Biophysical EPR Spectroscopy and Structural Proteomics: A Site-Directed Spin Labeling Approach
As the field of structural proteomics continues to grow, new insights into biochemical processes often result from a combination of structural and biophysical/biochemical information. The last decade has witnessed significant progress in X-ray crystallography of membrane proteins, and structures have now been reported for a number of membrane protein classes, including receptors, ion channels, pores and transporters. NMR spectroscopy has also provided useful information about structure and dynamics of membrane proteins. However, sometimes high resolution techqniques are insufficient to study complex biological assemblies, and lower resolution biophysical characterization techniques (such as IR, EPR, FRET and EM) may provide the only means to study such systems. Examples of these systems include membrane protein complexes and natively unstructured proteins.
Our lab utilizes X-band EPR spectroscopy and a technique called site-directed spin labeling (SDSL). The developments of loop-gap resonator technology and advances in modern molecular biology have allowed for this technique to gain its recent popularity and power for studying complex biomolecular assemblies. Through the application of biophysical site-directed spin labeling schemes (including other physical characterization techniques), questions concerning membrane protein structure/conformational changes, interaction of natively unstructured proteins with other proteins, membrane translocation phenomenon and conformational changes in mRNA are being investigated.
SDSL is tremendously useful for studying membrane proteins because they can be examined in their native membrane, in reconstituted lipid bilayers or other membrane mimetic systems. SDSL is also used to study the interactions of peptides with membrane bilayers. Additional advantages for using SDSL to study large biological systems include a relatively high sensitivity (nanomole quantities) and effectively no molecular weight limitation. In most cases, site-specific labeling strategies are required. For proteins, site-directed mutagenesis is used to manipulate DNA to introduce a unique cysteine residue at a desired location. After protein expression, the cysteine is chemically modified by reaction with a sulfhydryl-specific nitroxide spin label (Figure 1). Recently, developments have been made in site-specific labeling schemes in RNA.
Generally, three types of data are extracted from SDSL-EPR experiments. First, the dynamics of the nitroxide or the macromolecule to which it is attached are revealed in the EPR line shape. Line shape analysis provides information about backbone dynamics, conformational changes, and local secondary structure. When small peptides are spin-labeled, changes in dynamics correlate with binding to a larger system such as a bilayer or a larger protein assembly or RNA. Second, collision-parameters between the nitroxide and a paramagnet in the environment are used to generate solvent accessibility profiles. These profiles are used to determine secondary structure elements and orientation within membranes and at membrane surfaces. Third, dipolar broadening of line shapes from two nitroxides provides distance information. The distances between the two nitroxides become constraints for 3-D structure determination, as was elegantly demonstrated for the SNARE complex.
Figure 2. Figure of T4 Lysozyme showing specific sites where the R1 spin label was incorporated. Spectra are color coded for the individual sites showing how the EPR line shape changes as a function of local protein structure. (Image taken from personal communication with Wayne L. Hubbell)
List of Research Interests:
conformational changes in mRNA Riboswitches,
the association and translocation of Cell-Penetrating Peptides,
dynamics and "structure" of natively unstructured proteins and peptides
orientations of protein on/at membrane surfaces,
mechanism of colicin infection and translocation,
membrane protein structure, dynamics, and conformational changes.