文章基本信息

标题：Cooperative folding of a polytopic α-helical membrane protein involves a compact N-terminal nucleus and nonnative loops
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作者：Wojciech Paslawski ; Ove K. Lillelund ; Julie Veje Kristensen 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：26
页码：7978-7983
DOI：10.1073/pnas.1424751112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceHow a protein folds in a membrane is a problem of central biological significance. Although extensively investigated for globular proteins, there are very limited data available for membrane proteins due to the difficulties of finding a tractable model system. We present a study of the folding of a six-transmembrane helix protein, the rhomboid protease GlpG, which folds according to a two-state model in a membrane-mimicking mixed micelle surfactant system. By recording the kinetics of folding and unfolding of 69 GlpG mutants and performing an extensive {phi}-value analysis, we propose a folding mechanism and discuss its possible interpretations and implications. These data serve as an excellent starting point for computational studies of membrane protein folding mechanisms and kinetics. 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 {phi}-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 {phi}-values, which are sometimes taken to be indicative of nonnative interactions in the transition state. Mutations in transmembrane helices 3-6 yielded predominantly {phi}-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 {phi}-values remains to be elucidated.
关键词：GlpG ; membrane protein ; rhomboid ; folding ; kinetics