Table 1.

Kinetic parameters for the interaction of chemically synthesized and biosynthetic RBD proteins with H-Ras at 25°C

Reaction	kass/M−1·s−1	kdiss/s−1	Kd/nM
1. csRBD/H + H-Ras.mantGppNHp	2·107	6.6	330
2. cs[L91D,L-O]RBD/H + H-Ras.mantGppNHp	1.7·107	7.0	410
3. cs[L91L-O]RBD/H + H-Ras.mantGppNHp	1.8·107	6.8	380
4. RBD/H + H-Ras.mantGppNHp†	2.1·107	6.4	300
5. cs[L91D,L-O]RBD/H + H-Ras.GppNHp	2.8·107	11.4*	400
6. cs[L91L-O]RBD/H + H-Ras.GppNHp	3.4·107	10.3*	300
7. [L91W]RBD/H + H-Ras.GppNHp†	3.6·107	14	380

Reactions 1–4: Kinetics monitored by using the mant-fluorescence of H-Ras.mantGppNHp. Reactions 5 and 6: Kinetics monitored by using the N²-methyl-7-azatryptophan fluorescence of the artificial RBD proteins. The dissociation rates were obtained by displacement reactions as described (8). Reaction 7: Kinetics monitored by using the Trp-fluorescence of the [L91W]RBD/H mutant. 

^*Because of the slow second phase that sometimes also occurred in the displacement experiments we chose k_obs1 for k_diss, because it correlates well with our previous results for the dissociation rate of the RBD-Ras interaction. 

^†As published in ref. 8.