Biophysical/Organic Chemistry

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, usually by optical means, such as fluorescent readout or photochemical release of messenger substances. We have developed fluorescence probes that change their color in response to Ca2+, Na+, pH, membrane potential, cAMP, and gene transcription in single living cells. Fura-2 is an example (see accompanying structure and spectra), which detects Ca2+ concentrations of 10-7 to 10-6 M inside living cells with a spatial resolution of a micron or so and a temporal resolution of a fraction of a second. Recently we have also designed and produced pairs of molecules that work together to detect the voltage across cell membranes with a much better combination of speed and sensitivity than previously available. Membrane voltage and Ca2+ are the two most ubiquitous and important signals that neurons use internally to transmit information, so the ability to see their fluctuations is essential for analyzing the workings of the brain, which is still the most sophisticated and complex molecular assembly known.

Although the ion and membrane potential indicators were built by traditional organic synthesis, which is still a major activity in the laboratory, we are also active in protein engineering. For example, in collaboration with Susan Taylor's group, we labeled cyclic AMP-dependent protein kinase with dual fluorescent tags so that the enzyme becomes a fluorescent sensor for cyclic AMP. Imaging of this enzyme shows that remarkable spatial gradients of cyclic AMP are generated in neurons that are being trained to modify their synaptic properties. Another approach is based on a naturally fluorescent protein from jellyfish, the Green Fluorescent Protein, which we have mutagenized to alter its color in both possible directions, towards blue and yellow. Elucidation of the crystal structure of GFP will enable further rational engineering of this fascinating protein, which spontaneously synthesizes a heterocyclic fluorophore inside itself and tunes the wavelengths using side chains of neighboring amino acids. Molecular biology and organic synthesis offer many other areas of synergistic collaboration. For example, the most important long-term consequences of cellular signaling are changes in gene expression, which we can now see in individual living cells. We engineered the enzyme-lactamase to be a reporter enzyme and synthesized fluorescent cephalosporins as substrates that change color when the gene of interest is expressed. This technology has not only major research applications but is also of industrial importance as a way of testing the cellular effects of large numbers of potential drugs or toxins.

A complementary area of interest is the use of light not just to see dynamic biochemistry but to perturb it in a controlled manner. Here the key is the design and synthesis of molecules that photochemically release or absorb messenger substances such as Ca2+ , cyclic GMP, and nitric oxide (NO). Recently we have used such "caged" messengers to show that a major form of information storage in the brain is triggered by the coincidence of Ca2+ and NO, which then act through cyclic GMP and protein phosphorylation to modify the synapses between neurons.