Assigning membrane binding geometry of cytochrome C by polarized light spectroscopy.
In this work we demonstrate how polarized light absorption spectroscopy (linear dichroism (LD)) analysis of the peptide ultraviolet-visible spectrum of a membrane-associated protein (cytochrome (cyt) c) allows orientation and structure to be assessed with quite high accuracy in a native membrane environment that can be systematically varied with respect to lipid composition. Cyt c binds strongly to negatively charged lipid bilayers with a distinct orientation in which its alpha-helical segments are on average parallel to the membrane surface. Further information is provided by the LD of the pi-pi( *) transitions of the heme porphyrin and transitions of aromatic residues, mainly a single tryptophan. A good correlation with NMR data was found, and combining NMR structural data with LD angular data allowed the whole protein to be docked to the lipid membrane. When the redox state of cyt c was changed, distinct variations in the LD spectrum of the heme Soret band were seen corresponding to changes in electronic transition energies; however, no significant change in the overall protein orientation or structure was observed. Cyt c is known to interact in a specific manner with the doubly negatively charged lipid cardiolipin, and incorporation of this lipid into the membrane at physiologically relevant levels was indeed found to affect the protein orientation and its alpha-helical content. The detail in which cyt c binding is described in this study shows the potential of LD spectroscopy using shear-deformed lipid vesicles as a new methodology for exploring membrane protein structure and orientation.
Caesar
Christina E B
CE
Chalmers University of Technology, Department of Chemical and Biological Engineering, Division of Physical Chemistry, SE-412 96 Gothenburg, Sweden.
Esbjörner
Elin K
EK
Lincoln
Per
P
Nordén
Bengt
B
D000818:Animals; D002308:Cardiolipins / Q000378:metabolism; D002942:Circular Dichroism; D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism*; D006736:Horses; D008027:Light; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009206:Myocardium / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010713:Phosphatidylcholines / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013057:Spectrum Analysis
D002308:Cardiolipins; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; C060037:1-palmitoyl-2-oleoylglycero-3-phosphoglycerol; D045304:Cytochromes c; C028694:1-palmitoyl-2-oleoylphosphatidylcholine