We plan to study three membrane transport systems: bacteriorhodospsin, melibiose permease and ADP/ATP tranlocato. To study the molecular mechanism of proton transport by bacteriorhodopsin, we will desing and produce several mutants, witch will be analysed using several spectroscopic methods, especially by FTIR. Different strategies will be followed: first, the proton transfer process in the extracellular region will be analysed. The objective is to identify the groups that act as proton acceptors or donors, and to know how the signals between Asp85 and the proton release group are transmitted: what are the role of Arg82 and that of the water network? Second, the protonation changes that presumably occur in the retinal envioronment, and its relationship with the changes that occur in the extracellular region during proton transport will be studied (changes in Asp212 or in water molecules?). Third, the proton transfer mechanisme in the cytoplasmatic region will bestudied, with the objective of knowing how the proton junps across distances of 10-12A where apparently there are no ionisable groups. Fourth, the caution binding sites will be analysed by using several techniques (FTIR,EXAFS,AFM) with the objective of getting new data about their locatiion and function. Our objective in the study of the melibiose permease and the ADP/ATP tranlocator is similar in both: to establish the structural changes and/or athe protonation changes that occur because sobstrate binding. This will be done by using several FTIR techniques: difference spectra; H/D exchange kinetics; changes in the helices orientation.
|Effective start/end date||19/12/00 → 19/12/03|