Anne Gershenson |
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| Assistant Professor of Biophysical Chemistry Ph.D., University of Michigan, 1996 781-736-2548 gershenson@brandeis.edu |
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Proteins are dynamic molecules whose motions are important for folding, function and stability. Our laboratory uses optical techniques, particularly fluorescence, to study protein conformation and folding in real time. We are interested in elucidating the relationships between protein dynamics and other protein properties such as protein function and stability. Questions of interest include: What are the time scales and pathways for large-scale conformational changes associated with function or folding? How do protein dynamics affect stability? How does protein stability affect protein-protein interactions? How do different regions in proteins communicate; for example, how do mutations far from the active site modulate enzyme catalytic activity?
For fluorescence studies, observed fluorophores may be native to the protein, such as the amino acids tyrosine and tryptophan, or extrinsic covalently attached labels. Changes in lifetimes, excitation & emission spectra and anisotropy (polarization) of such fluorophores can be used to monitor protein conformation and dynamics. In addition, Förster resonance energy transfer (FRET) between donor and acceptor fluorophores can be used as a molecular ruler to directly measure changes in distance between the fluorophores.
Fluorescence Spectroscopy of Single Molecules
Bulk fluorescence studies measure the average values for a population while single molecule techniques allow measurements to be performed on individual fluorescently labeled proteins. Single molecule experiments can reveal details about a population such as conformational substates and whether all members behave identically. Single molecule methods can also be used to monitor the time course of conformational changes, such as those involved in protein function and protein folding. We use fluorescence based single molecule microscopy techniques and a single molecule setup constructed in our laboratory to identify conformational substates, to follow functionally important protein conformational changes, and to identify conformational intermediates. For these experiments we specifically label proteins with bright, organic fluorophores.
Figure 1. Conformational changes of the protease, bovine trypsin, and the serpin α1-proteinase inhibitor resulting in protease inhibition. Notice the loss of trypsin secondary structure resulting from interactions with the serpin. For the single pair Förster resonance energy transfer (spFRET) experimental data shown below, the donor fluorophore is located at trypsin residue 113 and the acceptor fluorophore is located at residue 232 on α1-proteinase inhibitor. The encounter complex structure is from Protein Databank (pdb) file 1oph (Dementiav, et al, J.Biol. Chem, 2003) and the covalent complex structure is from pdb file 1ezx (Huntington, et al, Nature 2000). The figure was generated using Molscript (Kraulis, P.J., J. Appl. Crystall. 1991)
Studying Metastable Proteins
Inhibitory serpins regulate serine and cysteine proteases involved in inflammation, immune responses and blood clotting. The unique serpin inhibitory mechanism requires formation of a covalent bond between the protease and serpin, translocation of the protease relative to the serpin, remodeling of the serpin structure and deformation of the protease structure (Fig. 1). The goal of our research is to determine the conformational heterogeneity of protease-serpin inhibitory complexes, to follow the formation of single complexes in real time and to determine how mutations affect the conformations and formation of these complexes. Single molecule experiments on trypsin (protease)-α1proteinase inhibitor (serpin, also known as α1-antitrypsin) complexes diffusing in solutions indicate that protease-serpin complexes can have multiple conformations and that the relative populations of these conformations can be manipulated by changing the stability of the serine protease.
Figure 2. (A) Raw data, at 1 kHz, from diffusing tyrpsin-α1proteinase inhibitor covalent complexes. The donor is located on trypsin and the acceptor is located on α1proteinase inhibitor. The arrows indicate single molecules in the focal volume. (B) An spFRET histogram for bovine tyrpsin-α1proteinase inhibitor showing a single conformation for the covalent complex centered at 28 percent efficiency. The peak centered at 0 percent efficiency arises from complexes that are labeled only with donor fluorophores or in which the acceptor has photobleached.
Serpins are metastable proteins, proteins which when perturbed assume a new, more stable fold. Other metastable proteins include viral fusion proteins that facilitate the entry of enveloped viruses such as influenza and HIV into cells. We are also using fluorescence techniques to study the HIV envelope proteins gp41 and gp120, the HIV proteins associated with membrane fusion.
Selected References
Gershenson A, Schauerte JA, Giver L, Arnold FH (2000) Tryptophan phosphorescence study of enzyme flexibility and unfolding in laboratory-evolved thermostable esterases. Biochemistry, 39: 4658-4665.
Spiller, B., Gershenson, A., Arnold, F.H. & Stevens, R.C. (1999) A structural view of evolutionary divergence. Proc. Natl. Acad. Sci., USA, 96: 12305-12310.
Giver, L., Gershenson, A., Freskgard, P.-O. & Arnold, F. H. (1998) Directed evolution of a thermostable esterase. Proc. Natl. Acad. Sci., USA, 95: 12809-12813.
Gershenson, A., Gafni, A. & Steel, D.G. (1998) Comparisons of the time-resolved absorption and phosphorescence from the tryptophan triplet state in proteins in solution. Photochem. & Photobiol., 67: 391-398.