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

 

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

 

Rebecca Fenn
Predoctoral Fellow

 

email:becks@stanford.edu

 

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

 

Erica Raffauf
Predoctoral Fellow

 

email:

 

Rebecca Maile Weisinger
Predoctoral Fellow

 

email: rmweis@stanford.edu

 

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

Alumni

Where Am I Now?

 

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

 

 

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

 

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

 

Joshua Silverman
(Former Predoctoral Fellow in the Harbury lab)

Avidia Research Institute
2450 Bayshore Drive
Mountain View, CA 94043

email: josh.silverman@avidia.org

 

 Lab Photos (more to come shortly)

Harbury lab ski trip 2005

Holiday Party Winter 2001

 

 

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Last Updated 10/12/05