The trials and tribulations of membrane protein folding in vitro

Great strides into understanding protein folding have been made since the seminal work of Anfinsen over 40 years ago, but progress in the study of membrane protein folding has lagged behind that of their water soluble counterparts. Researchers in these fields continue to turn to more advanced techniques such as NMR, mass spectrometry, molecular dynamics (MD) and single molecule methods to interrogate how proteins fold. Our understanding of β-barrel outer membrane protein (OMP) folding has benefited from these advances in the last decade. This class of proteins must traverse the periplasm and then insert into an asymmetric lipid membrane in the absence of a chemical energy source. In this review we discuss old, new and emerging techniques used to examine the process of OMP folding and biogenesis in vitro and describe some of the insights and new questions these techniques have revealed.

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BamA Alone Accelerates Outer Membrane Protein Folding In Vitro through a Catalytic Mechanism

Biochemistry ◽

10.1021/acs.biochem.5b00950 ◽

2015 ◽

Vol 54 (39) ◽

pp. 6009-6011 ◽

Cited By ~ 20

Author(s):

Ashlee M. Plummer ◽

Karen G. Fleming

Keyword(s):

Protein Folding ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Catalytic Mechanism ◽

Membrane Protein Folding

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SecY alterations that impair membrane protein folding and generate a membrane stress

The Journal of Cell Biology ◽

10.1083/jcb.200611121 ◽

2007 ◽

Vol 176 (3) ◽

pp. 307-317 ◽

Cited By ~ 42

Author(s):

Nobuyuki Shimohata ◽

Shushi Nagamori ◽

Yoshinori Akiyama ◽

H. Ronald Kaback ◽

Koreaki Ito

Keyword(s):

Protein Folding ◽

Membrane Proteins ◽

Membrane Protein ◽

Signal Recognition Particle ◽

Membrane Vesicles ◽

In Vitro Translation ◽

Membrane Protein Folding ◽

Protein Biogenesis ◽

Simple Reduction

We report on a class of Escherichia coli SecY mutants that impair membrane protein folding. The mutants also up-regulate the Cpx/σE stress response pathways. Similar stress induction was also observed in response to a YidC defect in membrane protein biogenesis but not in response to the signal recognition particle–targeting defect or in response to a simple reduction in the abundance of the translocon. Together with the previous contention that the Cpx system senses a protein abnormality not only at periplasmic and outer membrane locations but also at the plasma membrane, abnormal states of membrane proteins are postulated to be generated in these secY mutants. In support of this notion, in vitro translation, membrane integration, and folding of LacY reveal that mutant membrane vesicles allow the insertion of LacY but not subsequent folding into a normal conformation recognizable by conformation-specific antibodies. The results demonstrate that normal SecY function is required for the folding of membrane proteins after their insertion into the translocon.

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