Our laboratory studies molecular structures, assembly
mechanisms and function of biological macromolecular
machines with physical biochemistry methods. Nuclear
magnetic resonance is used extensively to characterize
both the component biomacromolecules and the detailed
interaction paths involved in forming functional complexes.
High field 2D, 3D, and 4D heteronuclear NMR techniques
are particularly important in determining the structures
of these complexes and characterizing the protein-protein
and protein-DNA interactions associated with assembling
them.
One of the systems currently being studied in detail
is the MetJ repressor, a methionine regulatory system
in E. coli. The mechanism by which the protein dimer,
activated by SAM, sequentially binds to 2 to 5 tandem
DNA recognition sites to form the competent repressor
complex is of particular current interest. The nucleotide
excision repair pathway for identifying and replacing
damaged DNA is also currently under investigation
in this laboratory. We are specifically interested
in the structures of the initial recognition complexes
involving the repair proteins XPA, RPA and XPC, HR23B
with lesioned DNA, and we are also investigating the
molecular mechanisms by which they are formed. The
molecular processes associated with the assembly of
the various components in the correct order to develop
an effective repair complex are being probed by NMR
and molecular modeling.
Along with the investigation of specific systems,
we are also developing biophysical methods to study
increasingly large and complex biological assemblies
often represented in functional biological systems.
These include measuring long-range dipolar contacts
for cross relaxation to define distance constraints
for structure determination and the use of stable
isotope labeling strategies to create relatively rare
spin systems for solution NMR studies. 800 MHz, 600
MHz, and 500 MHz NMR spectrometers are used in these
studies. A CMB trainee is currently, actively engaged
in studying both the MetJ and DNA repair systems and
is involved in the generation and stable isotope labeling
of the component proteins as well as the NMR characterization
of the complexes.
