Grabbing yeast iso-1-cytochrome c by Cys102: an effective approach for the assembly of functionally active metalloprotein carpets.
We report an approach for immobilizing iso-1-cytochrome c from Saccharomyces cerevisiae on oxygen exposing surfaces derivatized with SH-terminated silanes. The SH moieties from silanes have been brought to react with the partially buried Cys102, forming an intermolecular disulfide bond which anchored covalently cytochrome c to the surface. The presence of a single cysteine residue on the protein surface imparted a well-defined orientation to the molecular edifice. Molecular constructs obtained with native cytochrome c and with a cysteine-depleted mutant (C102T) have been investigated by means of scanning force microscopy under liquid, which was performed to assay the quality of the molecular carpet, showing that the native protein formed a robust monolayer at the surface, whereas only a negligible amount of physisorbed molecules were detected in the case of a mutant. UV-vis absorption spectroscopy was performed to confirm that immobilization takes place via the Cys102 residue. Linear sweep voltammetric measurements showed retention of the redox activity of the covalently immobilized cytochrome c, confirming the viability of the proposed immobilization method for obtaining monolayers of redox active molecules.
Gerunda
Mimmo
M
INFM National Center of nanoStructures and bioSystems at Surfaces-S3, University of Modena and Reggio Emilia, Via G. Campi 213/A, I-41100, Italy.
Bortolotti
Carlo A
CA
Alessandrini
Andrea
A
Sola
Marco
M
Battistuzzi
Gianantonio
G
Facci
Paolo
P
D003545:Cysteine / Q000737:chemistry*; D045304:Cytochromes c / Q000737:chemistry* / Q000235:genetics; D004800:Enzymes, Immobilized; D008667:Metalloproteins / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics; D012821:Silanes / Q000737:chemistry*; D013499:Surface Properties
C014059:CYC1 protein, S cerevisiae; D004800:Enzymes, Immobilized; D008667:Metalloproteins; D029701:Saccharomyces cerevisiae Proteins; D012821:Silanes; D045304:Cytochromes c; D003545:Cysteine