Probing the folding and unfolding of wild-type and mutant forms of bacteriorhodopsin in micellar solutions: evaluation of reversible unfolding conditions.
Wild-type and mutant forms of bacteriorhodopsin (sbR) from Halobacterium salinarium, produced by Escherichia coli overexpression of a synthetic gene, were reversibly unfolded in 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 3-[(3-cholamidopropyl)dimethylamino]-2-hydroxyl-1-propane (CHAPSO), and sodium dodecyl sulfate (SDS) mixed micelles. To study the effect on protein stability by substitutions on the hydrophobic surface with polar residues, the unfolding behavior of a G113Q, G116Q mutant [sbR(Q2)] was compared to the wild-type sbR [sbR(WT)]. sbR(Q2) was more sensitive to SDS-induced unfolding than sbR(WT) under equilibrium conditions, and kinetic experiments showed that sbR(Q2) was more sensitive to acid-induced denaturation and thermal unfolding than sbR(WT). Since the mutations in sbR(Q2) were on the detergent-embedded hydrophobic surface of sbR, protein destabilization by these mutations supports the concept that the membrane-embedded segments are important for the stability of sbR. Our experiments provide the basis for studying the thermodynamic stability of sbR by evaluating reversible folding and unfolding conditions in DMPC/CHAPSO/SDS mixed micelles.
Chen
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Department of Biochemistry and Molecular Biophysics, Columbia University, New York 10032, USA.
Gouaux
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D019943:Amino Acid Substitution / Q000235:genetics; D001436:Bacteriorhodopsins / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002793:Cholic Acids / Q000378:metabolism; D003902:Detergents / Q000378:metabolism; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D019791:Guanidine; D019617:Halobacterium salinarum; D006863:Hydrogen-Ion Concentration; D008823:Micelles; D017354:Point Mutation; D011489:Protein Denaturation / Q000235:genetics; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D014508:Urea
D002793:Cholic Acids; D003902:Detergents; D008823:Micelles; D011993:Recombinant Fusion Proteins; D012996:Solutions; D012967:Sodium Dodecyl Sulfate; D001436:Bacteriorhodopsins; C048531:chapso; D014508:Urea; D019791:Guanidine; D004134:Dimyristoylphosphatidylcholine