Our general research objectives are to understand biological processes
and phenomena in terms of physical mechanism. The fields and methodologies used
in our work are interdisciplinary, ranging from physics to chemistry and
molecular biology. To this end different techniques and tools are used, e.g.
X-ray crystallography, optical spectroscopy, magnetic resonance (EPR, ENDOR),
protein chemistry, recombinant DNA techniques and computational methods. The
specific problems that we are currently addressing are:
1.) The mechanism of
conversion of electromagnetic energy (light) into chemical energy, i.e.
photosynthesis. This process is mediated by an integral membrane
protein-pigment complex called the reaction center (RC). Our work focuses on
the structural and functional aspects of the RC from the photosynthetic
bacterium Rb. sphaeroides. Our specific aims are: a.) determine and
understand the mechanism of electron transfer kinetics between the co-factors
of the RC; b.) determine the proton pathway from solution to the secondary
quinone, QB, in the interior of the RC; c.) determine the kinetics
of the various steps of the proton transfer chain; d.) study the effect of site
directed mutagenesis on electron and proton transfer kinetics; e.) determine
the structure, at the highest possible X-ray resolution, of native and mutant
RC’s.
2.) The structure of the
cyt c2 = RC complex. In Rb. sphaeroides exogenous cytochrome
c2 forms a transient complex with the RC and functions as a
secondary electron donor to the oxidized bacteriochlorophyll dimer of the RC.
We have crystallized the transient cyt c2:RC complex and are in the
process of determining its three dimensional structure. We are also studying
the dynamics of the docking process as well as the interactions between cyt c2
and the RC in the complex.
3.) Electronic Structure of
Biomolecules. In addition to a knowledge of the spatial structure (obtained
primarily by X-ray diffraction), a knowledge of the electronic structure of the
reactants in the RC is important in understanding electron transfer kinetics.
Since the various donors and acceptors in the RC have unpaired electrons, EPR
and ENDOR are the technique of choice in these investigations. For example from
the EPR spectrum of QA-.QB-., the
exchange interaction between the two electrons, on the primary (QA-)
and secondary quinone (QB-), can be obtained. This is an
important parameter to understand electron transfer from (QA-)
to (QB-).
4.) Crystallization of
Biological Macromolecules, with special emphasis on proteins. Our emphasis is
to understand the detailed mechanisms leading to the formation of single
crystals, required for structure determination by X-ray diffraction. In the
past we have investigated the crystallization process on an easily
crystallizable, water soluble, model compound, lysozyme. We plan to extend the
methodology to other proteins, including integral membrane proteins. The
long-term objective is to arrive at a systematic procedure to obtain high
quality crystals for X-diffraction studies.
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