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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.

Electron Transfer Probes of Peptide/Protein Backbone Conformation

Single molecule polyproline isomerization is studied by fluorescence quenching, induced by short-ranged electron transfer between TMR(5-carboxytetramethylrhodamine) and DMPD(dimethyl-p-phenylenediamine). To do this, we have prepared a polyproline(n=2,3) peptide with DMPD at its carboxylic end and TMR at its amino end. The electron transfer efficiency is measured by TCSPC(time-correlated single photon counting) in which the acceptor fluorescence lifetime is comparatively quenched according to the proximity of donor molecule. This allows us to measure local protein or peptide conformation at the level of a few residues. It also allows us to investigate the role of conformation in biological electron transfer.

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

Awards & Honors