Table I. Rates of 5-HT4R peptide oxidation calculated from radiolytic footprinting experiments.
Rates include the seven 5-HT4R peptides for which comparative rates of oxidation were calculated (see supplemental Fig. S6).The fraction of unoxidized peptides was calculated from the ratio of the chromatographic area under the ion signals for the unoxidized peptides to the sum of the unoxidized peptides and their radiolytic products (22, 24). Dose-response curves are presented as unmodified fractions versus exposure periods. The equation Y = Y0e−kt (where Y and Y0 are the fractions of unmodified peptide at times t and 0 ms, and k is the first-order rate constant) was fit to the fraction of unmodified peptide. The values are obtained from three replicates with four to five time points per experiment.
| Peptide | Range | Domain | Oxidized amino acids | Mass shift | Oxidation rate |
|
|---|---|---|---|---|---|---|
| Bound | Free | |||||
| Da | s−1 | |||||
| VMPFGA | 73–78 | TM2 helix | M742.58 | 16 | 3.2 ± 0.5 | 8.5 ± 2.3 |
| RNKMTPLRIAL | 130–140 | ICL2 loop and TM4 helix | M133 | 16 | 0.98 ± 0.0011 | 7.9 ± 1.9 |
| MVNKPYAIT | 187–195 | ECL2 loop | M187 and or Y192 | 16 or 32 | 6.7 ± 0.86 | 8.7 ± 2.2 |
| QMLQRAGASSESRPQSA | 227–243 | ICL3 loop | M228 | 16 | 23 ± 9.1 | 9.0 ± 2.7 |
| LQRAGASSESRPQSADQHSTHRMRTETKA | 236–257 | ICL3 loop | M251 | 16 | 47 ± 10 | 43 ± 13 |
| YINSGLN | 302–308 | TM7 helix | Y3027.43 | 16 | 0 | 1.1 ± 0.9 |
| AQPSDTLLQMA | 383–393 | C terminus (T7 Tag) | M392 | 16 | 108 ± 17 | 54 ± 11 |