2019
DOI: 10.1126/science.aaw8208
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Watching helical membrane proteins fold reveals a common N-to-C-terminal folding pathway

Abstract: To understand membrane protein biogenesis, we need to explore folding within a bilayer context. Here, we describe a single-molecule force microscopy technique that monitors the folding of helical membrane proteins in vesicle and bicelle environments. After completely unfolding the protein at high force, we lower the force to initiate folding while transmembrane helices are aligned in a zigzag manner within the bilayer, thereby imposing minimal constraints on folding. We used the approach to characterize the fo… Show more

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Cited by 66 publications
(127 citation statements)
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References 66 publications
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“…In single molecule folding experiments, GlpG refolding rates increased from approximately 15% to approximately 60% when the bilayer curvature was increased by reducing vesicle size, thereby presumably reducing the barrier to insertion into the head group region. 70 GalP also preferred higher lateral pressures: optimal GalP refolding in DOPE/ DOPC vesicles was observed as the DOPE fraction was increased up to 60%. 99 Similarly, incorporation of KcsA into DOPE/DOPC vesicles increased as the fraction of PE increased.…”
Section: Bilayer Mechanics and Membrane Protein Insertionmentioning
confidence: 95%
See 1 more Smart Citation
“…In single molecule folding experiments, GlpG refolding rates increased from approximately 15% to approximately 60% when the bilayer curvature was increased by reducing vesicle size, thereby presumably reducing the barrier to insertion into the head group region. 70 GalP also preferred higher lateral pressures: optimal GalP refolding in DOPE/ DOPC vesicles was observed as the DOPE fraction was increased up to 60%. 99 Similarly, incorporation of KcsA into DOPE/DOPC vesicles increased as the fraction of PE increased.…”
Section: Bilayer Mechanics and Membrane Protein Insertionmentioning
confidence: 95%
“…68,69 Similarly, GlpG refolding rates improved as the negatively charged DMPG concentration in DMPC/ CHAPSO bicelles increased, reaching an approximately sevenfold increase at a DMPG fraction of 30% compared to 0% DMPG. 70 Membrane charge can also play a practical role in defining the orientation of proteins in artificial liposomes. The N-terminus of proteorhodopsin has many negatively charged residues, and the C terminus has many positively charged residues.…”
Section: Lipid Charge and In Vitro Reconstitutionmentioning
confidence: 99%
“…For example, recent experiments on membrane proteins have revealed previously hidden states using high resolution AFM cantilevers; 49 similarly, novel magnetic tweezers experiments on membrane proteins using standing bicelles have revealed the individual folding pathways and directionality of two distinct membrane proteins, occurring one a-helix at a time. 50 A key, striking finding in the field, enabled by the combined use of OT and polyproteins, was the discovery that proteins need to mechanically unfold to go through the ClpX proteasomal machinery. 51 Along similar lines, using a combination of OT and fluorescence particle-tracking, it was very recently discovered that the ClpB disaggregase translocates protein loops at forces in excess of B50 pN.…”
Section: State-of-the-art Applications New Directions and Final Concmentioning
confidence: 99%
“…EmrE: The previously described Nout-Cout oriented EmrE(Cout) version carrying mutations T 28 R, L 85 R and R 106 A was engineered in a pETDuet-1 vector (13). A series of constructs was designed by inserting nucleotides downstream of EmrE(Cout) coding for a variable LepBderived linker sequence (between 4 and 34 residues), the 9-residue long HA tag, the 17residue long E. coli SecM AP, and a 23-residue long C-terminal tail.…”
Section: Cloning and Mutagenesismentioning
confidence: 99%
“…Point mutations in an upstream TMH can affect the pulling force generated by downstream TMHs in a highly position-dependent manner, suggestive of residue-specific interactions between TMHs during the membrane-integration process. Complementing in vitro unfolding/folding studies (27,28), real-time FRET analyses (15), chemical crosslinking (29), structure determination (30), and computational modeling (31), high-resolution in vivo FPA can help identify the molecular interactions underlying cotranslational membrane protein biogenesis with single-residue precision. For constructs with N≥298, the C-terminal tail is 75 residues long.…”
mentioning
confidence: 99%