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Published on: April 28, 2023
Site-directed nanoparticle labeling of cytochrome c
Marie-Eve Aubin-Tam1, Wonmuk Hwang, Kimberly Hamad-Schifferli
1Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Summary
Site-specific labeling of proteins with nanoparticles is crucial for nanomedicine. Attaching nanoparticles to specific protein sites, especially core regions, can cause structural damage, impacting protein function.
Area of Science:
- Bioconjugation chemistry
- Nanomedicine
- Protein engineering
Background:
- Bioconjugation of nanoparticles and proteins is challenging, often leading to protein denaturation and loss of function.
- The protein-nanoparticle interface is poorly understood, hindering nanoparticle applications in nanomedicine.
- Previous studies explored nanoparticle ligand and material effects, but systematically studied the impact of labeling site on protein structure.
Purpose of the Study:
- To investigate the effect of labeling site on protein structure and function during nanoparticle bioconjugation.
- To understand how site-specific attachment of gold nanoparticles to cytochrome c impacts its structural integrity.
Main Methods:
- Site-specific labeling of cytochrome c with negatively charged gold nanoparticles using covalent thiol-Au bonds.
- Controlled attachment site selection through cysteine mutations of surface residues.
- Probing protein structural changes using circular dichroism and molecular dynamics simulations.
Main Results:
- Protein unfolding was most severe when nanoparticles attached to the N- and C-terminal foldon of cytochrome c.
- Attachment near charged residues exacerbated structural damage due to salt-dependent electrostatic interactions.
- Molecular dynamics simulations revealed site-dependent local to global structural perturbations.
Conclusions:
- The labeling site is a critical design criterion for developing functional nanoparticle-protein conjugates.
- Understanding the impact of labeling location is essential for minimizing structural damage and preserving protein function.
- This study provides insights into optimizing bioconjugation strategies for nanomedicine applications.
