1. Figure 1. Structure of microcystin
Figure 1. Structure of microcystin. Microcystin has the general structure of cyclo(D-Ala-X-D-MeAsp-Y-Adda-D-Glu-Mdha-), . Different kinds of microcystin can vary at the X and Y residues. Microcystin-LR has Leu at the X position and arginine at the Y position. The double bond of Mdha forms a covalent bond with cysteine 273 of PP1 and cystein 266 of PP2A.

2. Acidic Groove C-terminal groove Hydrophobic groove
Figure 2a. Structure of microcystin-LR (Red) bound to the active site of protein phosphatase-1 (grey). The yellow residue is cysteine 273, which forms a covalent bond with the Mdha group in microcystin.
Hydrophobic
groove

3. RVXF Motif Binding Site
Figure 2b. The RVXF motif binding site of protein phosphatase-1. This is located on the opposite side of the active site, which allows association of proteins with RVXF motifs even when microcystin is bound to the active site.
RVXF Motif
Binding Site

4. 121.04min 0 50 100 150 Absorbance (206nm) Retention Time (min) 0.300
0.200
0.100
0.000
121.04min
Absorbance (206nm)
Figure 3a. Reverse-phase HPLC chromatogram of microcystin-LR standard.
Column: microbore Vydac C18; Conditions: linear AB gradient (0.3%B/minute), where solvent A is 0.1% TFA / water and solvent B is 0.1% TFA / acetonitrile; Flow rate:0.2mL/minute; Detection: 206nm.
Retention Time (min)

5. 121.27 min Absorbance (206nm) 0 50 100 150 Retention Time (min) 0.300
0.200
0.100
0.000
min
102.74
Absorbance (206nm)
100.65
107.34
135.64
98.07
Figure 3b. Reverse-phase HPLC chromatogram of pooled microcystin fractions.
Column: microbore Vydac C18; Conditions: linear AB gradient (0.3%B/minute), where solvent A is 0.1% TFA / water and solvent B is 0.1% TFA / acetonitrile; Flow rate:0.2mL/minute; Detection: 206nm.
Retention Time (min)

6. 98.71
113.89;
min
0.400
0.200
0.000
Absorbance (206nm)
96.26
Figure 3c. Reverse-phase HPLC chromatogram of microcystin pool after reaction with aminoethanethiol.
Column: microbore Vydac C18; Conditions: linear AB gradient (0.3%B/minute), where solvent A is 0.1% TFA / water and solvent B is 0.1% TFA / acetonitrile; Flow rate:0.2mL/minute; Detection: 206nm.
125.94
Retention Time (min)

7. Absorbance (206nm) 0 50 100 150 Retention Time (min) 0.200 0.100 0.000
98.72
Absorbance (206nm)
96.91
Figure 3d. Reverse-phase HPLC chromatogram of the supernatant after reacting aminoethanethiol-microcystin to NHS-activated CH Sepharose.
Column: microbore Vydac C18; Conditions: linear AB gradient (0.3%B/minute), where solvent A is 0.1% TFA / water and solvent B is 0.1% TFA / acetonitrile; Flow rate:0.2mL/minute; Detection: 206nm.
Retention Time (min)

8.
Markers
(kDa) 75 50 37 25 20 15 10
Figure 4. SDS PAGE gel of PP1 binding experiment , visualized with Coomassie blue staining.
Lane 1: Kaleidoscope markers (Bio-Rad)
Lane 2: PP1 that was used to bind to the resin
Lane 3: Control resin after incubation with PP1 (washed with 0.3M NaCl)
Lane 4: Microcystin-Sepharose after incubation with PP1 (washed with 0.3M NaCl)
Lane 5: Control resin after elution with 3M NaSCN
Lane 6: Microcystin-Sepharose resin after elution with 3M NaSCN
Lane 7: Elution with 3M NaSCN off the control resin
Lane 8: Elution with 3M NaSCN off the microcystin-Sepharose resin
Samples were denatured in SDS, boiled at 100oC for 5 minutes, then subjected to electrophoresis on a 12% polyacrylamide gel and stained with Coomassie blue.

9.
Markers
(kDA) 75 50 37 25 20 15 10
Figure 5. SDS PAGE gel of PP2A binding experiment, visualized with Coomassie blue staining.
Lane 1: Kaleidoscope markers (Bio-Rad)
Lane 2: PP1 that was used to bind to the resin
Lane 3: Control resin after incubation with PP2A
Lane 4: Microcystin-Sepharose after incubation with PP2A
Lane 5: Control resin after elution with okadaic acid
Lane 6: Microcystin-Sepharose resin after elution with okadaic acid
Lane 7: Elution with 3M NaSCN off the control resin
Lane 8: Elution with 3M NaSCN off the microcystin-Sepharose resin
Samples were denatured in SDS, boiled at 100oC for 5 minutes, then subjected to electrophoresis on a 12% polyacrylamide gel and stained with Coomassie blue.