The non-native conformations of cytochrome c in sodium dodecyl sulfate and their modulation by ATP.
To understand the interaction of cytochrome c (cyt c) with membranes, a systematic investigation of sodium dodecyl sulfate (SDS)-induced conformational alterations in native horse heart ferricytochrome c (pH 7.0) was carried out using heme absorbance, tryptophan fluorescence and circular dichroism (CD) spectroscopy. ATP interaction with membrane-bound cyt c is known to regulate the process of apoptosis. To understand the effect of nucleotide phosphates on membrane-bound cyt c, we also carried out studies of the interaction of ATP with cyt c in the presence of SDS. Fluorescence and UV-Vis data suggest that SDS induces two different transitions (F to C1, C1 to C2) in cyt c, one in the pre-micellar region and the other in the post-micellar region. The fluorescence data further indicated the increase in distance between Trp 59 and heme in the intermediates in both the regions, suggesting loosening up of cyt c on titration with SDS. The far-UV and near-UV CD data suggest partial loss of secondary and tertiary structure in C1, but complete loss of tertiary structure and no further loss of secondary structure in C2. On titration of C1 and C2 with ATP, the secondary structure is restored. However, the heme ligation pattern and heme exposure change only for C2, but not for C1 on the addition of ATP.
Department of Chemistry, Indian Institute of Technology, Delhi, India.
Unnati
U
Ahluwalia
Shahid M
SM
Nayeem
Shashank
S
Deep
D000255:Adenosine Triphosphate / Q000737:chemistry*; D000818:Animals; D002942:Circular Dichroism / Q000379:methods; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D045304:Cytochromes c / Q000737:chemistry* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry; D006736:Horses; D007700:Kinetics; D008823:Micelles; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods
D003574:Cytochrome c Group; D008823:Micelles; C076615:cytochrome c''; D012967:Sodium Dodecyl Sulfate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D045304:Cytochromes c