Determination of the Orientation of Adsorbed Cytochromecon Carboxyalkanethiol Self-Assembled Monolayers by In Situ Differential Modification

Abstract

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.

Probing Surface Properties of Cytochrome c at Au Bionanoconjugates

“…This was established based on differential modification of surface amino acids to determine the electrode face and the solution face of the protein. 50 The opposing face on the 3D structure was then considered to be facing the solution. For YCc, the surface chosen to interact with the AuNPs is the face presenting the Cys 102 residue, the opposite face being again in contact with the solution.…”

Experimental characterization of adsorbed protein orientation, conformation, and bioactivity

“…15,17 Some of the methods that can provide information on adsorbed protein orientation and tertiary (and quaternary) structure include fluorescence, [18][19][20][21] time-of-flight secondary-ion mass spectrometry, [22][23][24] nuclear magnetic resonance spectroscopy (NMR), 25,26 and amino acid labeling/mass spectrometry (AAL/MS). [27][28][29][30][31] Methods for the determination of secondary structure of adsorbed proteins include Fourier transform infrared spectroscopy, 32,33 surface enhanced Raman scattering, 34,35 and circular dichroism spectropolarimetry (CD). 27,[36][37][38][39] Unfortunately, as the size of the protein increases, many of the spectral signatures that are needed for tertiary structure determination using fluorescence and NMR overlap, introducing much subjectivity into the analyses, thus making it difficult to accurately interpret the configuration of the adsorbed protein.…”

“…In contrast, MS has shown great promise in characterizing the adsorbed configuration of both large and small proteins at a molecular level, especially when used with AAL. [27][28][29][30][31] After evaluating several of these types of methods, our group has specifically focused on the development and adaptation of CD and AAL/MS for the characterization of adsorbed protein orientation and structure, and we will therefore focus on these methods for this paper. Readers are encouraged to refer to the above cited references for the application of the other types of methods that are noted above.…”

The impact of urea-induced unfolding on the redox process of immobilised cytochrome c

“…In fact, the substitution of those Lys residues (Scheme 1) involved in the coordination with the iron atom [15] excludes the existence of a Lys-coordinated species. Moreover, since the same Lys residues are also responsible for the electrostatic binding of cytochrome c on negatively charged self-assembled monolayers (SAMs) [18,19], their substitution affects the kinetics of the interfacial electron transfer process, suggesting different haem orientations for recombinant non-trimethylated Saccharomyces cerevisiae iso-1-cytochrome c (ycc) and K72AK73AK79A with respect the electrode surface.…”