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PUBLICATIONS: Before
2004
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.
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.
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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