Assigning Membrane Binding Geometry of Cytochrome c by Polarized Light Spectroscopy
Journal article, 2009

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 a-helical segments are on average parallel to the membrane surface. Further information is provided by the LID 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 LID 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 LID 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 a-helical content. The detail in which cyt c binding is described in this study shows the potential of LID spectroscopy using shear-deformed lipid vesicles as a new methodology for exploring membrane protein structure and orientation.

optical

linear dichroism

protein secondary structure

peripheral proteins

ferricytochrome-c

mitochondrial-membrane

flow dichroism

lipid-membranes

circular-dichroism spectra

rotatory dispersion

simple polypeptides

Author

Christina Hagelin

SuMo Biomaterials

Chalmers, Chemical and Biological Engineering, Physical Chemistry

Elin Esbjörner Winters

Chalmers, Chemical and Biological Engineering, Physical Chemistry

Per Lincoln

Chalmers, Chemical and Biological Engineering, Physical Chemistry

Bengt Nordén

Chalmers, Chemical and Biological Engineering, Physical Chemistry

Biophysical Journal

0006-3495 (ISSN) 1542-0086 (eISSN)

Vol. 96 8 3399-3411

Subject Categories

Physical Chemistry

Chemical Sciences

DOI

10.1016/j.bpj.2009.01.025

More information

Latest update

8/18/2020