(A) Reductive denaturation of non-mannosylated (blue) and C-mannosylated (green) UNC-5 TSR2 monitored using CD spectroscopy at a wavelength of 229 and 228 nm, respectively. Measurements of three non-mannosylated (gray-shaded squares) and C-mannosylated (gray-shaded circles) UNC-5 TSR2 samples were averaged and fitted (Figure 5—source data 1). Average half-life values are depicted in the right plot (error bars show standard deviation). (B) Modeled structures of UNC-5 TSR2 lacking disulfide bridges without C-mannosylation (top) and with two C-mannoses on Trp5 and Trp8 (bottom) at the starting point of each simulation followed by aligned Trp-Arg ladder structures from three simulation replicates after 100 and 200 ns simulation time. Tryptophans are depicted in light-blue, mannoses in green and arginines in salmon. (C) Final structures of non- and C-mannosylated UNC-5 TSR2 after each 200 ns simulation, colored using a blue-white-red gradient according to the calculated root-mean-square fluctuation (RMSF) values (blue ≤1 Å; red ≥5 Å) (Figure 5—figure supplement 1, Figure 5—source data 2). Cys26 and Cys38 residues, that are involved in the upper disulfide bridge of the TSR, are labeled with one or two asterisks, respectively.