Studies of molecular structures, assembly mechanisms and function of biological macromolecular machines with physical biochemistry methods.
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 structure, antigenecity, and immunogenicity of the highly conserved MPER domain of the HIV-1 gp41 coat protein trimer on the viral membrane. This is an important target for AIDS vaccine design. The MetJ repressor in the methionine regulatory system of bacteria is also currently under investigation. 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. Genomic, genetic and bioinformatic analysis along with biochemical and molecular biology approaches are being used to characterize the systems biology of MetJ repressor pathways across bacterial species.
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, 700 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.