TABLE 1.
Toxin | Chymotrypsin digestiona | CD spectrumb | Effect of DTT on toxin activity
|
Competitione | |
---|---|---|---|---|---|
SDS-PAGEc | Pore formationd | ||||
Cry1Aa | Partial | Wild type | − | − | NAf |
L126C mutant | NDg | ND | − | − | ND |
R127C mutant | ND | ND | ++ | − | ND |
E128C mutant | ND | ND | ++ | + | ND |
E129C mutant | Minor | Wild type | Trace | − | Complete |
M130C mutant | ND | ND | ++ | − | ND |
R131C mutant | Minor | Wild type | Trace | − | Strong |
I132C mutant | Partial | Lower | − | + | Strong |
Q133C mutant | ND | ND | ++ | − | ND |
F134C mutant | ND | ND | + | − | ND |
N135C mutant | Minor | Wild type | Trace | − | Complete |
M137C mutant | ND | ND | ++ | − | ND |
N138C mutant | Minor | Wild type | − | − | Strong |
S139C mutant | Minor | Lower | ++ | + | Strong |
A140C mutant | Partial | Wild type | Trace | − | ND |
L141C mutant | ND | ND | + | − | ND |
T142C mutant | Minor | Wild type | − | + | Strong |
A144C mutant | Partial | Wild type | − | − | Strong |
I145C mutant | ND | ND | Trace | + | ND |
P146C mutant | ND | ND | Trace | + | ND |
L147C mutant | Minor | Wild type | Trace | − | None |
A149C mutant | ND | ND | − | − | ND |
V150C mutant | Minor | Lower | − | + | Strong |
Apparent amounts indicate minor or partial digestion of the toxin to a fragment of about 55 kDa.
Compared with the Cry1Aa spectrum. “Wild type”, the spectrum of the mutant was similar to that of Cry1Aa; “Lower,” the α-helix content of the protein was decreased.
The presence of bands corresponding to a 120-kDa protein, characteristic of homodimers, was revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of dithiothreitol (DTT) in amounts that were substantial (++), moderate (+), or trace. The absence of homodimer bands is indicated by a minus.
The presence of disulfide bridges as revealed by the effect of dithiothreitol on pore formation. The addition of dithiothreitol had either no effect (−) or produced increased (+) toxin activity.
Ability of each mutant to inhibit the formation of pores by Cry1Aa.
NA, not applicable.
ND, not determined.