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Principal Investigator: Pehr. A. B. Harbury
Associate Professor of Biochemistry
For more information on the type of research
done in my lab please see our research site. For
more information on me you can also check out my
Searle Scholar profile at http://www.searlescholars.net/people/1999/harbury.html.
I am a recent winner of the prestigious MacArthur
Foundation Award, read more about me and my work at
http://www.macfound.org/programs/fel/fellows/harbury_pehr.htm
email: harbury@cmgm.stanford.edu
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F. Edward Boas
Predoctoral Fellow
For more information on me, please see my
website at http://b435-boas.stanford.edu/home/index.html.
Recent successes in the computational protein
design field include the design of a family of
right-handed alpha-helical bundle proteins with no
precedent in Nature, and the design of mutants of a
bistable integrin protein that adopt a single
conformation. In both of these examples, the target
structure was specified in advance, the design was
performed by a computer program, and the designed
sequence was validated experimentally, with a
crystal structure or functional assay.
Inspired by these examples and others, we would
like to develop a general computational method for
creating small-molecule binding sites in proteins.
Accomplishing this goal would put several exciting
applications on the horizon. For example, a protein
designed to bind the transition state of a chemical
reaction will catalyze that reaction. A protein
designed to switch conformation upon binding could
serve as a biosensor.
email: boas@stanford.edu
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Rebecca Fenn
Predoctoral Fellow
email:becks@stanford.edu
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Warham L. Martin
Postdoctoral Scholar
Understanding protein function is a core task of
biochemistry. Knowledge of a protein's
three-dimensional structure is necessary for the
complete understanding of protein function. The
determination of complete three-dimensional protein
structures requires using either X-ray
crystallography or NMR. These techniques involve
expensive equipment, specialized practioners, and
are time, reagent, and effort intensive. They have
been the exclusive domain of specialized, focussed
laboratories. My project is to bring PROTEIN
STRUCTURE to the PEOPLE!
The MPAX technology developed in the Harbury lab
is a tool for probing the solvent accessible
surface of any protein under almost arbitrary
conditions. We intend to extend this technology
into a tool for determining protein structure. The
MPAX method, (J Biol Chem 2002 Aug
23;277(34):30968-75) involves coexpressing the
protein of interest in E.coli with cysteine
tRNA synthase and cysteine tRNA with a non-cysteine
anticodon. Inducing these genes generates a pool of
protein with cysteines inserted (~1% frequency) at
these codons. In one expression one can generate a
pool of mutant proteins which may be analyzed
collectively. The misincorporated cysteines may be
probed with any of a variety of thiol-specific
chemistries. Silverman probed the solvent
accessibility of the misincorporated thiols and
developed both gel and mass spectrometry based
readouts of the data. We are extending this
methodology. We have synthesized heterofunctional
chemical crosslinkers to probe the protein
structure around surface exposed misincorporated
cysteines. They contain an a-iodoamide for thiol
reactivity, a perfluoroaryl azide for a UV-gated
generation of a highly reactive nitrene radical,
and an affinity handle containing vicinal diols
that bind to phenyl boronate. Crosslinked peptides
may be identified by mass spectrometry. By
identifying which segments of a protein crosslink
on the protein surface we will be able to acquire
an extensive set of distance constraints concerning
the protein structure. We will use these
constraints restrain the ROSETTA de novo
protein-folding algorithm. The ROSETTA algorithm is
capable of determining the structure of some small
proteins from sequence information alone. When
constrained with NMR data ROSETTA can generate
quite accurate structures. We expect to generate a
set of constraints similar in size to that obtained
in NMR experiments. Using this strategy of
misincorporation, crosslinking, mass spectrometry,
and ROSETTA we hope to make protein structure
generation accessible to the whole biological
research community. Vive le revolution!
email: warham@stanford.edu
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Erica Raffauf
Predoctoral Fellow
email:
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Rebecca Maile Weisinger
Predoctoral Fellow
email: rmweis@stanford.edu
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Jarrett Wrenn
Predoctoral Fellow
My research is focused on expanding the "DNA
Display" technology being developed in the lab to
interesting chemical systems. These systems include
non-biological polymers and non-protein/non-nucleic
acid biological polymers. Currently, I am adapting
N-substituted polyglycine ("peptoid") synthesis to
be compatible with DNA Display. In addition, I have
made progress towards enzymatic polyketide
synthesis in a DNA Display format. The long-term
goal is to evolve useful molecules out of
interesting and diverse polymer libraries.
email: sjwrenn@stanford.edu
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Alumni
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Where Am I
Now?
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Rama
Balakrishnan
(Former Postdoctoral Fellow in the Harbury lab)
Scientific Curator
300 Pasteur Drive, Room M339
Department of Genetics
School of Medicine
Stanford University
Stanford, California 94305-5120
Email: rama@genome.stanford.edu
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David Halpin
Predoctoral Fellow
SELEX and phage display are biological methods
used to identify DNA, RNA, and polypeptide ligands
to proteins of medicinal importance. The two
techniques derive their unrivaled selective power
from iterative amplification of biopolymer
libraries, interposed by selection for binding to a
protein target. Rare, promising compounds evolve
over multiple generations from a constantly
dividing biopolymer population. The restriction of
these methods to DNA, RNA and polypeptides severely
limits their utility in drug design. To overcome
this limitation, we are encoding the synthesis of
small molecule combinatorial libraries with DNA
"genes". By differential hybridization during the
course of a traditional split and pool
combinatorial synthesis, the DNA sequence of each
gene is read out and translated into a unique small
molecule structure. The ability to genetically
encode diverse classes of organic compounds permits
in vitro evolution of small molecules, and opens
numerous avenues to drug discovery and the
development of molecular tools.
Location: Postdoctoral Fellow
Rebecca Heald's lab
University of California, Berkeley
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James Havranek
(Former Predoctoral Fellow in the Harbury lab)
Location: Postdoctoral Fellow
David Baker's lab
University of Washington
A long-term goal of our lab is to engineer
proteins computationally that perform a useful
function via molecular recognition or catalysis.
Despite a demonstrated ability to redesign proteins
for stability, current design algorithms must
overcome a number of significant obstacles before
they are capable of engineering for function. My
research is centered on addressing three
limitations of computational protein design. First,
I developed a rapid continuum electrostatics model
suitable for protein design algorithms. This
component of protein energetics is crucial for
function, yet has been neglected in most design
efforts because accurate treatments tend to be
prohibitively costly in terms of computing time.
Second, I have developed a design framework which
simultaneously addresses both positive and negative
design. This allows for the design of proteins that
possess specificity. For instance, a protein may be
designed that will tightly bind a small molecule
but not chemically similar 'decoys'. Finally, I
have developed a parametric model that describes
the backbone of a (ß/alpha) 8 barrel protein.
This allows us to explore the backbone space of
this fold efficiently, by varying the parameters of
the models. We are currently designing proteins
using backbones generated by this model, rather
than backbones taken from experimentally determined
structures.
email: havranek@u.washington.edu
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Joshua Silverman
(Former Predoctoral Fellow in the Harbury lab)
Avidia Research Institute
2450 Bayshore Drive
Mountain View, CA 94043
email: josh.silverman@avidia.org
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Lab Photos (more to come
shortly)
Harbury
lab ski trip 2005
Holiday
Party Winter 2001
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