Table 2.
Best-fit values for the pH-dependent 13C chemical-shift changes and pKa values of Y76 and Y98 of PYP
| Residue | Nucleus∗ | δ0 (ppm) | Δδ (ppm) | pKa | nH |
|---|---|---|---|---|---|
| Y76 | Cδ | 132.47 | – | – | – |
| Cε | 117.1 ± 0.1 | 2.23 ± 0.04 | 10.14 ± 0.02 | 1.24 ± 0.06 | |
| Cγ (at Hβ2/3) | 127.8 ± 0.1 | −3.77 ± 0.08 | 10.21 ± 0.02 | 1.16 ± 0.04 | |
| Hβ | 3.003 | – | – | – | |
| Hδ | 6.05 | – | – | – | |
| Hε | 6.46 ± 0.04 | 0.162 ± 0.004 | 10.1 ±0.03 | 1.3 ± 0.1 | |
| Global fit† | Cδ | 132.47 ± 0.02 | −0.08 ± 0.02 | ||
| Cε | 117.1 ± 0.1 | 2.30 ± 0.04 | 10.20 ± 0.03 | 1.18 ± 0.06 | |
| Cγ | 127.7 ± 0.1 | −3.73 ± 0.06 | |||
| Y98 | Cδ | 133.00 | – | – | – |
| Cε | 118.6 ± 0.1 | 2.8 ± 0.1 | 10.32 ± 0.03 | 1.12 ± 0.08 | |
| Cγ (at Hβ2/3) | 129.5 ± 0.1 | −4.8 ± 0.1 | 10.20 ± 0.01 | 1.54 ± 0.06 | |
| Hβ | 3.15 | – | – | – | |
| Hδ | 7.231 ± 0.004 | 0.157 ± 0.04 | 10.43 ± 0.03 | 1.05 ± 0.05 | |
| Hε | 6.85 ± 0.01 | 0.28 ± 0.01 | 10.37 ± 0.04 | 1.14 ± 0.1 | |
| Global fit† | Cδ | 133.00 ± 0.02 | −0.23 ± 0.02 | ||
| Cε | 118.6 ± 0.1 | 2.5 ± 0.1 | 10.21 ± 0.03 | 1.48 ± 0.08 | |
| Cγ | 129.5 ± 0.1 | −4.8 ± 0.1 |
Cδ, Cε, Hδ, and Hε resonances were monitored by 13C-1H HSQC spectroscopy; Cγ and Hβ resonances were recorded by CG(CB)HB spectroscopy.
For global fitting, the four titrations pertaining to the same Tyr ring (probed at CγHβ2, CγHβ3, CδHδ, and CεHε) were combined and fitted simultaneously to a model with one pKa and one nH for that Tyr. Standard deviations (SDs) for the best-fit parameters resulting from the individual fits were estimated by analysis of the χ2 function (43). For the global-fit parameters, the SDs were estimated via a Monte Carlo protocol in which estimated SDs per data point (0.05 in pH and 0.03 ppm in chemical shift) were used. See text for further discussion about the accuracy of these results.