James A. Brozik, Ph.D
Washington State University
Department of Chemistry
Pullman, WA 99164-4630
Telephone: (509) 335-3746 (Office)
Fax: (509) 335-8867
Our biophysical work utilizes kinetically resolved single molecule imaging, fluorescence correlation spectroscopy, ensemble spectroscopy, and electrochemistry to answer basic questions about how dynamical changes in biochemical state and protein structure are connected to biochemical function. In this regard, we combine both biophysical and materials chemistry to understand, in detail, how the machine-like properties of membrane proteins are related to the structure of the individual subunits, the conformations of different protein assembly, the allosteric motions of the different subunits relative to one another, and to determine the free energy changes that drive transitions between different structural states and conformations of the assembly (i.e. their machine-like properties). In this effort we have concentrated on gramicidin, bacteriorhodopsin, the serotonin type-3 receptor (5HT3), cytochrome P450 (2C9 and 3A4; with Dr. Jeff Jones, WSU), and cytochrome P450 reductaces (with Dr. Jeff Jones, WSU). The second project addresses the mechanics and mechanism of HIV-1 Reverse Transcriptase (with Dr. David Keller, UNM). For this study we have developed fluorescence probes and single molecule imaging techniques as well as much faster two channel (two different colors or two separate polarizations) time-correlated-single-photon-counting experiments to study the chemical and structural dynamics of single enzyme / protein / function and the function of more complex protein assemblies.
Above: The graphic demonstrates how an individual Annexin V molecule (a membrane protein) will diffuse through a biomimetic membrane as the membrane undergoes a phase transition. Data was collected with a technique that utilized single molecule fluorescence. K. R. Poudel, D. J. Keller, and J. A. Brozik; Soft Matter, 8, 11285-11293, 2012
An interest in new and improved biosensors that probe small structural changes, substrate binding, fast kinetic processes, identification of biological molecules or particular structural states of biological molecules, and the states of biological membranes, is naturally derived from our biophysical research. To capitalize on this natural extension of our current research we have implemented a synergistic research program solely devoted to biosensor development. Our group’s primary interest in this area is; (1) the development of luminescence transduction schemes based on distance dependent phenomena, (2) development of new amplification schemes based upon a membrane disruption that is triggered by an analyte binding event, (3) bioadhesion to surfaces and surface bound receptors, and (4) new DNA and RNA barcoding technologies.
Above: A recently synthesized fluorescent analog of serotonin (5-hydroxytryptamine; 5HT*) and its binding curve to solubilized 5HT3R receptors. E. I. Silva-Lopez, A. O. Barden, and J. A. Brozik; Bioorganic and Medicinal Chemistry Letters, http://dx.doi.org/10.1016/j.bmcl.2012.11.082, 2012
Instrument Design and Fabrication:
Much of our research hinges on our ability (and even the adaptability) to design and build new optical based experiments. Throughout the lab’s history, we have designed and built time-resolved luminescence experiments that span from 100 fs to sec using photon counting, time-correlated-single-photon-counting, luminescence upconversion, and fluorescence correlation spectroscopy; a time-resolved infrared experiment in the nanosecond time regime; luminescence and Raman imaging experiments using home built spectrometers and high precision stages; a variety of microscopes that are suitable for single molecule fluorescence detection (confocal imaging with APD detection and wide-field through objective TIRF microscopy with ICCD and EMCCD cameras); cryogenic fiber optic based cryostats that are suitable for temperature dependent experiments from 2.1 K to room temperature; steady state luminescence spectroscopy with single photon counting detection; and Raman spectroscopy using CCD cameras. The optics lab is very adaptable. Experiments can be torn down and new experiments built as the need arises.
Above: The graphic above demonstrates how an individual 5HT3R receptors (a membrane protein) can be corralled and undergo hop-diffusion within a biomimetic membrane that has a BSA passivation layer. K. R. Poudel, D. J. Keller, and J. A. Brozik; Langmuir, 27(1), 320-327, 2011
Our material science program is generally centered around the self-assembly of lipid bilayers on solid-supported planar substrates and nano-porous silica beads that are suitable platforms for biosensors and for biophysical studies of membrane proteins.