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Analysis of the secondary structure of the cys-less yeast mitochondrial citrate transport protein and four single-cys variants by circular dichroism.

Abstract：

:Utilizing cysteine scanning mutagenesis, with functional Cys-less citrate transport protein (CTP) serving as the starting template, we previously demonstrated that four single-Cys mutants located in transmembrane domains III and IV, rendered the CTP nonfunctional. The present investigations assess and quantify the secondary structure of the Cys-less CTP and the four single-Cys mutants, both in the absence and presence of citrate, via circular dichroism (CD) spectroscopy. In detergent micelles, highly purified Cys-less CTP contained approximately 50% alpha-helix and approximately 20% beta-sheet. The CD spectra of the G119C, E122C, R181C, and R189C mutants in detergent micelles were virtually superimposable with that of the functional Cys-less CTP, thereby suggesting that the wild-type residues, rather than affecting structure, may assume important mechanistic roles. Exogenously added citrate caused a significant change in the CD spectra of all solubilized CTP samples. Analyses of the spectra of the Cys-less CTP indicated an approximately 10% increase in its alpha-helical content in the presence of citrate. The conformational changes effected by the addition of substrate were less pronounced with the single-Cys mutants. Studies of the Cys-less CTP reconstituted in liposomes indicated that while the CD spectra was red-shifted, the net secondary structure of the reconstituted carrier is approximately equivalent to that of the transporter in detergent micelles, and displayed a response to added citrate. In combination, the above studies indicate that purified Cys-less CTP in either sarkosyl micelles or in liposomes, and the four inactive single-Cys mutants in sarkosyl micelles, retain native-like structure, and thus represent ideal material for detailed structural characterization.

journal_name

J Bioenerg Biomembr

journal_title

Journal of bioenergetics and biomembranes

authors

Cascio M,Mayor JA,Kaplan RS

doi

10.1023/B:JOBB.0000047325.48943.71

keywords:

subject

Has Abstract

pub_date

2004-10-01 00:00:00

pages

429-38

issue

eissn

0145-479X

issn

1573-6881

pii

495538

journal_volume

pub_type

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Analysis of the secondary structure of the cys-less yeast mitochondrial citrate transport protein and four single-cys variants by circular dichroism.