Troy C. Messina, Ph.D.

Honors and Awards

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Mattie Allen Broyles Inaugural Year Research Chair (2007-2008)

NIH Ruth L. Kirschstein NRSA Post-Doctoral Fellow (2004-2007)

Molecular Biophysics Minisymposium Poster Prize (2007)

Molecular Biophysics Minisymposium Poster Prize (2006)

Best Student Paper Award, SPIE 2001: MEMS Components and Applications to Industry (2001)

National Honor Society (1991)

Experience

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Assistant Professor
Centenary College of Louisiana
Department of Physics
August 2007 to present

I am an assistant professor of physics at a liberal arts college in Shreveport, LA. The college maintains a high level of research goals involving undergraduates in addition to traditional liberal arts education, with a student to teacher ratio of twelve across the college. Our department develops the latest pedagogical techniques, e.g., inquiry and experienced based learning. In addition, Centenary College also has one of the first biophysics degree programs introduced at a liberal arts institution.

NIH Ruth L. Kirschstein NRSA Post-Doctoral Fellow
Rutgers, the State University of New Jersey
Department of Chemistry and Chemical Biology
August 2003 to present

I have been using time-correlated single photon counting (TCSPC) for biophysical kinetics experiments. I employ a femto- and picosecond mode Ti-Al₂O₃ laser for high temporal resolution of protein dynamics in solution. In bulk solutions, measurements are made for analyzing time-dependent fluorescence, rotational anisotropy, and Stokes shift. These measurements can provide detail about protein motions due to solvation, ligand-binding, and protein folding and fluctuations. These experiments provide a foundation for studying single molecules with confocal laser microscopy. I have been primarily interested in the chemosensory protein galactose/glucose binding protein for ligand-binding studies and ß-lactoglobulin (an amyloid-forming protein). Using unique fluorescent labeling schemes, the conformational, structural, and functional kinetics can be studied. In addition, the use of micro- and nanofluidics helps confine molecules to the microscope confocal volume without hindering natural behavior. I am implementing for the first time hidden Markov models (HMM) to analyze photon streams, which make maximum use of the information provided by these experiments.

Post-Doctoral Research Associate
Princeton University
Department of Physics
February 2003 to September 2003

I developed electronic sensing techniques for biological discrimination without the need for chemical or physical sample alteration. My primary research focus was in dielectric spectroscopy, where we employed microfluidic tectonics to create novel parallel-plate devices to study biological materials (e.g., DNA, proteins, and E. coli cells) over the range 0.05-40 GHz. Other research was aimed at multi-analyte detection for sizing of molecular and cellular biological materials using the Coulter resistive pulse technique.

Graduate Research Assistant
The University of Texas at Austin
Department of Physics
January 1997 to January 2003

Yttrium and lanthanum were discovered in 1996 to transition from a metallic mirror to a transparent insulator upon hydrogenation to the trihydride phase (YH₃ and LaH₃). I have looked into steric effects upon this transition by substituting scandium for yttrium. Scandium maximally forms dihydride (ScH₂), and therefore, does not exhibit this transition. My experience includes ultra-high vacuum, electron beam evaporation film deposition, optical spectroscopy, SQUID magnetometry, electrical transport measurements, NMR (both conventional and force microscopy), x-ray diffraction, iodometric titration for oxygen stoichiometry, solid-state reaction, and thermal gravimetric analysis.

Engineering Intern
International Sematech/International 300mm Initiative
Austin, Texas
May 1997 to April 2002

From June 1997 to May 2002 I conducted research as an intern for the International 300mm Initiative (I300I) and International Sematech (ISMT) in parallel with my dissertation studies. These companies are consortia from the semiconductor industry set forth to eliminate cost-bearing and technological obstacles within the industry. My research included analysis of production capability of 300 mm silicon wafers and metrology tools as compared to state-of-the-art 200 mm technology, developing mathematical models for the International Technology Roadmap for Semiconductors (ITRS) Starting Materials division, as well as thermal stability and wet etch capability of potential high-k dielectric materials alternatives ZrO2 and HfO2 to replace SiOx. The wet etch studies enabled ISMT to file for a patent on the film removal process that we developed.

