Determination of the orientation of adsorbed cytochrome C on carboxyalkanethiol self-assembled monolayers by in situ differential modification.
The contact domain utilized by horse cytochrome c when adsorptively bound to a C(10)COOH self-assembled monolayer (SAM) was delineated using a chemical method based on differential modification of surface amino acids. Horse cytochrome c was adsorbed at low ionic strength (pH 7.0, 4.4 mM potassium phosphate) onto 10 microm diameter gold particles coated with HS(CH(2))(10)COOH SAMs. After in situ modification of lysyl groups by reductive Schiff-base methylation, the protein was desorbed, digested using trypsin, and the peptide mapped using LC/MS. Relative lysyl reactivities were ascertained by comparing the resulting peptide frequencies to control samples of solution cytochrome c modified to the same average extent. The least reactive lysines in adsorbed cytochrome c were found to be 13, 72, 73, 79, and 86-88, consistent with a contact region located up and to the left (Met-80 side) of the solvent-exposed heme edge (conventional front face view). The most reactive lysines were 39, 53, 55, and 60, located on the lower backside. The proposed orientation features a heme tilt angle of approximately 35-40 degrees with respect to the substrate surface normal. Factors that can complicate or distort data interpretation are discussed, and the generality of differential modification relative to existing in situ methods for protein orientation determination is also addressed.
Xu
Jishou
J
Department of Chemistry, North Carolina State University, Raleigh, NC 27695-8204, USA.
Bowden
Edmond F
EF
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001671:Biochemistry / Q000379:methods*; D002853:Chromatography, Liquid; D045304:Cytochromes c / Q000737:chemistry* / Q000378:metabolism*; D004566:Electrodes; D006046:Gold; D006736:Horses; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D008745:Methylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012545:Schiff Bases; D014357:Trypsin
D012545:Schiff Bases; D006046:Gold; D045304:Cytochromes c; D014357:Trypsin; D008239:Lysine