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: How do protein
dynamics affect stability? How do different regions in proteins
communicate; for example, how do mutations far from the
active site modulate enzyme catalytic activity? What are
the time scales and pathways for large scale conformational
changes associated with function or folding?
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,
fluorescence 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.
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. Bulk studies are conducted using commercially
available fluorimeters while single molecule experiments
involve laser-based confocal microscopy and analysis techniques
such as fluorescence correlation spectroscopy and photon
counting histograms.
In addition to fluorescence and other optical techniques
such as circular dichroism, a number of molecular biology
techniques are used in our laboratory. By generating series
of closely related protein variants using directed evolution,
we can distinguish between functional and non-functional
changes in protein dynamics. In directed evolution, the
gene of interest is randomly mutated using mutagenic PCR
(polymerase chain reaction) and mutated proteins with functional
differences (for example, increases in activity and/or thermostability)
are identified by screening libraries. Positive variants
are sequenced, characterized and used to parent the next
generation. This process results in families of proteins
with functional differences whose sequences differ by only
a few, identified amino acids. By studying entire families,
functional changes in dynamics may be identified.
Selected Publications
Arnold FH, Wintrode PL, Miyazaki K, Gershenson A., (2001)
How enzymes adapt: lessons from directed evolution. Trends
Biochem Sci. 26(2):100-6.
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.
Last review: July 6, 2005. E-mail comments or questions
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