Change of apocytochrome c translocation across membrane in consequence of hydrophobic segment deletion.
Wild-type apocytochrome c and its hydrophobic segment deleted mutants, named delta28-39, delta72-86 and delta28-29/72-86 were constructed, expressed and highly purified respectively. Insertion ability into phospholipid monolayer, inducing leakage of entrapped fluorescent dye fluorescein sulfonate (FS) from liposomes, and translocation across model membrane system showed that the wild-type apoprotein and delta28-39 almost exhibited the same characteristics, while mutants with segment 72-86 deletion did not. Furthermore, CD spectra, intrinsic fluorescence emission spectra, and the accessibility of the protein to the fluorescence quenchers: KI, acrylamide and HB demonstrated that the segment 72-86 deletion has a significant effect on the conformational changes of apocytochrome c following its interaction with phospholipid. On the basis of these results it is postulated that the C-terminal hydrophobic segment 72-86 plays an important role in the translocation of apocytochrome c across membrane.
Wang
Xiaoping
X
National Laboratory of Biomacromolecules, Institute of Biophysics, The Chinese Academy of Sciences, Beijing.
Han
Xuehai
X
Jia
Songtao
S
Yang
Fuyu
F
D020106:Acrylamide / Q000494:pharmacology; D000818:Animals; D001059:Apoproteins / Q000235:genetics / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002645:Chickens; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000235:genetics / Q000378:metabolism*; D045304:Cytochromes c; D005452:Fluoresceins / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D008081:Liposomes; D008567:Membranes, Artificial; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D010743:Phospholipids / Q000378:metabolism; D011193:Potassium Iodide / Q000494:pharmacology; D011487:Protein Conformation; D017510:Protein Folding; D021381:Protein Transport / Q000502:physiology*; D017384:Sequence Deletion / Q000502:physiology*
D001059:Apoproteins; D003574:Cytochrome c Group; D005452:Fluoresceins; D006454:Hemoglobins; D008081:Liposomes; D008567:Membranes, Artificial; D010743:Phospholipids; D011193:Potassium Iodide; D020106:Acrylamide; C110302:sulfonefluorescein; D045304:Cytochromes c