Contract Engineer
Xidex Corporation
Austin, Texas
July 2000 to December 2000

As part of a collaboration with my graduate research advisor, I was contracted to design, construct, and use a chemical vapor deposition (CVD) system for growth of carbon nanotubes (CNTs). The CNTs were grown using a precursor catalyst method, and analyzed with scanning electron microscopy (SEM). The growth conditions were tailored for growing a single, straight CNT on an ultrasenstive crystal silicon cantilever for scanning force microscopy. The nanometer diameter of the CNT provides higher resolution force microscopy than standard commercial cantilevers.
Teaching Assistant
University of Texas at Austin
January 1997 to May 2002

I have taught and graded seven semesters of physics courses for science, engineering, and non-science students. The courses were Conceptual Physics, Pseudoscience, and Mechanics and Electricity – Magnetism.

Publications

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T. C. Messina and D. S. Talaga, "Shallow free energy landscapes remodelled by ligand binding in glucose/galactose binding protein," Biophys. J. accepted (2007).

T. C. Messina, C. W. Miller, and J. T. Markert, "Observation of steric quenching of the switchable mirror effect in Y_1-zSc_zH_x'', Phys. Rev. B accepted (2007).

T. C. Messina, H. Kim, J. T. Giurleo, D. S. Talaga, "Hidden Markov Model Analysis of Multichromophore Photobleaching," J. Phys. Chem. B 110, 10.1021/jp063367k (2006).

J.-H. Choi, T. C. Messina, J. Yan, G. I. Drandova, and J. T. Markert, “Thermal Conductivity and ⁸⁹Y NMR of Ca_2+xY_2-xCu₅O₁₀”, J. of Magn. Mag. Mat. 272, 970-971 (2004).

T. C. Messina, L. N. Dunkleberger, G. A. Mensing, A. S. Kalmbach, R. Weiss, D. Beebe, L. L. Sohn, “A Novel Sensor for Biological Discrimination”, The 7th International Conference on Miniaturized Chemical and BioChemical Analysis Systems, Squaw Valley, CA, USA, 5-9 October 2003.

C.W. Miller, U. Mirsaidov, T.C. Messina, J.T. Markert, “External Field Effects on Characteristics of Magnetically-Capped Oscillators for Magnetic Resonance Force Microscopy”, J App. Phy.
93, 6572 (2003).

T. C. Messina, C. W. Miller, J. T. Markert, “Steric Effects in the Metal-Insulator (Mirror-Transparent) Transition in YH_x”, J. Alloys and Compounds 356-357181 (2003).

J.T. Markert, T.C. Messina, B. Dam, J. Huijbregste, J.H. Rector, and R. Griessen, “Infinite-Layer Copper-Oxide Laser-Ablated Thin Films: Substrate, Buffer-Layer, and Processing Effects”, IEEE Transactions on Applied Superconductivity, 13, 2684 (2003).

G. I. Drandova, T. C. Messina, J. T. Markert, “NMR of ⁸⁹Y in the Copper-Oxide Spin-Chain Compound Ca_2+xY_2-xCu₅O₁₀”, J. Low Temperature Physics 131, 305 (2003).

K. Mochizuki, J.-H. Choi, T. C. Messina, Y. Ando, K. Nakamura, J. T. Markert, “Extreme Smallness of the Transverse Force on Moving Vortices”, Physica C 388-389, 705 (2003).

J. Barnett, D. Riley, T. Messina, P. Lysaght, “Wet Etch Enhancement of HfO₂ Films by Implant Processing”, Solid State Phenomena 92, 11 (2003).

P. S. Lysaght, P. J. Chen, R. Bergmann, T. Messina, R. W. Murto and H. R. Huff, “Experimental Observations of the Thermal Stability of High-k Gate Dielectric Materials on Silicon”, Journal of Non-Crystalline Solids, 303, 54 (2002).

