Brian R. Crane
The Structural Chemistry of Biological Timing
Research in the Crane Group is directed towards understanding interactions among proteins, electrons, and photons. Specifically, we are interested in how metalloenzymes stabilize transient intermediates during catalysis, how protein structure controls long-range electron transfer, and how photo and redox processes are used in biological information transfer. To correlate protein structure directly with reactivity we combine genetic and chemical manipulation of proteins, atomic-resolution structure determination, and novel photochemically triggered experiments in single crystals.
Structures of Metalloenzyme Activated States
Catalytically key metalloenzyme redox states can be difficult to characterize because they are often unstable and generated transiently in situ. We uniformly stimulate chemical reactions in single protein crystals by electron transfer to and from transition metal active centers at rates where important species can be observed by time-resolved crystallography or captured by cryocrystallography. Systems of interest include the production of nitric oxide by mammalian nitric oxide synthases, a heme-peroxide intermediate important in the generation of reactive oxidants for biosynthesis and detoxification, and intermediates in the six-electron reductions of sulfite to sulfide and nitrite to ammonia by sulfite and nitrite reductases.
Evolutionary Constraints on Protein Electron Transfer
Although specific protein structure can influence electronic coupling between donor and acceptor redox sites and thereby facilitate biological electron transfer, the relative importance of electronic coupling as a control mechanism is less clear. We intend to use genetic selections for competent oxidoreductases to optimize linkages between catalytic and electron-supplying protein domains. Successful recombinant molecules capable of supporting growth under the selection conditions will be characterized to determine the requirements for functional electronic communication across protein interfaces.
Light and Redox Sensing
The ability to sense and respond to the environment is a primary requirement of any living organism. We are interested in the biophysical mechanisms that allow organisms to monitor energy in their surroundings. Specifically, we are studying proteins involved in bacterial taxis and mammalian circadian clocks. In these systems, light or reducing energy is trapped by cofactors within sensory proteins. Through unknown mechanisms this captured energy is transduced to the production of new interactions among response proteins within the cell. We aim to determine structures of sensory proteins in different redox states and in association with target response proteins. We also intend to characterize electron transfer mechanisms that allow energy conversion among components of signaling pathways.
Photoinduced electron transfer in single protein crystals. (A) Crystal structure of the blue-copper protein azurin chemically modified with a ruthenium photosensitizer. (B) Transient absorption trace of electron transfer between the azurin Cu and Ru metal centers in a single crystal. On excitation with 480 nm light the Ru2+ complex injects an electron into the protein Cu2+ center. Ru3+ formation produces a bleach at 430 nm, which then recovers during the back electron transfer from Cu1+ to Ru3+.