Bacteriophage coat proteins have a mature phase in which they are assembled about the phage DNA, but also a phase in which they are in the E. coli membrane prior to final assembly. We are using FTIR spectroscopy to study secondary structure in the membrane phase. In one of the two cases considered thus far (pf1), the membrane-bound structure is highly similar to the mature (phage assembled) structure, which is essentially all alpha helix. In the case of the m13 phage, this is not the case as much beta sheet is present. Our work will shed light on the still murky but very important issue of membrane protein secondary structure and its relation to the amino-acid sequence, and to the nature of the lipid in which the protein is incorporated.
Cell membranes generally have rather high electric fields within them, but their structures are almost never probed in the presence of such a field. We are developing strategies for doing this using FTIR and (in collaboration with Karl Ludwig) x-ray diffraction. Small peptides that are thought to change their orientations from surface to transmembrane in the presence of a field appear to be the most attractive candidates for study.
In association with M. El-Batanouny and C. Willis, we are currently assembling a Brillouin Spectrometer to study biomolecules and assemblies of biomolecules, a field in which this technique has been applied only sparingly. Brillouin spectroscopy is inelastic light scattering that probes low-lying excitations of the scatterer (typically low-lying phonons). We intend to apply the technique to membrane and protein phase changes and dynamics. It should be noted that the large-scale biologically-significant internal motions in proteins are typically on a timescale that may make them accessible to Brillouin techniques. We are prepared to support this experimental program with theoretical calculations and molecular simulations.
Energy Transduction, Ion Transport and Signal
Recognition
K. Rothschild
Membrane proteins facilitate many key cellular processes including energy transduction, ion transport, and signal recognition. Because these proteins are often difficult to crystallize for x-ray analysis, little is known about their structures and molecular mechanisms. For this reason, a combination of advanced spectroscopic and recombinant DNA techniques are being developed to investigate how these membrane proteins function.
A key focus of research is the light-driven proton pump, bacteriorhodopsin. Since the early 1970's, this protein has become a focus for understanding the molecular mechanisms of active ion transport and energy transduction in biological systems. Two other proteins being investigated are rhodopsin, the primary receptor in vision, and acetylcholine receptor, which is involved in the transmission of the nerve impulses.
A central approach is the use of Fourier transform infrared (FTIR) difference spectroscopy. We have shown that this method can be used to detect small conformational changes in membrane proteins. By combining this approach with site-directed mutagenesis, we have been able to construct a model for the key molecular events that occur during proton pumping in bacteriorhodopsin. A more advanced method for assigning bands and conducting structure-function studies is also under development which involves the site-directed isotope labeling of proteins using in vitro expression and supressor tRNAs. In a related approach (see below), the substitution of non-native amino acids is being used to produce new forms of membrane proteins that will be useful in biotechnology.
Biomembrane Technology & Biomolecular Photonics
K. Rothschild
The design of a new generation of materials based on biomembrane components holds promise in diverse areas including optical recording media, chemical sensors, nanometer lithography, energy transducers, and enzyme catalysis. A variety of membrane proteins, including bacteriorhodopsin, rhodopsin, and acetylcholine receptor, exhibit active properties such as energy transduction, active and passive transport, chemical sensing, voltage channel gating, signal transduction, and self-assembly which could find important uses in such materials. However, future progress will depend on the development of new methods for engineering such biomolecules so that they are suitable for use in biomolecular devices including enhanced stability and the ability to exist in a solid-state environment.
In this project, we are developing new methods based on molecular genetics and advanced biophysical techniques which will provide the capability to modify membrane proteins at the molecular level. Key among these techniques will be site-directed non-native amino acid replacement (SNAAR). This approach will provide a new dimension in protein engineering, enabling the replacement of native amino acid residues with custom designed residues. Current studies in collaboration with G. Jones in the Chemistry Department are aimed at incorporating photoactive non-native residues that will alter the electro-optical properties of bacteriorhodopsin. In this regard, recent studies have demonstrated that films produced from bacteriorhodopsin can be used for optical information processing.
We have also demonstrated recently that self-assembled biomembranes can be used as molecular templates for nanostructure material fabrication. In one set of experiments in collaboration with N. Clark and K. Douglas at the University of Colorado, 2-dimensionally crystalline S-layers were used as templates to produce patterned thin metal films with nanometer-sized holes and wires. In a different study (in collaboration with C. Safinya at the University of California Santa Barbara), we recently reported the discovery that dry films of bacteriorhodopsin are structurally stable up to 140oC. Studies are being conducted to produce heat-proof arrays of other proteins.