Research / Clinical Summary

Roger Tsien, PhD Professor, Pharmacology / Chemistry & Biochemistry Cancer Biology Program Contact by Email

Naturally Fluorescent Proteins
The overall goal of my laboratory is to understand how living cells and neuronal networks process information. Our preferred approach is through the rational design, synthesis, and use of new molecules to detect and manipulate intracellular biochemical signals. We build both small synthetic molecules and genetically encoded macromolecules, which preferably work together in synergy. One major approach has been based on the Green Fluorescent Protein (GFP) from jellyfish. GFP has revolutionized much of molecular and cell biology because it provides the first genetic means to encode strong visible fluorescence. We improved the spectral purity and brightness of the native protein and created mutants with blue, cyan, and yellow fluorescence. These mutants can monitor dynamic protein interactions such as association of nuclear hormone receptors with coactivator proteins, homotrimerization of plasma membrane receptors that trigger cell suicide, and aggregation of lipid-anchored proteins into membrane 'rafts'. We have also engineered chimeric fluorescent proteins that monitor important intracellular signals such as Ca2+, cyclic AMP, and cyclic GMP. We are trying to extend this concept to other kinase and phosphatase activities and to apply these transfectable, targetable indicators in many biological systems.

Organic Synthetic Tags Targeted by Molecular Biology
Although GFP and its descendants are enormously useful, they still have many limitations. Longer wavelengths were first achieved in a somewhat analogous red fluorescent protein from coral. Recently we determined the structure of its red fluorophore by mass spectrometry, but the protein still needs considerable re-engineering. Another problem is that all these proteins contain over 200 amino acids and can only confer fluorescence, so it would be desirable to engineer a much smaller peptide motif, ideally only a few amino acids long, which could be labeled in vivo with designed small organic molecules. Such a system would combine molecular biology's precise but flexible targeting with organic chemistry's versatility to introduce properties such as photochemical reactivity, paramagnetism, crosslinking, etc. We showed that a motif containing four cysteines could indeed be labeled in live mammalian cells with dye molecules containing two appropriately spaced arsenic atoms. Now we are developing a second motif without cysteines to be recognized by a different set of dyes, to permit simultaneous labeling or crosslinking of two different proteins and to avoid the oxidizability and potential toxicity of the tetracysteine-biarsenical combination.

Towards Imaging Specific mRNAs in Intact Animals.
Among the most important long-term consequences of cellular signaling are changes in gene transcription. We are developing nonoptical readouts such as positron emission tomography, magnetic resonance imaging, or ultrasound, to noninvasively image gene expression in live opaque organisms. We also are interested in approaches to link endogenous mRNA levels to reporter RNAs to enable us to image mutant vs. normal mRNAs, for example in tumors.

The projects in this laboratory encompass many disciplines ranging from organic synthesis, engineering and directed evolution of protein and nucleic acids, optical spectroscopy and photochemistry, computerized microscopy and image processing, cell biology, and neurobiology.


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SELECTED PUBLICATIONS

Quantitation of Transcription and Clonal Selection of Single Living Cells Using Beta-lactamase as Reporter. With G. Zlokarnik, P.A. Negulescu, T.E. Knapp, L. Mere, N. Burres, L. Feng, M. Whitney, and K. Roemer, Science 279, 84 (1998).

Specific Covalent Labeling of Recombinant Protein Molecules Inside Live Cells. With B.A. Griffin and S.R. Adams. Science 281, 269 (1998).

The Green Fluorescent Protein. Annual Review of Biochemistry 67, 509 (1998).

Circular Permutation and Receptor Insertion into Green Fluorescent Proteins. With G.S. Baird and D.A. Zacharias. Proc. Natl. Acad. Sci. USA 96, 11241 (1999).

A New Cell-permeable Fluorescent Probe for Zn2+. With G.K. Walkup, S.C. Burdette, and S.J. Lippard. J. Amer. Chem. Soc. 122, 5644 (2000).

The Structure of the Chromophore within DsRed, a Red Fluorescent Protein from Coral. With L.A. Gross, G.S. Baird, R.C. Hoffman, and K.K. Baldridge. Proc. Natl. Acad. Sci. USA 97, 11990 (2000).

Spatiotemporal dynamics of guanosine 3',5'-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator. With A. Honda, S.R. Adams, C.L. Sawyer, V. Lev-Ram, and W.R.G. Dostmann. Proc. Natl. Acad. Sci. USA, 98, 2437 (2001).

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