Although we use a complement of techniques in our research including x-ray crystallography, cryo-electron microscopy and mass spectrometry, the strength of the laboratory is in the use and development of spin and fluorescence labeling. These techniques allow the determination of the static structure of proteins and the mapping of conformational dynamics involved in function.
Both spin and fluorescence labeling rely on cysteine mutagenesis to incorporate the reporter group into the protein sequence. We use bacterial and insect cell systems to express recombinant proteins. Five HPLCs and/or FPLCs are available for purification and subsequent labeling of the protein.
Labeled proteins are then analyzed by EPR or fluorescence to lead structural and dynamic information.
EPR analysis of spin labeled proteins results in a set of structural restraints that describes, in a native-like setting, local environments as well as aspects of the global fold of the protein. Spin label accessibility and mobility can be used to determine secondary structure location and topology. Distance measurements between pairs of spin labels in the range from 5-60Å reflect the relative packing of domains and secondary structures. In cases where these parameters are obtained in various conformational intermediates of the protein, they allow for a detailed mapping of structural changes involved in function. There are relatively few limits on the size and environment of the protein, particularly when compared to X-ray crystallography and NMR spectroscopy. Recently, a Rosetta approach to determine protein structure was implemented in collaboration with Jens Meiler's lab.
Similar information can be obtained from analysis of fluorescently labeled proteins.