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Site-specific Labeling of Active Proteins with Gold Nanoparticles

  • Marie-Eve Aubin-Tam (a1) and Kimberly Hamad-Schifferli (a2)


Covalent conjugation of nanoparticles to proteins is challenging as proteins have numerous residues to which the nanoparticle can non-specifically adsorb. This is problematic as non-specific adsorption is known to denature the protein, altering its structure and thus compromising protein activity. We study site specific gold nanoparticle labeling of two enzymes, Ribonuclease S and Cytochrome c, with the goal of understanding conditions that minimize non-specific adsorption and optimize protein structure and activity. Ribonuclease S is a two-piece protein made of S-peptide and S-protein. 3nm gold nanoparticle is attached to a mutated cysteine residue on the S-peptide. The altered enzymatic activity of gold labeled Ribonuclease S is determined using RNA substrate with a fluorophore-quencher couple. Cytochrome c is linked to 1.5nm nanoparticles with ligands having neutral, negatively, or positively charged endgroups, through covalent attachment of gold with a specific surface cysteine residue. The labeled protein is characterized by circular dichroism spectroscopy and UV-visible absorption. For both proteins, agarose gel electrophoresis was used to determine optimal reaction stoichiometry and also probe non-specific adsorption between the nanoparticle and protein.



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[1] Gole, A., Dash, C., Soman, C., Sainkar, S. R., Rao, M., and Sastry, M., “On the Preparation, Characterization, and Enzymatic Activity of Fungal Protease-Gold Colloid Bioconjugates,” Bioconjugate Chemistry, vol.12, pp. 684690, 2001.
[2] Djalali, R., Chen, Y.-f., and Matsui, H., “Au Nanowire Fabrication from Sequenced Histidine-Rich Peptide,” JACS, vol.124, pp. 1366013661, 2002.
[3] Hainfeld, J. F. and Powell, R. D., “New Frontiers in Gold Labeling,” Journal of Histochemistry & Cytochemistry, vol.48, pp. 471480, 2000.
[4] Hamad-Schifferli, K., Schwartz, J. J., Santos, A. T., Zhang, S. G., and Jacobson, J. M., “Remote Electronic Control of DNA Hybridization through Inductive Coupling to an Attached Metal Nanocrystal Antenna,” Nature, vol.415, pp. 152155, 2002.
[5] Brown, K. R., Fox, A. P., and Natan, M. J., “Morphology-Dependent Electrochemistry of Cytochrome c at Au Colloid-Modified SnO2 Electrodes,” JACS, vol.118, pp. 11541157, 1996.
[6] Keating, C. D., Kovaleski, K. M., and Natan, M. J., “Protein:Colloid Conjugates for Surface Enhanced Raman Scattering: Stability and Control of Protein Orientation,” J. Phys. Chem. B, vol.102, pp. 94049413, 1998.
[7] Jiang, X., Jiang, J., Jin, Y., Wang, E., and Dong, S., “Effect of Colloidal Gold Size on the Conformational Changes of Adsorbed Cytochrome c: Probing by Circular Dichroism, UV-Visible, and Infrared Spectroscopy.,” Biomacromolecules, vol.6, pp. 4653, 2005.
[8] Verma, A. and Rotello, V. M., “Surface Recognition of Biomacromolecules Using Nanoparticle Receptors,” Chemical Communications, vol.3, pp. 303312, 2005.
[9] Jana, N. R. and Peng, X., “Single-Phase and Gram-Scale Routes toward Nearly Monodisperse Au and Other Noble Metal Nanocrystals,” J. Am. Chem. Soc., vol.125, pp. 1428014281, 2003.
[10] Weare, W. W., Reed, S. M., Warner, M. G., and Hutchison, J. E., “Improved Synthesis of Small (dcore 1.5 nm) Phosphine-Stabilized Gold Nanoparticles,” JACS, vol.122, pp. 1289012891, 2000.
[11] Kelemen, B. R., Klink, T. A., Behlke, M. A., Eubanks, S. R., Leland, P. A., and Raines, R. T., “Hypersensitive substrate for ribonucleases,” Nucleic Acids Res., vol.27, pp. 36963701, 1999.
[12] Sreerama, N. and Woody, R. W., “Computation and Analysis of Protein Circular Dichroism Spectra,” Numerical Computer Methods, vol.383, pp. 318351, 2004.
[13] Aubin, M.-E., Morales, D. G., and Hamad-Schifferli, K., “Labeling Ribonuclease S with a 3nm Au nanoparticle by two-step assembly,” Nano Letters, vol.5, pp. 519522, 2005.
[14] Dwyer, J. J., Dwyer, M. A., and Kossiakoff, A. A., “High Affinity RNase S-Peptide Variants Obtained by Phage Display Have a Novel “Hot-Spot” of Binding Energy,” Biochemistry, vol.40, pp. 1349113500, 2001.
[15] Aubin-Tam, M.-E. and Hamad-Schifferli, K., “Gold nanoparticle-cytochrome c complexes: the effect of nanoparticle ligand charge on protein structure,” Langmuir, vol.21, pp. 1208012084, 2005.


Site-specific Labeling of Active Proteins with Gold Nanoparticles

  • Marie-Eve Aubin-Tam (a1) and Kimberly Hamad-Schifferli (a2)


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