Core Facilities and Major Equipment

Bioexpression and Fermentation Facility (BFF)
Proteomics and Mass Spectrometry (PAMS) Facility
CCRC NMR Spectroscopy Facility
South Eastern Regional Collaborative Access Team (SER-CAT)

CMS has established core facilities with specialized equipment for the production and characterization of metal centers in metallobiomolecules. Facilities for optical spectroscopy (absorption/CD/MCD) and magnetochemistry, vibrational spectroscopy (resonance Raman and FTIR), EPR spectroscopy, ICP-MS metal analysis and small scale metalloprotein production (BioXpress Laboratory) have been established at a total cost of more than $5.5M using a combination of NSF, NIH, DOE, University of Georgia Research Foundation and Georgia Research Alliance funding.  CMS faculty also have access to the Bioexpression and Fermentation Facility for large scale production of metalloproteins, the Small Molecule and Macromolecular X-ray Diffraction facilities for determining structures of metalloproteins and synthetic active-site analogs, the Proteomics and Mass Spectrometry Facility, Biomolecular NMR facilities, and Stopped Flow and Freeze Quench Kinetic Facilities. A summary of the utility of these facilities for metallobiochemistry research is given below and complete descriptions of the specific instrumentation in these facilities are given in Facilities and Major Equipment.

The BioXpress Laboratory provides a fully equipped molecular biology laboratory for small scale recombinant expression of wild-type and variant forms of metalloproteins in E. coli (1-10L) and purification of the resultant proteins. This facility is located on the 6th floor of the Chemistry building. The BioXpress Laboratory is a satellite facility of the Bioexpression and Fermentation Facility (BFF) that is located in the Davidson Life Science Complex. The BFF offers large scale recombinant protein expression and purification (100-500 L) using a range of recombinant expression systems (E.coli, mammalian, yeast and baculovirus). The Adams group maintains a state-of-art ICPMS Metal Analysis Facility in the Davidson Life Science Complex which is available to CMS members for routine analyses of 54 metals in protein samples at sub-ppb levels.

The Proteomics and Mass Spectrometry (PAMS) Facility located in the Chemistry Department has several mass spectrometers that are useful for general purpose protein and peptide analysis, for example, measuring the molecular weight of denatured proteins by matrix assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry or a MALDI-TOF/TOF mass spectrometry. These are also useful for examining proteolytic digests of proteins, and can be used to determine or confirm the location of sequence modifications in a mutated protein. A 9.4 T quadrupole-Fourier transform mass spectrometer (FTMS) located in the Amster laboratory is well suited for making accurate mass measurements of intact, nondenatured metalloproteins subunits and holoproteins. These data provide direct measurements of the elemental stoichiometry of metal centers such as Fe-S clusters in a subunit, as well as subunit and metal cofactor stoichiometry in a holoprotein. The 4.7 tesla FTMS located in the PAMS facility is also capable of making these measurements for smaller proteins (<10 kDa).

Excellent facilities are available for structural characterization of metalloproteins or small molecules using NMR or X-ray crystallography. The Chemistry Department NMR facility contains 400 and 500 MHz NMR instruments that are used for routine small molecule and biomolecular studies, respectively. More detailed studies of protein structure and dynamics are generally carried out on a 600 MHz NMR with a triple resonance cryogenic probe that is located in the Davidson Life Science Complex. The Prestegard group maintains 800 and 900 MHz NMR instruments in the CCRC NMR Spectroscopy Facility, which are available for more specialized biomolecular applications. The Davidson Life Science Complex is also home to the Biomolecular X-ray Diffraction Core Facility with B. C. Wang as Director. This facility has several crystallization robots for rapid high-throughput screening of crystallization under aerobic and anaerobic conditions and four X-ray beams with area detectors available for data collection. The facility also manages the South Eastern Regional Collaborative Access Team (SER-CAT) at the Advanced Photon Source (APS) for the collection of high quality diffraction data suitable for obtaining metalloprotein structures at atomic-level resolution. A high throughput small molecule X-ray diffraction facility is located in the Chemistry Department under the direction of Greg Robinson.

