Activity of membrane proteins immobilized on surfaces as a function of packing density.
A systematic study of the influence of the packing density of proteins on their activity is performed with cytochrome c oxidase (CcO) from R. sphaeroides as an example. The protein was incorporated into a protein-tethered bilayer lipid membrane and CcO was genetically engineered with a histidine-tag, attached to Subunit II, and then tethered by an interaction with functionalized thiol compounds bound to a gold electrode. The packing density was varied by diluting the functionalized thiol with a nonfunctionalized thiol that does not bind to the enzyme. After attaching the CcO to the gold surface, a lipid bilayer was formed to incorporate the tethered proteins. The reconstituted protein-lipid bilayer was characterized by surface enhanced infrared reflection absorption spectroscopy (SEIRAS), electrochemical impedance spectroscopy, surface plasmon resonance, and atomic force microscopy. The activity of the proteins within the reconstituted bilayer was probed by direct electrochemical electron injection and was shown to be very sensitive to the packing density of protein molecules. At low surface density of CcO, the bilayer did not effectively form, and protein aggregates were observed, whereas at very high surface density, very little lipid is able to intrude between the closely packed proteins. In both of these cases, redox activity, measured by the efficiency to accept electrons, is low. Redox activity of the enzyme is preserved in the biomimetic structure but only at a moderate surface coverage in which a continuous lipid bilayer is present and the proteins are not forced to aggregate. Electrostatic and other interaction forces between protein molecules are held responsible for these effects.
Friedrich
Marcel G
MG
Max Planck Institute for Polymer Research, Ackermannweg 10, Mainz, Germany.
Kirste
Vinzenz U
VU
Zhu
Jiapeng
J
Gennis
Robert B
RB
Knoll
Wolfgang
W
Naumann
Renate L C
RL
D004563:Electrochemistry; D004583:Electrons; D021621:Imaging, Three-Dimensional; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D015394:Molecular Structure; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D020349:Surface Plasmon Resonance; D013499:Surface Properties
D008055:Lipids; D008565:Membrane Proteins; D009571:Nitrilotriacetic Acid