Introducing Mutations in the GB3 Protein to Understand Gold Nanoparticle Interactions
Sharkiesha Jackson1, M.d. Siddik Alom2, Y. Randika Perera2, Nicholas C. Fitzkee2
1Mississippi INBRE Research Scholar, Department of Chemistry, Alcorn State University, Lorman, MS
2Department of Chemistry, Mississippi State University, Mississippi State, MS
Understanding protein-gold nanoparticle (AuNP) interactions, especially understanding the binding competition among multiple proteins in the same solution, is a significant challenge. These interactions are vital when designing functionalized AuNPs, where nanoparticles are used as biological sensors and drug delivery vectors. In these applications, proteins in the biological environment can compete with the AuNP surface, interfering with the nanoparticle’s intended function. Various techniques have been employed to study this behavior, yet the biophysics of protein-surface binding remains insufficiently understood. We hypothesize that, using the right model system, it is possible to develop a predictive model for an amino acid’s contribution to AuNP binding. Therefore, the introduction of point mutations is being explored through polymerase chain reaction (PCR) based site-directed mutagenesis in the third IgG binding domain of Streptococcal protein G (GB3). Previous studies have suggested that GB3 is appropriate for mutagenesis because changing a particular residue, K13, can dramatically alter AuNP binding. Primers were designed, and PCR was performed to vary the residue at position 13. Agarose gel analysis was used to confirm PCR product formation, and after transformation into E. coli, the DNA sequence was determined to ensure successful mutagenesis. Using this approach, we have successfully developed a library of K13 GB3 variants, namely K13H, K13Q, and K13S. We have also optimized the primer sequence, and this sequence is being used to generate additional variants. Future work will study the binding of K13X relative to K13G on AuNPs, and this will reveal a numerical trend for each residue’s intrinsic binding affinity.