Thermodynamic studies of bacteriorhodopsin (BR) have been undertaken in order to investigate the factors that stabilize the structure of a membrane protein. The stability of the native, intact protein was compared to that of protein with retinal removed, and/or cleaved in one or two of the loops connecting the transmembrane helices. The stability was assessed using differential scanning calorimetry and thermal denaturation curves obtained from ultraviolet circular dichroism and absorption spectroscopy. Retinal binding and the loop connections were each found to make a small contribution to stability, and even a sample that was cleaved twice as well as bleached to remove retinal denatured well above room temperature. Removal of retinal destabilized the protein more than cleaving once, and about as much as cleaving twice. Retinal binding and the connections in the loops were found to stabilize BR in independent ways. Cleavage of the molecule into fragments did not reduce the intermolecular cooperativity of the denaturation. Dilution of the protein by addition of excess lipid in order to eliminate the purple membrane crystal lattice also did not alter the cooperativity. These results are used to compare the relative importance of various contributors to the stability of BR. Study holds ProTherm entries: 4032, 4033 Extra Details: membrane protein; retinal binding; loop connections;,purple membrane; cooperativity
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:24 p.m.
|Number of data points||6|
|Proteins||Bacteriorhodopsin ; Bacteriorhodopsin|
|Assays/Quantities/Protocols||Experimental Assay: dHcal buffers:Potassium carbonate: 50 mM, pH:9.0 ; Experimental Assay: Tm buffers:Potassium carbonate: 50 mM, pH:9.0 ; Experimental Assay: dHvH buffers:Potassium carbonate: 50 mM, pH:9.0 ; Experimental Assay: dHcal buffers:Potassium phosphate: 3 mM, pH:7.0 ; Experimental Assay: Tm buffers:Potassium phosphate: 3 mM, pH:7.0 ; Experimental Assay: dHvH buffers:Potassium phosphate: 3 mM, pH:7.0|
|Libraries||Mutations for sequence EAQITGRPEWIWLALGTALMGLGTLYFLVKGMGVSDPDAKKFYAITTLVPAIAFTMYLSMLLGYGLTMVPFGGEQNPIYWARYADWLFTTPLLLLDLALLVDADQGTILALVGADGIMIGTGLVGALTKVYSYRFVWWAISTAAMLYILYVLFFGFTSKAESMRPEVASTFKVLRNVTVVLWSAYPVVWLIGSEGAGIVPLNIETLLFMVLDVSAKVGFGLILLRSRAIFGEAEAPEPSAGDGAAATS|