M. D. Chabot, T.C. Messina, V. Manicevski, C. W. Miller, J. T. Markert, “Single-Crystal Silicon Triple-Torsional Micro-Oscillators for Use in Magnetic Resonance Force Microscopy”, SPIE-Int. Soc. Opt. Eng. Proceedings of Spie - the International Society for Optical Engineering 4559, 24 (2001).

J. T. Markert, T. C. Messina, B. Dam, J. Huijbregste, J. Rector, R. Griessen, “Observation of Step-Flow Growth in Laser-Ablated thin films of the T’-Phase compound Pr₂CuO₄”, Physica C, 341-348, 2355-56 (2000).

J. T. Markert, T. C. Messina, B. Dam, J. Huijbregste, J. H. Rector, R. Griessen, “Laser-Ablated Thin Films of Infinite-Layer Compounds and Related Materials”, Proceedings of SPIE 4058, 141 (2000).

T. Ono, G. A. Rozgonyi, C. Au, T. C. Messina, R. Goodall, H. R. Huff, “Oxygen Precipitation Behavior in 300mm Polished Czochralski Silicon Wafers,” J.Electrochem. Soc. 146, 3807
(1999).

H. R. Huff, D. McCormack Jr., C. Au, T. C. Messina, K. Chan, R. Goodall, “Current Status of 200mm and 300mm Silicon Wafers,” Proceedings of the Intl. Solid StateDevices and Materials (ISSDM ’97), Japan, p. 456 and Conference Abstracts p.575, (1997) also published in Jpn J. Appl Phys, 37, (1998) Pt.1, No.3B.

C. Au, T. C. Messina, R. Goodall, H. R. Huff, “Characterization of 300mm Polished Silicon Wafers,” Proceedings of the 8^th International Symposium on Silicon Materials and Technology, 1, p.641, (1998).

T. C. Messina, C. Au, S. Shih, Z. Yang, R. Goodall, H. R. Huff, “Current Status of 300mm Wafer Characterization,” Proceedings of the International Mechanical Engineering Conference and Exposition (IMECE ’98), p.825, (1998).

S. Shih, C. Au, Z. Yang, T. C. Messina, R. Goodall, H. R. Huff, “Characterization of 300mm Silicon-Polished and Epi Wafers,” Microelectronic Engineering 45, 169 (1999).

J. T. Markert, K. Mochizuki, T. C. Messina, B. C. Dunn, A. V. Elliott, “Studies of Infinite-Layer, T’-Phase, and 1-D Ladder Copper-Oxide Compounds,” Physics and Materials Science of High Temperature Superconductor, IV. Proceedings of the NATO Advanced Research Workshop. Kluwer Academic Publishers. 1997, pp.151.

New Thiol-Reactive Chromophores

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Sometimes commercially available chromophores are not perfectly suited for the experiment one wishes to do. In my case, I want an extremely solvatochromic chromophore so that I can perform a single covalent labeling near the binding center of GGBP that will report local motions corresponding to global protein rearrangements upon glucose/galactose binding. The chromophore needs to be excitable preferably in the blue to green spectrum (optimal for Ti:Al₂O₃ lasers). Nile Red is an excellent dye in this case, however, there is no commercially available form that is thiol-reactive (covalent bonding to cysteine amino acid sidechain).
About a year ago, I began synthesizing a nile red maleimide, but I had a lot of difficulty getting maleimide to link to nile red phenol. It took some trained organic chemists at Kent State University (S. Y. Kim et al., J. Phys. Chem. B 109, 24517 (2005).) about the same amount of time to figure out that a protecting group is necessary. A simple way of protecting the maleimide and later deprotecting it is through a Diels-Alder/Retro-Diels-Alder set of reactions. I have shown a rough schematic of the process below. I am now optimizing the set of reactions and preparing to attach the chromophore to GGBP for single molecule studies.