Metalloproteins are often unique in having active sites that can be selectively investigated in a protein macrocycle by virtue of the electronic and magnetic properties of the metal center. To this end, three complementary facilities have been established in the Chemistry Department under the direction of Michael Johnson, to investigate the electronic, magnetic and vibrational properties of chromophoric transition metal centers. The Optical Spectroscopy and Magnetochemistry Facility provides detailed assignments of electronic excited states in the UV/visible/NIR region based on three complementary approaches, i.e. absorption, circular dichroism, and variable-temperature magnetic circular dichroism (VTMCD) measurements. In many metalloenzymes, the electronic structure of transition metal active sites is as important as 3D structure for understanding catalytic activity. Since metalloproteins with paramagnetic chromophores invariably yield temperature-dependent MCD spectra, VTMCD provides a method of deconvoluting electronic transitions from diamagnetic and paramagnetic metal centers and detailed investigations of MCD intensity as a function of temperature (1.5-300 K) and applied field (0-6 T) (MCD magnetization plots) yield ground state electronic properties such as spin state, zero field splitting parameters and transition polarizations. Complementary ground state electronic information is available based on SQUID magnetometry studies and EPR studies. The EPR Facility enables quantitative measurement of the EPR spectra of paramagnetic transition metal centers in metalloproteins at X-band, Q-band and S-band frequencies at temperatures down to 2 K. This provides quantitative assessment of the nature and amount of each paramagnetic transition metal center along with information on spin states, ground state g-values, hyperfine coupling constants, and the extent of spin-spin interaction between metal centers. The Vibrational Spectroscopy Facility is primarily designed for resonance Raman studies of frozen chromophoric metalloproteins at temperatures down to 15 K and is equipped with CW Ar-ion, Kr-ion and dye lasers that facilitate excitation at any wavelength in the 400-760 nm region. Resonance Raman studies provide an exquisitely sensitive probe of the structure and ligation of metal centers in metalloproteins and a means of investigating excited state electronic structure via excitation profiles. The Vibrational Spectroscopy Facility also includes an FTIR spectrometer with Far-IR and cryogenic (15-300 K) capabilities, that is primarily used to assess metal-ligand stretching modes in model complexes and the vibrational frequencies of small molecules such as O2, CO, NO, CN- and N3- bound to metal sites in metalloproteins.

Spectroscopic studies of metalloenzymes are particularly useful for investigating catalytic mechanisms via kinetic studies to monitor the time course of the reaction and identify catalytic intermediates. Equipment for spectrophotometrically monitored stopped-flow kinetic studies on the ms time scale is available in the Phillips laboratory in the Chemistry Department. In addition an Update Freeze apparatus for kinetic studies on the ms timescale that can be monitored by EPR, resonance Raman or Mössbauer spectroscopy is available in the EPR Facility.

Not all spectroscopic studies are carried out in-house. Synchrotron radiation-based X-ray absorption (XAS) studies provide information of the valence state and coordination environment of metal centers in metallobiomolecules in solution. CMS faculty have established collaborations with Graham George’s group (University of Saskatchewan) for XAS studies. The ability of Mössbauer spectroscopy to characterize and quantify all types of Fe center in a sample, irrespective of valence state or coordination environment, makes it an essential technique for studying Fe-containing metalloproteins. Several CMS faculty collaborate with Carsten Krebs’ group at Penn State University for Mössbauer studies. ENDOR spectroscopy provides a valuable adjunct to EPR for investigating H-bonding interactions, identifying metal ligands and investigating metal substrate and inhibitor interactions in paramagnetic metalloproteins exhibiting EPR signals with unresolved hyperfine interactions. CMS faculty currently collaborate with Brian Hoffman’s group at Northwestern for ENDOR studies of metalloproteins. Finally, Nuclear Resonance Vibrational Spectroscopy (NRVS) is emerging as a valuable adjunct to resonance Raman and FTIR of investigating vibration modes involving Fe movement in Fe-containing metalloproteins. CMS faculty are currently collaborating with Steve Cramer’s group (UC Davis and Lawrence Berkeley National Laboratory) or Nicolai Lehnert’s group (University of Michigan) for NRVS studies.

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This website is maintained by Sharon Hayes. Last updated on 12/03/2013.

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