Fig. 1: The principle of the heme binding tag-based iCliPSpy assay.

a Bis-thiolate ligation of heme depends on dimerization of TNFα-(1-39). Cleavage within the TMD part L31-L39 of TNFα-(1-39) prevents dimerization by removing or disrupting the S³⁴XXS³⁷ motif and results in mono-thiolate ligation of heme by Cys30. Bis-thiolate and mono-thiolate-coordinated heme molecules differ in their absorbance spectra, so that TNFα-(1-39) (substrate for intramembrane-cleaving protease) and TNFα-(1-34) (product of intramembrane proteolysis) with bound heme show different colors. Because of increased heme binding affinity, we use TNFα-(1-39)-L31P instead of TNFα-(1-39) wt as heme binding tag. b MBP-TNFα constructs used in our studies [lacking the signal peptide (SP) of E. coli MBP shown at the top of the figure]. 1, MBP- TNFα-(1-39)-L31P with a poly-Asn linker was used in our initial study to characterize heme binding of TNFα²⁵. 2-4 MBP-TNFα fusions used in the present study all contain the peptide DAALAAAQTNAAA, which links MBP to TNFα-(1-39). The topology of MBP-TNFα-(1-39) fusion proteins is exemplary shown for construct 1. For co-production with I-CLiP RseP, construct 3 [named MBP_mut-TNFα-(1-39)-L31P] was used, which contains additional eight substitutions that further increase affinity for amylose, reduce the surface entropy and increase the crystal-packing interactions between adjacent MBP molecules³¹.