title: Stable folding core in the folding transition state of an alpha-helical integral membrane protein.
abstract: Defining the structural features of a transition state is important in understanding a folding reaction. Here, we use Φ-value and double mutant analyses to probe the folding transition state of the membrane protein bacteriorhodopsin. We focus on the final C-terminal helix, helix G, of this seven transmembrane helical protein. Φ-values could be derived for 12 amino acid residues in helix G, most of which have low or intermediate values, suggesting that native structure is disrupted at these amino acid positions in the transition state. Notably, a cluster of residues between E204 and M209 all have Φ-values close to zero. Disruption of helix G is further confirmed by a low Φ-value of 0.2 between residues T170 on helix F and S226 on helix G, suggesting the absence of a native hydrogen bond between helices F and G. Φ-values for paired mutations involved in four interhelical hydrogen bonds revealed that all but one of these bonds is absent in the transition state. The unstructured helix G contrasts with Φ-values along helix B that are generally high, implying native structure in helix B in the transition state. Thus helix B seems to constitute part of a stable folding nucleus while the consolidation of helix G is a relatively late folding event. Polarization of secondary structure correlates with sequence position, with a structured helix B near the N terminus contrasting with an unstructured C-terminal helix G.
authors.affiliation: School of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, United Kingdom.
authors.forename: Paul
authors.initials: P
authors.lastname: Curnow
authors.forename: Natalie D
authors.initials: ND
authors.lastname: Di Bartolo
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Moreton
authors.forename: Oluseye O
authors.initials: OO
authors.lastname: Ajoje
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: Saggese
authors.forename: Paula J
authors.initials: PJ
authors.lastname: Booth
mesh_terms: D000409:Alanine / Q000235:genetics; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism*; D019617:Halobacterium salinarum / Q000737:chemistry*; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis / Q000235:genetics; D009154:Mutation / Q000235:genetics; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D058767:Protein Unfolding
chemical_list: D001436:Bacteriorhodopsins; D000409:Alanine