Talaga, David

	Phone:	732-445-6359
	E-mail:
	FAX:	732-445-5312
	Lab:	732-445-2192
	Office:	Wright Rieman Labs 170
	Mail:	Chemistry & Chemical Biology, 610 Taylor Road, Piscataway, NJ 08854

Research Summary

The Talaga lab is focussed on studying the molecular-level basis for structural changes in proteins. We use a variety of biophysical techniques ranging from dynamic light scattering to atomic force microscopy to single molecule fluorescence. We are actively developing new methods in the area of single molecule fluorescence spectroscopy to increase our ability to understand the role of structural changes and fluctuations as they occur at "equilibrium" by allowing us to observe them in real time. A typical project in the Talaga group will include producing the protein of interest by expressing it in E. coli, characterizing the protein with traditional bulk measurements, and performing advanced single molecule measurements on the protein while it is engaged in the dynamic activity we are seeking to characterize. Some of the dynamic structure-function relationships we are investigating include:

• protein/peptide secondary structure fluctuations at the 1 or 2 amino acid level;
• protein folding and unfolding with particular emphasis on visualizing the transition state;
• protein misfolding and assembly into ß-amyloid fibrils;
• protein hydrophobic core fluctuations and their connection to protein stability;
• protein structural fluctuations and their role in substrate/ligand recognition.

Please click through to our Research Page for more details about our ongoing scientific activities.

Molecular-Level Mechanisms of Amyloidogenesis

The study of amyloid structure and growth has been motivated by their implication in many human diseases. There are ~20 diseases associated with excessive deposits of amyloid plaques in the affected tissue or organ including Alzheimer's disease (AD), Parkinson's disease (PD), type II diabetes, and spongiform encephalopathies. In these disease states, proteins that are normally soluble undergo aggregation to form various intermediates and amyloidogenic species. These species subsequently assemble to generate insoluble fibrils that accumulate in the affected tissues or organs. A detailed understanding of amyloid growth mechanisms will allow new approaches to the prevention of amyloid formation and better diagnostics for early detection of amyloidogenic diseases.

A molecular-level mechanism of how the different amyloid species interconvert is the goal of this project. There are many species of amyloid particles present physiologically. Our single molecule studies aim to classify the species involved in amyloid formation according to size, shape, kinetic reactivity, and monomer 2° and 3° structural information. A molecular-level mechanism of amyloid growth must include details as to when the protein misfold occurs and how it is influenced by the dynamics of protein structure. To determine the physical interactions and structural changes involved in the amyloid assembly mechanism, we study effect of environmental variables such as temperature, pH, helix promoting solvents, denaturants, and reducing agents. The environmental effect on aggregation is expected to be species-dependent reflecting a possible hierarchy of structural interactions.

Nanopore Measurements of Proteins

The ultimate goal of this research is to provide single molecule equivalents to the existing battery of gel electrophoretic methods. These nanopore methods aim to determine the number and type of proteins in samples the size of a single cell and provide real-time monitoring of the assembly of multi-protein structures.
The near-term objectives are to:
* use nanopores to distinguish proteins based on coarse-grain (10-30 aa segments) primary sequence differences,
* evaluate competing models of the translocation physics,
* determine nanopore geometry from AC impedance,
* optimize the nanopore geometries for protein identification,
* optimize nanopore physical/chemical treatments, and
* find nanopore assays that do not disrupt protein assemblies.

Protein Conformational Dynamics

Glucose/Galactose Binding Protein (GBP) is a receptor in the chemosensory pathway of bacterial chemotaxis. GBP consists of two domains, each of which contains a beta-sheet packed between alpha-helices. The binding cleft is between the two hinged domains. Signal transduction begins in the periplasmic compartment where GBP is located. Binding of glucose or galatose by GBP causes a large amplitude conformational change that encapsulates the ligand. This allows GBP to bind to a transmembrane receptor initiating the remainder of the chemosensory pathway that regulates the bacterial flagellar motor and determines swimming behavior of the cell in response to chemical atractants or repellents. GBP is also involved in the transport of glucose across the cell membrane. The chemotaxis pathway has several binding proteins that activate it, while the transport cassette is specific to GBP. We have been studying the conformational changes in GBP associated with glucose binding. We have found that conformational fluctuations move GBP amongst at least three structures, providing a possible mechanism by which GBP can adapt to the generic receptor for chemotaxis and the specific receptor for glucose transport.

Awards & Honors

• 2001 Research Innovation Award, Research Corporation
• 1997 NIH/NRSA Postdoctoral Fellowship
• 1996 UCLA Physical Chemistry Dissertation Award
• 1994 Bauer prize for excellence in research
• 1992 UCLA first year chemistry graduate student award
• 1990 Alpha Chi Sigma national award for outstanding professional service (Tutoring Program)
• 1987 Academic Olympiad Science Gold Medalist

Publications

He, X.; Giurleo, J. T.; Talaga, D. S. (2009) '“Role of small oligomers on the amyloidogenic aggregation free energy landscape.' J. Mol. Biol. (Under Review)

Talaga, D.S.; Li, J. (2009), 'Single-molecule protein unfolding in solid state nanopres' J. Amer. Chem. Soc. 131, 9287-9297.

Talaga, D.S. (2009), 'Information-theoretical analysis of time-correlated single-photon counting measurements of single molecules.' J. Phys. Chem. A, 113(17) 5251–5263 (Cover).

Giurleo, J. T. & Talaga, D. S. (2008), 'Global fitting without a global model: Regularization based on the continuity of the evolution of parameter distributions.', J. Chem. Phys. 128, 114114/1-114114/18.

Pronchik, J.; Giurleo, J. T. & Talaga, D. S. (2008), 'Separation and Analysis of Dynamic Stokes Shift with Multiple Fluorescence Environments: Coumarin 153 in Bovine beta -Lactoglobulin A.', J. Phys. Chem. B 112, 11422-11434.

Messina, T. C. & Talaga, D. S. (2007), 'Protein free energy landscapes remodeled by ligand binding.', Biophys. J. 93, 579-585.

Talaga, D. S. (2007), 'Markov processes in single molecule fluorescence.', Curr. Opin. Colloid Interface Sci. 12, 285-296.

Messina, T. C.; Kim, H.; Giurleo, J. T. & Talaga, D. S. (2006), 'Hidden Markov Model Analysis of Multichromophore Photobleaching.', J. Phys. Chem. B 110, 16366-16376.

Talaga, D. S. (2006), 'Information Theoretical Approach to Single-Molecule Experimental Design and Interpretation.', J. Phys. Chem. A 110, 9743-9757.

Andrec, M.; Levy, R. M. & Talaga, D. S. (2003), 'Direct determination of kinetic rates from single-molecule photon arrival trajectories using hidden markov models.', J. Phys. Chem. A 107, 7454-7464.

Talaga, D. S.; Lau, W. L.; Roder, H.; Tang, J.; Jia, Y.; DeGrado, W. F. & Hochstrasser, R. M. (2000), 'Dynamics and folding of single two-stranded coiled-coil peptides studied by fluorescent energy transfer confocal microscopy.', Proc. Natl. Acad. Sci. U. S. A. 97, 13021-13026.

Talaga, D. S. & Zink, J. I. (2001), 'Symmetry and Local Mode Coupling in Absorption and Raman Spectroscopy of Intervalence Electronic Transitions.', J. Phys. Chem. A 105, 10511-10519.