Characterization of Denatured States and Reversible Unfolding of Sensory Rhodopsin II.


Type
Article
Change log
Authors
Mitchell, James 
Klein-Seetharaman, Judith 
Nietlispach, Daniel  ORCID logo  https://orcid.org/0000-0003-4364-9291
Abstract

Our understanding on the folding of membrane proteins lags behind that of soluble proteins due to challenges posed by the exposure of hydrophobic regions during in vitro chemical denaturation and refolding experiments. While different folding models are accepted for soluble proteins, only the two-stage model and the long-range interactions model have been proposed so far for helical membrane proteins. To address our knowledge gap on how different membrane proteins traverse their folding pathways, we have systematically investigated the structural features of SDS-denatured states and the kinetics for reversible unfolding of sensory rhodopsin II (pSRII), a retinal-binding photophobic receptor from Natronomonas pharaonis. pSRII is difficult to denature, and only SDS can dislodge the retinal chromophore without rapid aggregation. Even in 30% SDS (0.998 ΧSDS), pSRII retains the equivalent of six out of seven transmembrane helices, while the retinal-binding pocket is disrupted, with transmembrane residues becoming more solvent exposed. Folding of pSRII from an SDS-denatured state harboring a covalently bound retinal chromophore shows deviations from an apparent two-state behavior. SDS denaturation to form the sensory opsin apo-protein is reversible. We report pSRII as a new model protein which is suitable for membrane protein folding studies and has a unique folding mechanism that differs from those of bacteriorhodopsin and bovine rhodopsin.

Description
Keywords
folding, kinetics, membrane proteins, retinal, Animals, Bacteriorhodopsins, Cattle, Kinetics, Protein Binding, Protein Conformation, Protein Denaturation, Protein Folding, Protein Refolding, Protein Structure, Tertiary, Protein Unfolding, Sensory Rhodopsins, Solvents
Journal Title
J Mol Biol
Conference Name
Journal ISSN
0022-2836
1089-8638
Volume Title
430
Publisher
Elsevier BV
Sponsorship
Biotechnology and Biological Sciences Research Council (BB/K01983X/1)
Biotechnology and Biological Sciences Research Council (BB/G011915/1)
BBSRC