The Rhoades lab studies protein folding, misfolding, and dynamics. We have a particular interest in
Intrinsically Disordered Proteins, or IDPs. IDPs are proteins that,
under physiological conditions (and in the absence of binding partners — although structure does not always
accompany binding, it often does), do not assume a single well-defined three-dimensional
structure. IDPs are exceptions to the general rule (sometimes called
Anfinsen's dogma) that protein sequence determines a unique structure,
in that IDPs rapidly interconvert between several energetically
Structural models of α-synuclein based on smFRET studies:
an ensemble of structures in solution (left),
and an extended helix when bound to large unilamellar lipid vesicles (right).
IDPs make up significant fractions of eukaryotic proteomes, and
tend to play important roles in biological processes as 'hubs' in
signalling networks. IDPs may assume different structures upon binding
to different partners, and this promiscuity confers upon IDPs a greater
capacity than folded proteins to integrate and transduce information.
Partly for this reason, IDPs are relatively likely to be involved in
disorders such as cancer and cardiovascular disease.
αS (green) is taken up into SH-SY5Y cells, but does not colocalize with mitochondria (red).
Intriguingly, several IDPs (and certain folded proteins) share a
propensity to form insoluble, ordered aggregates called amyloid. Amyloid
fibers and plaques are hallmarks of degenerative diseases such as
Alzheimer's (AD), Parkinson's (PD), and Type II Diabetes (T2D). Much of
our research focuses on three IDPs implicated in these widespread and
debilitating diseases: Tau, which forms tangles in the brains of
persons with AD (as well as in several other neurodegenerative conditions);
α-synuclein (αS), which forms amyloid plaques in
PD-affected brains; and islet amyloid polypeptide (IAPP), which
similarly forms amyloid plaques in the pancreas during the course of
Images showing the time course of lipid-induced tau aggregation. Tau/lipid aggregates (Rhodamine filter, top) also stain positively with the amyloid indicator dye thioflavin T (ThT filter, bottom).
There is emerging evidence that oligomeric intermediates in the
fiber formation process are more toxic than mature amyloid aggregates.
We are developing biophysical tools to study the conversion of these
monomeric, soluble IDPs into toxic oligomers and mature fibrils, with
the ultimate goal of developing therapies to modulate the disease
processes of AD, PD and T2D. More broadly, studies on Tau, αS and
IAPP may provide new insights into general principles of amyloid
formation pathways critical to a wide variety of protein misfolding
and lipid-induced aggregation monitored by FCS. Adapted from Elbaum-Garfinkle, Ramlall
& Rhoades (2010) Biophys J 98:2722-30
Other systems of interest include β- and γ-synuclein,
homologs of αS that may modulate αS oligomerization and (in
the case of γS) have been linked to breast cancer. We also study
catalysis-related dynamics of glutathione-S-transferases,
detoxification enzymes which play a crucial role in the response to
oxidative stress. In collaboration with groups at Yale and elsewhere, we
also study DNA-binding proteins, HIV virion assembly, the catalytic
cycle of fatty acid synthase, synaptic vesicle fusion, pattern formation
during zebrafish development, and the cytochrome P450 family of
Resources & Techniques
Much of our work involves single-molecule fluorescence
experiments conducted on our two home-built microscope setups.
Single-molecule spectroscopy eliminates ensemble averaging and thus
allows us to dissect the thermodynamics and kinetics of biochemical
processes with much greater detail than many traditional techniques. This is
especially useful when dealing with heterogeneous, dynamic systems such
as monomeric and oligomeric IDPs.
Fiber-coupled 561nm laser line
Our instrumentation consists of two inverted Olympus IX-71
microscopes, coupled to 488nm, 561nm and 630nm laser lines,
single-photon APD detectors, an EM-CCD camera with DV2 two-color accessory, scanning stages for
micron- and nanometer-scale positioning, and acousto-optical modulators
for alternating laser excitation (ALEX). We are capable of one- and
two-color confocal and surface-based fluorescence techniques, some of
Acousto-optic modulators (boxes in foreground) used for ALEX
smFRET (single-molecule Förster resonance energy transfer), in which the
efficiency of energy transfer between two different dyes (a donor and an
acceptor) serves as a sensitive "molecular ruler" of the distances
between them. When applied to individual protein molecules or small
complexes diffusing through a confocal volume, FRET can provide very
useful information on conformation, conformational changes and sample heterogeneity. We are
currently adding ALEX capabilities to one of our instruments. In this
scheme, the sample is illuminated by rapidly alternating the 488nm and 561 nm laser lines, allowing us to independently
probe the donor and acceptor fluorescence. This
provides a much broader dynamic
range of FRET measurements by allowing access to low FRET efficiency events.
- FCS (fluorescence correlation spectroscopy), in which the
autocorrelation decay rates of fluorescent particles diffusing through
the confocal volume are used to determine diffusion rates and hence
molecular dimensions. FCS can also measure the kinetics and extent of
conformational dynamics displayed by a protein.
FCS measures the diffusion rates of fluorescent particles through a focal volume, allowing us to distinguish protein in solution (red) from protein bound to vesicles (black)
- FFS (fluorescence fluctuation spectroscopy), a family of
techniques that includes photon-counting histograms and fluorescence
intensity distribution analysis, involves measuring the stoichiometry of
protein-protein and protein-vesicle complexes by analyzing the intensity
from multiple fluorophores in each complex.
488nm and 561nm lasers for two-color excitation
TIRF-M (total internal reflection fluorescence microscopy), in
which an evanescent electromagnetic field generated by total internal
reflection is used to excite fluorophores within a few hundred
nanometers of a surface. This allows us to selectively monitor particles
bound to a surface while eliminating background emission from fluors in
solution. We can use TIRF-M to measure binding and dissociation of
ligands to proteins or phospholipid membranes, and to monitor diffusion
of membrane-associated peptides in vesicles.
Structures sampled from a molecular dynamics simulation of IAPP in solution
Other analytical techniques we commonly use include ensemble
steady-state and lifetime fluorescence, circular dichroism, UV-vis
spectroscopy, mass spectrometry, dynamic light scattering, electron
microscopy and NMR. Instrumentation is available in our own lab, through
our colleagues in the MB&B department, and via Yale facilities including the
Chemical & Biophysical Instrumentation Center
and the Center for Molecular Discovery.
Additionally, we have facilities for protein expression in
E. coli, peptide synthesis (via the W.M. Keck Facility), protein
purification, and mammalian cell culture. We also
use the Yale High Performance Computing clusters and lab workstations
for molecular dynamics simulations (Gromacs), quantum chemistry
calculations (GAMESS), computational ligand docking (Autodock),
and Monte Carlo protein structural refinement
(Rosetta), to complement and extend our experimental measurements.