Supplementary data for "Molecular insights into the remarkable thermostability of Ts2631 endolysin" by Karolina Ciemińska, Anna-Karina Kaczorowska, Lukasz Pawel Kozlowski, Tadeusz Kaczorowski, Magdalena Plotka

In the antibiotic resistance crisis, novel solutions to combat bacterial infections are explored, one of which includes the use of endolysins, enzymes derived from bacteriophages that destroy peptidoglycan, a vital bacterial structure. Of particular interest are endolysins that exhibit exceptional thermostability. An example of such endolysin is Ts2631 from bacteriophage vB_Tsc2631 infecting extremophile Thermus scotoductus. The Ts2631 endolysin melting temperature, representing the point at which the protein is equally folded and unfolded, is between 99.8°C and 104.7°C, depending on the solvent. Proline, arginine and tryptophan are known to be involved in protein stabilization. Therefore, we obtained single substitution variants of Ts2631 endolysin by replacing aforementioned residues with alanine. The lytic activity of 31 variants was analyzed against Thermus thermophilus HB8 in turbidity reduction assay. Variants R51A, W53A and R82A revealed reduced activity at 60°C. Five other substitutions R20A, W102A, W109A, P140A, and W145A displayed decreased functionality after heating at 90°C. The thermodynamic stability of all proteins was analyzed using nano differential scanning fluorimetry (nanoDSF). The variants with reduced activity after heat treatment also exhibited significantly lower melting temperatures compared to the wild-type enzyme (ΔT_m up to -24°C). Another 24 substitution variants of conserved residues, whose activity was determined in Ts2631 endolysin previous studies, were included in the nanoDSF experiments. The study confirmed that changes of residues involved in direct Zn²⁺ binding (H30, H131 and C139) have a destabilizing effect on the structure of the Ts2631 endolysin. Substitutions of residues located mainly near the catalytic center of the protein lead to a decrease in its thermostability, which consequently affects the lytic activity of Ts2631. We also found that arginine R20 is involved in peptidoglycan binding. This is the first study of the thermostability mechanism of an endolysin isolated from extremophilic environment.