Heterodimerizing helices as tools for nanoscale control of the organization of protein-protein and protein-quantum dots

• Published in: Biochimie 2019-12, Vol.167, p.93-105
• Main Authors: Sztatelman, Olga, Kopeć, Katarzyna, Pędziwiatr, Marta, Trojnar, Martyna, Worch, Remigiusz, Wielgus-Kutrowska, Beata, Jemioła-Rzemińska, Małgorzata, Bzowska, Agnieszka, Grzyb, Joanna
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Cysteine - chemistry; Dimerization; Green Fluorescent Proteins - chemistry; Heterodimerizing helices; Hydrogen Bonding; Luminescent Proteins - chemistry; Models, Molecular; Nanohybrids; Protein Conformation, alpha-Helical; Protein Multimerization; Quantum dots; Quantum Dots - chemistry; Red Fluorescent Protein
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2019.09.015

In this study, we tested the possibility of creating complexes of two proteins by fusing them with heterodimerizing helices. We used the fluorescent proteins GFP and mCHERRY expressed with a His-tag as our model system. We added heterodimer-forming sequences at the C- or N- termini of the proteins, opposite to the His-tag position. Heterodimerization was tested for both helices at the C-terminus or at the N- terminus and C-terminus. We observed complex formation with a nanomolar dissociation constant in both cases that was higher by one order of magnitude than the Kds measured for helices alone. The binding of two C-terminal helices was accompanied by an increased enthalpy change. The binding between helices could be stabilized by introducing an additional turn of the helix with cysteine, which was capable of forming disulphide bridges. Covalently linked proteins were obtained using this strategy and observed using fluorescence cross-correlation spectroscopy. Finally, we demonstrated the formation of complexes of protein dimers and quantum dots. [Display omitted] •Heterodimerizing helices were used to drive association between fluorescent proteins.•Fusing helices to proteins markedly increased their dissociation constant.•Termodynamic properties of the complex depend on relative orientation of partners.•Adding a helix turn containing cysteine enabled to form stable and redox reversible complexes.•The complexes of protein dimers with quantum dots were obtained.