The physiological importance of chloride transport across cell membranes has been underscored recently by the demonstration that genetic defects in a single transmembrane protein involved in chloride ion transport cause cystic fibrosis. However, despite such advances in genetic information the detailed mechanisms of chloride transport by membrane proteins are understood only poorly, in large part because of a paucity of basic information about the dynamics of interactions between anions and their binding sites on transport proteins. We will apply time-resolved Fourier transform infrared (TR-FTIR) spectroscopy to the study of the active chloride transport by biological membranes. The model system to be studied is halorhodopsin (hR), a light-driven anion transport protein found in the membranes of several species of archaebacteria. Comparisons of TR-FTIR vibrational spectra from hR and its transient photointermediates will help to elucidate structural changes occurring during the functioning of this protein. The broad sensitivity of time-resolved FTIR difference spectroscopy should allow detection of transient structural changes in the chromophore, as well as localized conformational alterations of the peptide backbone; protonations or deprotonations of ionizable amino acid side chains; and alterations in non-covalent interactions of cationic residues with chloride or other anions. The time resolution of these measurements will be in the range of 1-10 mus. Spectra of isotope-labeled and mutant hR proteins will also be obtained, in order to assign roles for individual amino acids in various anion binding and release steps. The results should allow the development of a detailed mechanistic model for active chloride transport for this particular system, and the derivation of general principles applicable to mammalian anion transport proteins.
| Hutson, M S; Shilov, S V; Krebs, R et al. (2001) Halide dependence of the halorhodopsin photocycle as measured by time-resolved infrared spectra. Biophys J 80:1452-65 |
| Hutson, M S; Alexiev, U; Shilov, S V et al. (2000) Evidence for a perturbation of arginine-82 in the bacteriorhodopsin photocycle from time-resolved infrared spectra. Biochemistry 39:13189-200 |
| Dioumaev, A K; Braiman, M S (1997) Nano- and microsecond time-resolved FTIR spectroscopy of the halorhodopsin photocycle. Photochem Photobiol 66:755-63 |
| Braiman, M S; Dioumaev, A K; Lewis, J R (1996) A large photolysis-induced pKa increase of the chromophore counterion in bacteriorhodopsin: implications for ion transport mechanisms of retinal proteins. Biophys J 70:939-47 |
| Mitrovich, Q M; Victor, K G; Braiman, M S (1995) Differences between the photocycles of halorhodopsin and the acid purple form of bacteriorhodopsin analyzed with millisecond time-resolved FTIR spectroscopy. Biophys Chem 56:121-7 |
