Research

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 comparable structures.

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 T2D.

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 diseases.

Tau lipid-binding 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 drug-metabolizing enzymes.

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 which include:

• 
  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.