Caracterització de l'estructura i funció del transportador de melibiosa d'escherichia coli per espectroscòpia d'infraroig

Student thesis: Doctoral thesis

Abstract

MelB is an integral membrane of Escherichia coli (473 amino acids) that couples uphill transport of alpha- or beta-galactosides to the downhill inward movement of Na+, Li+, or H+. The coupling ions compete for the same binding site and enhanced the affinity of the co-transported sugar, with Na+ and Li+ as better activators than H+. The secondary structure topological model consist in 12 transmembrane segments with the N and C termini located in the cytoplasm. In this work, solubilized and reconstituted MelB in four different substrate conditions have been studied: Na+, H+, Na+ with melibiose and H+ with melibiose.
Firstly, MelB secondary structure analysis and quantification were performed by transmission infrared spectroscopy. Results revealed the existence of two type of alpha-helices which suffer conformational changes by different substrate binding, being Na+ the one who produces major changes. This result is coherent with the intrinsic triptophan conformational changes studied by fluorescence spectroscopy. MelB solubilized in denaturating detergents or warmed up in dodecil maltosid, states where the protein is not functional, outcome one only alpha helical band instead of two. This would indicate that the two alpha helical existence is very important for the normal protein function. The secondary structure quantification gave a 50% of alpha helices, 20% beta sheet, 17% reverse turns and 11% assigned to 310 helices, open loops or alpha helices. This assignation agrees with the 12 transmembrane helices topological model. The obtained D2O quantification reinforces these results.
Secondly, the H/D exchange technique by ATR-FTIR was applied, particularly useful to study membrane proteins dynamics properties widely unknown. Results shown that MelB is a relatively accessible protein, with and H/D exchange of 60% after 24 hours. The MelB H/D exchange in different substrate conditions comparison, suggested that melibiose addition significantly reduces its aqueous solvent accessibility. A deeper H/D exchange analysis at a secondary structure level, revealed conformational changes in different substrate conditions. Particularly, at the beta sheet and in less amount at the alpha helical level. Melibiose binding gives more protection to both types of structures. This technique was also applied to the MelB mutant, the R141C, in the same substrate conditions. The R141C mutant is capable of binding substrate but not of trasnlocating them on the other membrane site. The results indicate the mutant bind to H+ is about 10% more accessible to the aqueous solvent than the wild type MelB in the same condition. Substrates addition also produces conformational changes, in this case at the beta sheet, alpha helical and reverse turns level. Incubation with melibiose protects beta sheet against the aqueous solvent, too, although the Na/melibiose conditions is the most protected state.
Finally, the polarization technique by ATR-FTIR was applied to study if the different substrate binding also resulted into orientation changes of the transmembrane helices. By means of parallel and perpendicular polarized spectra the order parameter can be calculated, a necessary tool to calculate the orientation angle of the protein structures. The results demonstrate that the reconstituted protein is oriented. The two main bands principally correspond to helicoidal transmembrane segments. MelB without asubstrates (H+/MelB) shows an orientation angle of 26º and doesn't suffer any significant change by melibiose addition. On the contrary, when Na+ is bind to MelB the orientation angle is of 36º and the posterior melibiose addition reduces it about 5º.
Date of Award18 Jul 2003
Original languageCatalan
SupervisorEsteve Padrós Morell (Director)

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