Fig. 1: The principle of inserted peptide structure allosteric regulation (INSRTR) of protein function.

a Scheme of the INRSTR principle, unstructured inserted peptide maintains protein structure and function, CC dimer formation is triggered by the regulatory peptide. INS — inserted peptide, REG — regulatory peptide. b Molecular model of INSRTR firefly luciferase that maintains its structure with insertion of the unstructured CC-forming peptide (inserted peptide) into the loop. The addition of a regulatory peptide leads to CC formation and thus structural changes in firefly luciferase leading to its inactivation. Both modeled by AlphaFold2; fLuc-firefly luciferase. c Peptide N8 was genetically fused into firefly luciferase with different lengths of connecting linker peptides on both sides of the peptide sequence. Linker regions allow for flexibility around peptides which affects firefly luciferase activity as well as the degree of inhibition with the addition of a complementary peptide. d The extent of inhibition depends on CC pair affinity, with the affinity rank N7:N8 > P7A:N8 > P7:N8 > P7N:N8. The values in (b, c) are the means of four biological replicates ± SD and representative of three independent experiments on plasmid transfected HEK293T cells. Significance was tested with one-way ANOVA with Dunnett’s multiple comparisons test between the INSRTR variant and the addition of a REG peptide; P values 0.1234 (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***), < 0.0001 (****) (significance, confidence intervals, degrees of freedom, F and P values are listed in Supplementary Table S2).