Table 2.
Free sulfhydryl content of bMsrA
| Cysteines replaced | No. of Cys | Mass increase of monomer, Da*
|
Decrease in free thiols† | |
|---|---|---|---|---|
| l-Met(O) | l-Met | |||
| None (WT) | 4 | 81 /86 | 201 /197 | 2 |
| 72 | 3 | 138 /142 | 140 /144 | 0 |
| 107 | 3 | 35 /38 | 145 /153 | 2 |
| 218 | 3 | 44 /51 | 160 /157 | 2 |
| 227 | 3 | 49 /50 | 154 /158 | 2 |
| 72, 218 | 2 | 91 /92 | 90 /92 | 0 |
| 107, 218 | 2 | 1 /0 | 104 /100 | 2 |
| 107, 227 | 2 | 7 /0 | 102 /106 | 2 |
| 218, 227 | 2 | 78 /75 | 101 /100 | 0 |
| 72, 107, 227 | 1 | 48 /52 | 50 /53 | 0 |
| 107, 218, 227 | 1 | 45 /39 | 66 /72 | 0 |
*
The values indicate the observed increase in mass of the monomer of each variant upon treatment with MMTS from two independent experiments as described in Materials and Methods. The mass of each variant without pretreatment was used as the reference. The theoretical mass increases for the covalent addition of one to four methylthiol groups (—SCH3) from MMTS to cysteine are 47, 94, 141, and 188 Da, respectively.
† The difference in the number of free cysteine thiols upon treatment with l-Met(O) vs. l-Met. A value of 2 represents the formation of a disulfide bond. The disulfide bonds formed could be reduced with the addition of DTT (data not shown).