Kneirim et al. 10.1073/pnas.0710393104.
Table 1. Proton release of Gtα-derived peptides upon binding to light-activated rhodopsin
Peptide | Sequence | KD,μM | H+/MII | |
1. | Gtα(340-350) | NH2-IKENLKDCGLF-COOH | 83.1(1) | -0.53 |
2. | Gtα(340-350)K341L | NH2-ILENLKDCGLF-COOH | 0.39(1) | -0.49 |
3. | Ac-Gtα(340-350)K341L | Ac-NH -ILENLKDCGLF-COOH | -0.39 | |
4. | Gtα(340-350)E342N | NH2-IKNNLKDCGLF-COOH | 94.7(1) | -0.24 |
5. | Gtα(340-350)C347abu | NH2-IKENLKD(abu)GLF-COOH | -0.49 | |
6. | Gtα(340-350)HAA | NH2-VLEDLKSCGLF-COOH | 0.92(2) | -0.52 |
7. | Gtα(340-350)HAA K345A | NH2-VLEDLASCGLF-COOH | -0.53 | |
8. | Gtα(340-350)L349A | NH2-IKENLKDCGAF-COOH | >1,000(3) | 0.75 |
Abbreviations: abu, α-amino butyric acid; HAA, high-affinity analog.
KD values for rhodopsin binding were taken from:
1. Herrmann R, Heck M, Henklein P, Kleuss C, Wray V, Hofmann KP, Ernst OP (2006) Vision Res 46:4582-4593.
2. Martin EL, Rens-Domiano S, Schatz PJ, Hamm HE (1996) J Biol Chem 271:361-366.
3. Bartl F, Ritter E, Hofmann KP (2000) FEBS Lett 473:259-264.
As shown in Fig. 6 in the main paper, binding of C-terminal Gtα-derived peptides (peptides 1 and 2) to light-activated rhodopsin is accompanied by net proton release at high peptide concentration. To determine whether the protons originate from the peptide and/or rhodopsin, we examined proton release upon binding for different homologous peptides in which specific groups that could potentially act as proton sources were replaced (acidic or basic residues, including Cys) or chemically modified (terminal NH2); the strongly acidic C-terminal residue (pKa ≈ 3) is assumed to be at all times ionized and was not modified. H+/MII values were determined from proton transfer measurements in the presence of 1 mM Gtα-derived peptide using bromocresol purple (see Materials and Methods). Peptides 2-7 represent sequences where each of these groups, either singly or in groups, are replaced or modified; additional modifications relative to the native peptide (peptide 1) are also made as noted in bold. Formation of MII is unaffected by all peptides. Each peptide showed similar proton release, indicating that the protons released upon rhodopsin binding originate from rhodopsin. The nonbinding control peptide 8 shows very little proton release, i.e., only the proton uptake caused by MIIbH+ formation is observed; the small decrease of 0.25 protons is presumably caused by buffering.