title: Cooperative folding of a polytopic α-helical membrane protein involves a compact N-terminal nucleus and nonnative loops.
abstract: Despite the ubiquity of helical membrane proteins in nature and their pharmacological importance, the mechanisms guiding their folding remain unclear. We performed kinetic folding and unfolding experiments on 69 mutants (engineered every 2-3 residues throughout the 178-residue transmembrane domain) of GlpG, a membrane-embedded rhomboid protease from Escherichia coli. The only clustering of significantly positive ϕ-values occurs at the cytosolic termini of transmembrane helices 1 and 2, which we identify as a compact nucleus. The three loops flanking these helices show a preponderance of negative ϕ-values, which are sometimes taken to be indicative of nonnative interactions in the transition state. Mutations in transmembrane helices 3-6 yielded predominantly ϕ-values near zero, indicating that this part of the protein has denatured-state-level structure in the transition state. We propose that loops 1-3 undergo conformational rearrangements to position the folding nucleus correctly, which then drives folding of the rest of the domain. A compact N-terminal nucleus is consistent with the vectorial nature of cotranslational membrane insertion found in vivo. The origin of the interactions in the transition state that lead to a large number of negative ϕ-values remains to be elucidated.
authors.affiliation: Interdisciplinary Nanoscience Center, Department of Molecular Biology and Genetics, Center for Insoluble Protein Structures, Aarhus University, DK-8000 Aarhus C, Denmark;
authors.forename: Wojciech
authors.initials: W
authors.lastname: Paslawski
authors.affiliation: Interdisciplinary Nanoscience Center, Department of Molecular Biology and Genetics, Center for Insoluble Protein Structures, Aarhus University, DK-8000 Aarhus C, Denmark;
authors.forename: Ove K
authors.initials: OK
authors.lastname: Lillelund
authors.affiliation: Interdisciplinary Nanoscience Center, Department of Molecular Biology and Genetics, Center for Insoluble Protein Structures, Aarhus University, DK-8000 Aarhus C, Denmark;
authors.forename: Julie Veje
authors.initials: JV
authors.lastname: Kristensen
authors.affiliation: Interdisciplinary Nanoscience Center, Department of Molecular Biology and Genetics, Center for Insoluble Protein Structures, Aarhus University, DK-8000 Aarhus C, Denmark;
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: Schafer
authors.affiliation: Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
authors.forename: Rosanna P
authors.initials: RP
authors.lastname: Baker
authors.affiliation: Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
authors.forename: Sinisa
authors.initials: S
authors.lastname: Urban
authors.affiliation: Interdisciplinary Nanoscience Center, Department of Molecular Biology and Genetics, Center for Insoluble Protein Structures, Aarhus University, DK-8000 Aarhus C, Denmark; dao@inano.au.dk.
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Otzen
mesh_terms: D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C102898:GlpG protein, E coli; D008565:Membrane Proteins; D010450:Endopeptidases
keywords: GlpG; folding; kinetics; membrane protein; rhomboid