an inherent flexibility in the SH3 domain may assist interconversion of the closed, intramolecularly ligated state and the open, active state of Src family kinases. Furthermore, this type of previously undetectable, slow unfolding process may provide the basis for new mechanisms in which kinetics of local unfolding combines with thermodynamics to regulate enzymatic activity. The combination of hydrogen exchange and mass spectrometry appears to be the only general method capable of examining these slow unfolding processes.
 
Pubmed: 9398156

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