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Fourier transform IR spectroscopy was used to study protonation and the protein conformational changes that accompany the binding of retinal to an apoprotein state of bacteriorhodopsin in its native membrane. The hydrogen-bonding network associated with the proton pumping function of bacteriorhodopsin was found to be largely formed during these retinal-binding stages. The protonation behaviour seen between the apoprotein and regenerated states is the same as that seen during the photocycle, with the same amino acid residue regulating the protonation changes.
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Marti T Refolding of bacteriorhodopsin from expressed polypeptide fragments. J Biol Chem. 273:1998;9312-9322. In order to investigate the effects of the helix-connecting loops on the regeneration of functional bacteriorhodopsin, fragments of the protein were expressed in E. coli. This extends earlier studies carried out by other groups on the five-helix fragment (helices C-G) that can regenerate a functional protein with the addition of either a two-helix A-B fragment or individual helices A and B. Fragments of two, three, four or five of the transmembrane segments have been expressed. Each fragment was purified from the membrane and denatured in SDS. The complementary fragments A-B C-G, A-C D-G, A-D E-G and A-E F-G were then assembled in the presence of retinal in mixed detergent/lipid micelles. Each of the connecting loops was found to be dispensable for protein regeneration, although differences in stability of the final state were found. The CD and EF loops seem to contribute to helix specificity. Of particular interest is the fact that fragments with overlapping helices can form a functional protein, even with three redundant helices (i.e. a functional protein is regenerated from fragments A-E and C-G, with helices, C, D and E duplicated).
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A thorough characterisation of the individual α helices of bacteriorhodopsin in lipid vesicles. Polypeptides corresponding to each transmembrane α helix were synthesised and their stability and structure assessed in vesicles consisting of native H. salinarium phospholipids using circular dichroism spectroscopy, polarised Fourier transform IR spectroscopy, amide exchange experiments, protease protection and mass spectrometry. Five of the α helices, A-E, independently formed stable transmembrane α helices in the absence of the rest of the protein. A peripheral membrane-bound α/β conformation is preferred for helix, F, however, whilst helix G forms a membrane-associated β-sheet conformation.
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Hunt JF, Earnest TN, Bousche O, Kalghati K, Reilly K, Horváth C, Rothschild KJ, Engelman DM A biophysical study of integral membrane protein folding. Biochemistry. 36:1997;15156-15176. A thorough characterisation of the individual α helices of bacteriorhodopsin in lipid vesicles. Polypeptides corresponding to each transmembrane α helix were synthesised and their stability and structure assessed in vesicles consisting of native H. salinarium phospholipids using circular dichroism spectroscopy, polarised Fourier transform IR spectroscopy, amide exchange experiments, protease protection and mass spectrometry. Five of the α helices, A-E, independently formed stable transmembrane α helices in the absence of the rest of the protein. A peripheral membrane-bound α/β conformation is preferred for helix, F, however, whilst helix G forms a membrane-associated β-sheet conformation.
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A time-resolved circular dichroism study reporting the first observation of α-helix formation during the folding of a membrane protein. Bacteriorhodopsin was refolded from a SDS-denatured state in DMPC/DHPC lipid micelles. The apparent slow formation of the α helices (of over 30 amino acids, equivalent to more than one transmembrane α helix) from a disordered structure was observed, with a time constant of tens of seconds during the rate-limiting folding step. It was proposed that this α-helix formation corresponds to either the folding of a few amino acids at the ends of each transmembrane α helix in the interfacial region or the insertion and formation of larger segments of the N or C-terminal transmembrane α helices.
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The insertion of synthetic peptides with differing average hydrophobicities into neutral or anionic lipid micelles is investigated. Above a certain threshold hydrophobicity, peptides were found to insert spontaneously, whereas below this threshold, electrostatic interactions provided by charged phospholipids are essential for peptide insertion.
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Liu L-P, Deber CM Anionic phospholipids modulates peptide insertion into membranes. Biochemistry. 36:1997;5476-5482. The insertion of synthetic peptides with differing average hydrophobicities into neutral or anionic lipid micelles is investigated. Above a certain threshold hydrophobicity, peptides were found to insert spontaneously, whereas below this threshold, electrostatic interactions provided by charged phospholipids are essential for peptide insertion.
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note
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A of 6 for the insertion reaction. The protonation of one of the two aspartic acids in helix C is assumed to be responsible for this transition. Protonation/deprotonation of one of these aspartic acid residues, Asp96, is involved in the functional photocycle of bacteriorhodopsin, in which it is also accompanied by a conformational change of the protein. The spontaneous insertion of helix C and the photocycle of bacteriorhodopsin therefore both involve the coupling of protein structural changes and the protonation state of a transmembrane aspartic acid.
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Biochemistry
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Woolf TB Molecular dynamics simulations of individual α helices of bacteriorhodopsin in dimyristoylphosphatidylcholine. II. Interaction energy analysis. Biophys J. 74:1998;115-131.
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A transmembrane helix dimer: Structure and implications
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This paper describes the NMR structure of the dimer formed by the transmembrane domain of glycophorin A in SDS micelles. A peptide corresponding to residues 62-101 of glycophorin A was studied, with residues 73-96 forming the transmembrane α helices. The structure reveals that sidechain and backbone atoms of seven amino acids make favourable van der Waals' interactions, with extensive backbone-backbone contacts between the two helices being the result of the presence of two glycines positioned four residues apart.
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MacKenzie KR, Prestegard JH, Engelman DM A transmembrane helix dimer: structure and implications. Science. 276:1997;131-133. This paper describes the NMR structure of the dimer formed by the transmembrane domain of glycophorin A in SDS micelles. A peptide corresponding to residues 62-101 of glycophorin A was studied, with residues 73-96 forming the transmembrane α helices. The structure reveals that sidechain and backbone atoms of seven amino acids make favourable van der Waals' interactions, with extensive backbone-backbone contacts between the two helices being the result of the presence of two glycines positioned four residues apart.
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MacKenzie, K.R.1
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A biophysical characterisation of synthetic peptides corresponding to the six putative membrane-spanning segments of the N-terminal transmembrane domain of the cystic fibrosis transmembrane conductance regulator. The peptides were incorporated into lipid vesicles and Fourier transform IR spectroscopy revealed that they are largely α helical in nature. Although the transbilayer nature of these helices has not yet been investigated, the α-helical content is consistent with the two-stage model of protein folding and provides some of the first structural data on these transmembrane segments.
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Wigley WC, Vijayakumar S, Jones JD, Slaughter C, Thomas PJ Transmembrane domain of cystic fibrosis transmembrane conductance regulator: design, characterisation and secondary structure of synthetic peptides m1-m6. Biochemistry. 37:1998;844-853. A biophysical characterisation of synthetic peptides corresponding to the six putative membrane-spanning segments of the N-terminal transmembrane domain of the cystic fibrosis transmembrane conductance regulator. The peptides were incorporated into lipid vesicles and Fourier transform IR spectroscopy revealed that they are largely α helical in nature. Although the transbilayer nature of these helices has not yet been investigated, the α-helical content is consistent with the two-stage model of protein folding and provides some of the first structural data on these transmembrane segments.
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