To elucidate the structural and energetic basis of attractive protein interactions in the aging lens, we investigated the binding of destabilized mutants of betaB1-crystallin to the lens chaperones, alpha-crystallins. We show that the mutations enhance the binding affinity to alphaA- but not alphaB-crystallin at physiological temperatures. Complex formation disrupts the… Read More

A central step in understanding lens aging is to characterize the thermodynamic stability of its proteins and determine the consequences of changes in the primary sequence on their folding equilibria. For this purpose, destabilized mutations were introduced in betaB1-crystallin targeting the domain interface within the fold of a subunit. Global… Read More

A biochemical oscillator can be reconstituted in vitro with three purified proteins, that displays the salient properties of circadian (daily) rhythms, including self-sustained 24-h periodicity that is temperature compensated. We analyze the biochemical basis of this oscillator by quantifying the time-dependent interactions of the three proteins (KaiA, KaiB, and KaiC)… Read More

We demonstrate the feasibility and practical limitations of using steady-state anisotropy to determine distances from fluorescence homotransfer in the context of a protein of known crystal structure. Eight double mutants of T4 lysozyme spanning the distance range between 20 A and 50 A were labeled with a methanethiosulfonate derivative of… Read More

We have identified sequence and structural determinants of oligomer size, symmetry, and polydispersity in the small heat shock protein super family. Using an insertion mutagenesis strategy that mimics evolutionary sequence divergence, we induced the ordered oligomer of Methanococcus jannaschii Hsp16.5 to transition to either expanded symmetric or polydisperse assemblies. A… Read More

We present a novel hypothesis for the molecular mechanism of autosomal dominant cataract linked to two mutations in the alphaA-crystallin gene of the ocular lens. AlphaA-crystallin is a molecular chaperone that plays a critical role in the suppression of protein aggregation and hence in the long term maintenance of lens… Read More

We report an approach for determining the structure of macromolecular assemblies by the combined application of cryo-electron microscopy (cryo-EM) and site-directed spin labeling electron paramagnetic resonance spectroscopy (EPR). This approach is illustrated for Hsp16.5, a small heat shock protein that prevents the aggregation of nonnative proteins. The structure of Hsp16.5… Read More

In site-directed spin labeling, the relative solvent accessibility of spin-labeled side chains is taken to be proportional to the Heisenberg exchange rate (W(ex)) of the nitroxide with a paramagnetic reagent in solution. In turn, relative values of W(ex) are determined by continuous wave power saturation methods and expressed as a… Read More

We used site-directed spin-labeling and electron paramagnetic resonance spectroscopy to characterize the conformational motion that couples energy expenditure to substrate translocation in the multidrug transporter MsbA. In liposomes, ligand-free MsbA samples conformations that depart from the crystal structures, including looser packing and water penetration along the periplasmic side. Adenosine triphosphate… Read More

Mammalian small heat shock proteins (sHSP) form polydisperse and dynamic oligomers that undergo equilibrium subunit exchange. Current models of their chaperone activity hypothesize that recognition and binding of protein non-native states involve changes in the oligomeric state. The equivalent thermodynamic representation is a set of three coupled equilibria that includes… Read More