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Probing folded and unfolded states of outer membrane protein a with steady-state and time-resolved tryptophan fluorescence

Kim, Judy E. and Arjara, Gitrada and Richards, John H. and Gray, Harry B. and Winkler, Jay R. (2006) Probing folded and unfolded states of outer membrane protein a with steady-state and time-resolved tryptophan fluorescence. Journal of Physical Chemistry B, 110 (35). pp. 17656-62. ISSN 1520-6106. PMCID PMC2519049. doi:10.1021/jp061991r.

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Steady-state and time-resolved fluorescence measurements on each of five native tryptophan residues in full-length and truncated variants of E. coli outer-membrane protein A (OmpA) have been made in folded and denatured states. Tryptophan singlet excited-state lifetimes are multiexponential and vary among the residues. In addition, substantial increases in excited-state lifetimes accompany OmpA folding, with longer lifetimes in micelles than in phospholipid bilayers. This finding suggests that the Trp environments of OmpA folded in micelles and phospholipid bilayers are different. Measurements of Trp fluorescence decay kinetics with full-length OmpA folded in brominated lipid vesicles reveal that W102 is the most distant fluorophore from the hydrocarbon core, while W7 is the closest. Steady-state and time-resolved polarized fluorescence measurements indicate reduced Trp mobility when OmpA is folded in a micelle, and even lower mobility when the protein is folded in a bilayer. The fluorescence properties of truncated OmpA, in which the soluble periplasmic domain is removed, only modestly differ from those of the full-length form, suggesting similar folded structures for the two forms under these conditions.

Item Type:Article
Related URLs:
URLURL TypeDescription CentralArticle
Gray, Harry B.0000-0002-7937-7876
Winkler, Jay R.0000-0002-4453-9716
Additional Information:© 2006 American Chemical Society. ReceiVed: March 30, 2006; In Final Form: June 17, 2006. Publication Date (Web): August 16, 2006. We thank Lukas K. Tamm at the University of Virginia for providing us with plasmids pET1102 and pET1103; Dennis Rinehart (University of Virginia), Thomas Surrey (EMBL Heidelberg), and William Ja for assistance with OmpA isolation and purification; and Jennifer C. Lee and Kate Pletneva for helpful discussions. This work was supported by NIH (NRSA postdoctoral fellowship to J.E.K.; NRSA training grant to G.A.; GM-068461 to J.R.W.), the Department of Energy (J.R.W., DE-FG02-02ER15359), the Ellison Medical Foundation (Senior Scholar in Aging to H.B.G.), and the Arnold and Mabel Beckman Foundation.
Funding AgencyGrant Number
NRSA Postdoctoral FellowshipUNSPECIFIED
Department of Energy (DOE)DE-FG02-02ER15359
Ellison Medical FoundationUNSPECIFIED
Arnold and Mabel Beckman FoundationUNSPECIFIED
Issue or Number:35
PubMed Central ID:PMC2519049
Record Number:CaltechAUTHORS:20150424-152416156
Persistent URL:
Official Citation:Probing Folded and Unfolded States of Outer Membrane Protein A with Steady-State and Time-Resolved Tryptophan Fluorescence Judy E. Kim, Gitrada Arjara, John H. Richards, Harry B. Gray, and Jay R. Winkler The Journal of Physical Chemistry B 2006 110 (35), 17656-17662 DOI: 10.1021/jp061991r
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:56978
Deposited By: George Porter
Deposited On:24 Apr 2015 22:37
Last Modified:10 Nov 2021 21:06

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