Citrate synthase, in its rate-determining step, abstracts a proton from the methyl group of acetylCoenzyme A to form a carbon-carbon double bond. It is fascinating that these different fluorescent proteins are nevertheless based on the same Xaa-Tyr-Gly peptide.įor many years we worked to determine structure function relationships in citrate synthase, which is the entry to the citric acid cycle and is found in every organism examined. Crystal structures were determined of related fluorescent proteins from corals that fluoresce yellow, orange and red, enabling multicolor reporting of a variety of cellular processes. Furthermore, the color of the protein can be modified by changing the environment or internal structure of the chromophore, which is derived from the primary sequence (Xaa)65-Tyr66-Gly67. Using structure-based genetic engineering techniques we successfully constructed visual pH indicators, halide (chloride) concentration indicators and sensors that report on the thiol/disulfide redox potential within cells. GFP and its red, yellow and blue cousins are enormously popular as visible tag for proteins of interest or as a marker for gene expression. Since 1996 we have worked with Green Fluorescent Protein, which spontaneously rearranges itself to become fluorescent, absorbing blue light and re-emitting green light. Studies of TlpA, TlpB and TlpD are ongoing in both laboratories. Much subsequent work has, for the first time, led to a rather complete understanding of how a receptor (TlpB, see illustration) can sense pH, allowing the bacteria to navigate away from the low-pH interior of the stomach to the lining, where they can induce inflammation, ulcers and even cancerous transformations. Crystal structures were determined for TlpA and TlpB, two of the three critical chemotaxis receptors. In collaboration with Karen Guillemin’s group, we are interested to understand the structure and function of sensory proteins that allow the bacterium Helicobacter pylori to thrive within the hostile environment of the human stomach. In the laboratory, chemists and biologists collaborate with physicists to achieve a broader intellectual basis for the research.
The primary techniques we use are mutagenesis, x-ray crystallography and spectroscopy, but occasionally we perform computer modeling of enzyme active sites and other properties of proteins. Our group uses an interdisciplinary approach in applying physical techniques to the study of biological molecules, especially the structure, function, and interaction of enzymes, chemoreceptors and fluorescent proteins.
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