Analysis of the secondary structure of the cys-less yeast mitochondrial citrate transport protein and four single-cys variants by circular dichroism.
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.
Cascio
Michael
M
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Mayor
June A
JA
Kaplan
Ronald S
RS
D019943:Amino Acid Substitution; D001665:Binding Sites; D002352:Carrier Proteins / Q000032:analysis / Q000737:chemistry*; D002942:Circular Dichroism; D019343:Citric Acid / Q000737:chemistry*; D003545:Cysteine / Q000032:analysis / Q000737:chemistry*; D005656:Fungal Proteins / Q000032:analysis / Q000737:chemistry*; D008081:Liposomes / Q000032:analysis / Q000737:chemistry*; D008928:Mitochondria / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D005656:Fungal Proteins; D008081:Liposomes; C020723:citrate-binding transport protein; D019343:Citric Acid; D003545:Cysteine