Molecular Dynamics Simulations of GGBP

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Using IMPACT, I am performing umbrella-potential sampling and replica exchange molecular dynamics simulations of GGBP. In the absence of crystallographic data, one can steer the protein conformation and systematically search for and minimize the energy of conformational structures along a sensible pathway. Once the minimized structures are obtained, molecular dynamics simulations will populate the structures, and from the relative populations, one can extract the potential of mean force on a two-dimensional (theta, phi) coordinate system. Shown in the image are contour plots of the populations (~ PMFs) of ribose binding protein (RBP), which is nearly homologous to GGBP. The top contour is ribose-free and the bottom contour is ribose-bound. There are clearly three distinct populations as we observed in bulk fluorescence measurements of GGBP. The contour plots are from Prof. Ron Levy's publication:

Ravindranathan, K. P.; Gallicchio, E.; Levy, R. M., "Conformational Equilibria and Free Energy Profiles for the Allosteric Transition of the Ribose-binding Protein", Journal of Molecular Biology 353, 196–210 (2005). Molecular Biology 1965, 12, 88–118.

Reconstructing Langevin Dynamics with HMM

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I am currently using HMM to analyze Langevin dynamics on a double-well potential. These HMM use lifetime and the known potential of mean force to reconstruct the photon trajectory for a protein labeled with two chromophores. The goal is to identify limits of barrier heights, friction coefficients, Forster distance, and potential curvature for trajectory reconstruction.

(a) Potential of mean force used to describe the protein dynamics dictated by a FRET fluorophore pair (donor and acceptor) distance attached to a protein. The residence probability distribution function is overlaid.

(b) Simulated photon trajectories recorded by donor (red) and acceptor (blue) channels.

(c-d) Microtime (the time lag between the laser pulse and the arrival of the photon) for each photon recorded in donor channel (c) and acceptor channel (d).

(e) Reconstructed donor-acceptor distance trajectory using hidden Markov model (HMM) with evenly spaced 21 states across the donor-acceptor distance.

(f) “True” donor-acceptor distance trajectory, generated by Langevin dynamics simulation on (a).

Glucose/Galactose Binding Protein

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Enteric bacteria use glucose/galactose binding protein (GGBP) in separate pathways to actively transport methylgalactosides across the cell membrane and to chemically sense them as part of the swimming regulatory scheme. Crystallographic and bulk steady-state experiments have been reported for GGBP. Binding of glucose has been described both by a single and by multiple binding constants. GGBP undergoes large dynamic structural fluctuations that are decreased, but not eliminated upon binding of glucose. Thermodynamic characterisation of the structural changes associated with ligand recognition and protein-complex docking can be difficult and detailed understanding of the role of conformation in ligand binding and delivery to the cytosol or activation of the methyl accepting chemotaxis protein Trg are not well characterised.

Here we show that GGBP fluctuates between at least three conformations with the relative weights of those conformations being modulated by the binding of glucose. Each structure has different binding affinity and thermodynamic properties. The single binding site of GGBP was considered to have a single association constant, our results suggest that the binding constant is conformationally dependent. Moreover, the ligand binding does not induce the conformational change, rather, it biases the distribution of conformation by stabilising the high-affinity receptor-competent structures. Computational predictions for the closely related ribose binding protein show qualitatively similar results.12 The presence of two high-affinity binding structures is suggestive of the different membrane receptors to which GGBP must bind to provide either active transport or chemotaxis. Conformational plasticity is increasingly becoming recognised as an important issue in drug resistance. GGBP is an ABC transporter system which are common targets for therapeutics. An inhibitor for flexible targets like GGBP would need to interact with all parts of the binding-competent conformational ensemble. Glucose/galactose binding protein (GGBP) is a component of the the β-methyl galactoside chemosensory transport system in Escherichia coli responsible for delivery of glucose and galactose to periplasmic membrane receptor proteins. Previous studies of GGBP have shown large-scale conformational changes upon ligand binding.

