FIG 4.

Activity of MS-DesK mutants. (A) Sequences of the N terminus of MS-DesK variants, with point mutations that eliminate (K10L) or duplicate (L11K) the positive charge. WT, wild type. (B) Cells expressing MS-DesK variants were grown at 37°C and transferred to 25°C at an OD at 525 nm (OD₅₂₅) of 0.3. β-Galactosidase activity was assayed every hour in independent triplicates. The data shown are expressed as averages of the results from three independent experiments and correspond to 4 h after the cold shock; error bars represent the standard deviations for each experimental repetition (10). (C) Proteoliposomes containing MS-DesK or its variants were incubated with [γ-³²P]ATP at 25°C, and the autokinase activity was determined using an ADP-Glo kinase assay. The total amount of each MS-DesK variant was determined by densitometry; equal amounts of protein were used for each reaction. (D) The corresponding relative initial velocities were calculated from the slopes of the curves shown in panel C and compared, considering MS-DesK as 100%. (E and F) The phosphatase activity of MS-DesK variants was determined as described for Fig. 3E. (G) Relative initial velocities were calculated from the slope of the curves shown in panel F, taking the activity of MS-DesK as 100%.