Protein folding

Proteins are known to adopt specific (but dynamic) three-dimensional structures that allow them to carry out their function.  It is well recognized that this process doesn't occur by a random search but is instead a biased search.  We are interested in understanding the factors that guide the protein to the correct structure or set of structures. 
    As part of efforts toward understanding this process we are interested in obtaining structural snapshots of the earliest stages of a protein folding reaction.  Over the past few years we have been focusing on using spectroscopic probes monitoring the geometric and hydrodynamic dimensions of a protein in real time during a folding reaction.  Two complementary experiment techniques that we use and find promising are time-resolved Forster resonant energy transfer (trFRET) and small-angle x-ray scattering (SAXS).  These techniques, especially when combined with rapid mixing techniques, reveal information on the point-to-point distance distributions (trFRET) and low-resolution structural information about partially folded intermediates that transiently populate as the protein folds to the native state.

Protein association, misfolding and disease

    Protein misfolding and aggregation have been implicated in a number of human diseases.  One of the proteins that we study, human superoxide dismutase (hSOD1), has been implicated in amyotrophic lateral sclerosis, also known as Lou Gehrig's disease.  As part of an effort led by Bob Matthews at UMass, we have been studying the thermodynamics of hSOD1 to better understand the factors leading to aggregation.  By mapping out the energy landscape of the protein from thermodynamic and kinetic studies, we can better understand whether mutations lead to a preferential increase in the populations of monomeric species or unfolded species, both of which may be more prone to aggregation or perhaps gain of function than the native dimeric species.

Funding support (Thanks!):

Single-molecule fluorescence and FCS

    Another area that we are interested in is using single-molecule fluorescence methods for studying protein folding reactions and protein-protein interactions.  Over the past year we have built a single-molecule microscope with both 2-photon and 1-photon excitation capability that can operate in FIFO (or time-tagged time-resolved mode, as some prefer to call it).  There are a number of advantages to collecting data with this approach.  With a recent upgrade to 4-card TCSPC detection system, one can monitor dynamics down to ps resolution.  The system we are using was built by an undergraduate in the lab, Danielle Jacobsen, with ~$20K of funds (not including the TCSPC card).

Recent publications

The folding free-energy surface of HIV-1 protease: insights into the thermodynamic basis for resistance to inhibitors.

Noel AF, Bilsel O, Kundu A, Wu Y, Zitzewitz JA, Matthews CR.  J Mol Biol. 2009 Apr 10;387(4):1002-16. Epub 2009 Jan 6.

Microsecond acquisition of heterogeneous structure in the folding of a TIM barrel protein.

Wu Y, Kondrashkina E, Kayatekin C, Matthews CR, Bilsel O.  Proc Natl Acad Sci U S A. 2008 Sep 9;105(36):13367-72. Epub 2008 Aug 29.