We discuss results of bulk steady-state and time-resolved fluorescence measurements of this protein. The protein has a point mutation at residue Leucine 255 to Cysteine (L255C) and is conjugated with acrylodan. The N-terminus is labeled with a long-lived (~ 1 µs) ruthenium complex. Using a two-state binding model to analyze fluorescence anisotropy data and fitting the data globally over the ligand titration, we are able to extract time scales for local dye reorientation, rotational diffusion of the protein, and ligand binding constant. The results indicate up to 40% concentration of the "closed" (glucose-bound) protein conformation exist at 0 M glucose, evidence that proteins are continuously sampling their thermodynamically available conformational space. Global analysis of the fluorescence lifetime, time-resolved anisotropy, and dynamic Stokes shift indicates that there are three distinct states of the protein the free energy of which is modified by the binding of glucose. This suggests a dynamic conformational sampling model of ligand binding where the ligand stabilizes the closed conformation(s) with respect to the other conformation(s). This picture requires barriers of in the apo form of the protein with the barrier to opening increasing due to the stabilization of the closed state(s). This result confirms a theoretical prediction made on a related system, ribose binding protein.

In collaboration with the group of Ron Levy we have begun umbrella-potential sampling and replica exchange molecular dynamics simulations of GGBP to better characterize the heterogeneity of structures associated with the holo and apo forms of the protein.

References

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Prof. David S. Talaga -- Post-Doctoral Advisor
talaga@rutchem.rutgers.edu
Department of Chemistry and Chemical Biology
610 Taylor Road
Piscataway, NJ 08854
tel: 732.445.6359
fax: 732.445.5312

Prof. John T. Markert -- Graduate Advisor
markert@physics.utexas.edu
Department of Physics, 1 University Station
University of Texas, Austin, TX 78712
tel: 512.471.1978
fax: 512.471.9637

Prof. Edward W. Castner -- Collaborator
castner@rutchem.rutgers.edu
Department of Chemistry and Chemical Biology
610 Taylor Road
Piscataway, NJ 08854
tel: 732.445.2564
fax: 732.445.5312

Dr. Howard R. Huff -- Internship Manager
Howard.huff@sematech.org
International Sematech, 2706 Montopolis Dr., Austin, TX 78741
tel: 512.356.3334
fax: 512.356.7640

Dr. Randal K. Goodall -- Internship Manager
Randy.Goodall@sematech.org
International Sematech, 2706 Montopolis Dr., Austin, TX 78741
tel: 512.356.0000
fax: 512.356.7640

Dr. Paul F. McClure -- Contract Manager
pfm@xidex.com
Xidex Corp., 8906 Wall St., Suite 105, Austin, TX 78754
tel: 512.339.0608
fax: 512.339.9497

Hidden Markov Model Analysis of Multichromophore Photobleaching

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The interpretation of single-molecule measurements is greatly complicated by the presence of multiple fluorescent labels. However, many molecular systems of interest consist of multiple interacting components. We address this issue using multiply-labeled dextran polymers that we intentionally photobleach to the background on a single molecule basis. Hidden Markov models allow unsupervised analysis of the data to determine the number of fluorescent subunits involved in the fluorescence intermittency of the 6-carboxy-tetramethylrhodamine labels by counting the discrete steps in fluorescence intensity. The Bayes information criterion allows us to distinguish between hidden Markov models that differ by number of states, i.e., number of fluorescent molecules. We determine information-theoretical limits and show via Monte Carlo simulations that the hidden Markov model analysis approaches these theoretical limits. This technique has resolving power of one fluorescing unit up to as many as 30 fluorescent dyes with the appropriate choice of dye and adequate detection capability. We discuss the general utility of this method for determining aggregation-state distributions as could appear in many biologically important systems and its adaptability to general photometric experiments.

Education

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The University of Texas at Austin

1991-1996 Bachelor of Science in Physics

1997-1999 Master of Arts in Physics

1999-2002 Ph.D. in Physics