Cooperative folding of a polytopic α-helical membrane protein involves a compact N-terminal nucleus and nonnative loops

Wojciech Paslawski, Ove K. Lillelund, Julie Veje Kristensen, Nicholas P. Schafer, Rosanna Baker, Sinisa Urban, Daniel E. Otzen

Research output: Contribution to journalArticle

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 membraneembedded 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 denaturedstate-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.

Original languageEnglish (US)
Pages (from-to)7978-7983
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume112
Issue number26
DOIs
StatePublished - Jun 30 2015

Fingerprint

Cluster Analysis
Membrane Proteins
Peptide Hydrolases
Pharmacology
Escherichia coli
Mutation
Membranes
Proteins

Keywords

  • Folding
  • GlpG
  • Kinetics
  • Membrane protein
  • Rhomboid

ASJC Scopus subject areas

  • General

Cite this

Cooperative folding of a polytopic α-helical membrane protein involves a compact N-terminal nucleus and nonnative loops. / Paslawski, Wojciech; Lillelund, Ove K.; Kristensen, Julie Veje; Schafer, Nicholas P.; Baker, Rosanna; Urban, Sinisa; Otzen, Daniel E.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 112, No. 26, 30.06.2015, p. 7978-7983.

Research output: Contribution to journalArticle

Paslawski, Wojciech ; Lillelund, Ove K. ; Kristensen, Julie Veje ; Schafer, Nicholas P. ; Baker, Rosanna ; Urban, Sinisa ; Otzen, Daniel E. / Cooperative folding of a polytopic α-helical membrane protein involves a compact N-terminal nucleus and nonnative loops. In: Proceedings of the National Academy of Sciences of the United States of America. 2015 ; Vol. 112, No. 26. pp. 7978-7983.
@article{e3171d18122341b58451cb7128165f5a,
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 membraneembedded 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 denaturedstate-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.",
keywords = "Folding, GlpG, Kinetics, Membrane protein, Rhomboid",
author = "Wojciech Paslawski and Lillelund, {Ove K.} and Kristensen, {Julie Veje} and Schafer, {Nicholas P.} and Rosanna Baker and Sinisa Urban and Otzen, {Daniel E.}",
year = "2015",
month = "6",
day = "30",
doi = "10.1073/pnas.1424751112",
language = "English (US)",
volume = "112",
pages = "7978--7983",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "26",

}

TY - JOUR

T1 - Cooperative folding of a polytopic α-helical membrane protein involves a compact N-terminal nucleus and nonnative loops

AU - Paslawski, Wojciech

AU - Lillelund, Ove K.

AU - Kristensen, Julie Veje

AU - Schafer, Nicholas P.

AU - Baker, Rosanna

AU - Urban, Sinisa

AU - Otzen, Daniel E.

PY - 2015/6/30

Y1 - 2015/6/30

N2 - 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 membraneembedded 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 denaturedstate-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.

AB - 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 membraneembedded 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 denaturedstate-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.

KW - Folding

KW - GlpG

KW - Kinetics

KW - Membrane protein

KW - Rhomboid

UR - http://www.scopus.com/inward/record.url?scp=84937881485&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84937881485&partnerID=8YFLogxK

U2 - 10.1073/pnas.1424751112

DO - 10.1073/pnas.1424751112

M3 - Article

VL - 112

SP - 7978

EP - 7983

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 26

ER -