Brozik, James A.

Research

Brozik Group Website

 

Biophysics

Our biophysical work utilizes dynamic single molecule imaging, fluorescence correlation spectroscopy, and patch-clamp electrochemistry to answer basic questions about how dynamical changes in protein structure are correlated to biochemical function.
The first project addresses the mechanics and mechanism of HIV-1 Reverse Transcriptase. For this study we have developed fluorescence probes and single molecule imaging techniques as well as a much faster two channel (two different colors or two separate polarizations) time-correlated-single-photon-counting experiment to study the chemical and structural dynamics of single enzyme / protein / function and the function of more complex protein assemblies.
The second project probes the basic mechanisms, structure-function relationships, and thermodynamic principles of membrane bound proteins (biological ion channels in particular). In this project we have developed experimental techniques to simultaneously monitor or identify the different structural states of individual membrane bound proteins within lipid bilayers (through single molecule fluorescence spectroscopy) while simultaneously measuring ion current. Single channel recordings are accomplished with a micro fabricated on-chip patch clamp electrochemical cells and lipid coated nanoporous beads. In this effort we have concentrated on gramicidin, bacteriorhodopsin, and the serotonin type 3 receptor (5HT3).

 

Material Science

Our material science program is generally centered on understanding the physical properties and basic design principles necessary to build molecular scale machines. This research thrust area has two major goals:

1. understanding the structural and electronic principles of energy conduction and the charge transfer properties of molecular assemblies and conjugated molecules, and
2. understanding the basic chemical physics necessary to produce mechanical motion from an electromagnetic stimulus.

In addition to the synthetic molecular machine studies we are also engaged in developing self-assembly techniques on solid-supported planar substrates and nano-porous silica beads that are suitable platforms in biosensors and for biophysical studies of membrane proteins.

 

Biosensors

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 the development of luminescence transduction schemes based on distance dependent phenomena and in the development of new amplification schemes based upon a membrane disruption that is triggered by an analyte binding event.

 

Instrument Design and Fabrication

Much of our research hinges on our ability (and even adaptability) to design and build new optical based experiments. In this regard we have designed and built time-resolved luminescence experiments that span from 100 fs to sec using photon counting, time-correlated-single-photon-counting, and luminescence upconversion; a time-resolved infrared experiment in the nanosecond time regime; electronic and vibrational imaging experiments using home built spectrometers and high precision stages; a variety of microscopes that are suitable for single molecule fluorescence detection; single molecule fluorescence experiments combined electrochemical detection; 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.

Publications

• Elsa I. Silva-Lopez, Adam O. Barden, and James A. Brozik* “Near Native Binding of a Fluorescent Serotonin Conjugate to Serotonin Type-3 Receptors”, Bioorganic and Medicinal Chemistry Letters, http://dx.doi.org/10.1016/j.bmcl.2012.11.082, 2012.Lance E. Edens, James A. Brozik, and David J. Keller* “Course-Grained Model DNA: Structure, Sequences, Stems, Circles, Hairpins” Journal of Physical Chemistry B, DOI: 10.1021/jp3009095, ASAP, http://pubs.acs.org/doi/pdf/10.1021/jp3009095, 2012.

 

• Kumud R. Poudel, David J. Keller, and James A. Brozik* “The Effect of a Phase Transition on Single Molecule Tracks of Annexin V in Cushioned DMPC Assemblies”, Soft Matter, 8, 11285-11293, 2012.

 

• Kumud R. Poudel, Jeffery P. Jones, and James A. Brozik* “A Guide to Tracking Single Transmembrane Proteins in Supported Lipid Bilayers” Methods in Molecular Biology, Springer Press, In Press: Scheduled Publication Date is December 2012.

 

• Kumud R. Poudel, David J. Keller, and James A. Brozik* “Single Particle Tracking Reveals Corralling of a Transmembrane Protein in a Double-Cushioned Bilayer Assembly” Langmuir, 27(1), 320-327, 2011.

 

 

• Christopher M. Hartshorn, Cayla M. Jewett, and James A. Brozik* “Molecular Effects of a Nanocrystalline Quartz Support Upon Planar Lipid Bilayers” Langmuir, 26(4), 2609-2617, 2010.

 

• Ryan W. Davis, Adrean Flores, Todd Barrick, Jason M. Cox, Susan M. Brozik, Gabriel P. Lopez, and James A. Brozik* “Nanoporous Microbead Supported Bilayers: Stability, Physical Characterization, and Incorporation of Functional Transmembrane Proteins” Langmuir 23, 3864-3872, 2007.

 

• Theodore P. Ortiz, Jason A. Marshall, Lauren A. Meyer, Ryan W. Davis, Jed C. Macosko, David J. Keller, and James A. Brozik* “Stepping Statistics of Single HIV-1 Reverse Transcriptase Molecules During DNA Polymerization”. Journal of Physical Chemistry B 109 (33), 16127 – 16131, 2005.

 

• David J. Keller* and James A. Brozik, “Framework Model for DNA Polymerases”. Biochemistry 44 (18), 6877 – 6888, 2005.

 

• Hialong Lu, Jed C. Macosko, James A. Brozik, and David J. Keller* “Motor Forces of HIV Reverse Transcriptase Closing of the Fingers Domain Generates Motor Forces in the HIV Reverse Transcriptase”. Journal of Biological Chemistry 279, 54529-54532, 2004.

 

• Ryan W. Davis, Elizabeth L. Patrick, Lauren A. Meyer, Theodore P. Ortiz, Jason A. Marshall, David J. Keller, Susan M. Brozik, and James A. Brozik* “The Thermodynamic Properties of Single Ion Channel Formation: Gramicidin”. Journal of Physical Chemistry B, 108, 15364-15369, 2004.