Extended Data Fig. 5: Regulation of NAL1 Proteolytic Activity: The Dual Impact of Residue 233.
From: The catalytic triad of rice NARROW LEAF1 involves H234
a-d, Proteolytic activity assays were performed with a series of H233 mutants of NAL1SPIKE. The left panel displays a comparison of proteolytic activity between the wild type (NAL1SPIKE) and the mutants, while the right panel presents a histogram representing the residual content (%) of the substrate (FZP51-244). The integrated densities of various bands were scanned and calculated using Quantity One software from Bio-Rad. Data show mean ± SD from three independent experiments. Black circles represent individual data points. All differences between each mutant and the wild-type (NAL1SPIKE) were analysed within the same degradation time by two-tailed paired t-tests. Means with p < 0.05 are indicated. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. All proteolytic activity assays were carried out according to the procedures outlined in the Methods section, utilizing three different degradation time intervals, including 1 h, 3 h, and 6 h. CK represents the substrate (FZP51-244) alone. The triangle symbol indicates a decrease in the substrate (FZP51-244), while the round symbol signifies an increase in the proteolyzed substrate. Uncropped gel images are available as source data. The H233 mutants are categorized as follows: (a) H233R and H233K (positively charged residues), (b) H233D and H233E (negatively charged residues), (c) H233N and H233Q (polar uncharged residues), and (d) H233Y and H233W (hydrophobic residues).
