Pulse dipolar EPR spectroscopy reveals buffer modulated cooperativity of metal templated protein dimerization

06 June 2022, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Self-assembly of protein monomers directed by metal ion coordination constitutes a promising strategy for designing supramolecular architectures complicated by the non-covalent interaction between monomers. Herein, two pulse dipolar electron paramagnetic resonance spectroscopy (PDS) techniques, double electron-electron resonance (DEER) and relaxation-induced dipolar modulation enhancement (RIDME), were simultaneously employed for studying the CuII-templated dimerization behavior of a model protein (Streptococcus sp. Group G, Protein G B1 domain) in both phosphate and Tris-HCl buffers. A cooperative binding model could simultaneously fit all data and demonstrate cooperativity of protein dimerization across α-helical double-histidine motifs in presence of Cu(II) is strongly buffer modulated, representing a platform for highly tunable buffer-switchable templated dimerization. Hence, PDS enriches the family of techniques for monitoring binding processes, supporting the development of novel strategies for bioengineering structures and stable architectures assembled by an initial metal-templated dimerization.


Metal-templated dimerization
cooperative binding model

Supplementary materials

Supplementary Material
I Experimental Procedures: Construct Design, Expression and Purification; EPR Sample Preparation; Pulse EPR Measurement Parameters; Continuous-Wave EPR Measurement Parameters; Pulse Dipolar EPR and Data Processing and Validations; Continuous-Wave EPR Data Processing; Analytical Cooperative Binding Model; In Silico Modelling of Metal Templated Dimer. II Results and Discussion: Screening of Dimer Formation for Different Constructs and Templates; Inversion Recovery Measurements; Validated PELDOR and RIDME Measurements; Continuous-Wave EPR Measurements; Estimating K_D and Cooperativity (α) Parameters; In Silico Modelling of Metal Templated Dimer. III References


Comments are not moderated before they are posted, but they can be removed by the site moderators if they are found to be in contravention of our Commenting Policy [opens in a new tab] - please read this policy before you post. Comments should be used for scholarly discussion of the content in question. You can find more information about how to use the commenting feature here [opens in a new tab] .
This site is protected by reCAPTCHA and the Google Privacy Policy [opens in a new tab] and Terms of Service [opens in a new tab] apply.