TABLE 1.

Summary of macroscopic dissociation constants for Ras/Sos complexes measured by fluorescence

Protein variants used for each titration experiment as well as a brief description of the expected functional effect of mutation are indicated.

Description of expected effect of mutation used	Sos variant presenta	Ras variant added	Measured Kd
			μm
None	SosCat	H-Ras·GDP	54 ± 4
None	SosCat	H-Ras·GTPγS	5.0 ± 0.8
None	SosCat	H-Ras·GppNp	6.0 ± 0.9
None	SosCat	H-Ras·GppCp	8.0 ± 1.0
Is not expected to bind at catalytic site	SosCat	H-RasY64A·GTPγS	10 ± 1
Binding expected to be hindered at allosteric site	SosCatW729E	H-Ras·GTPγS	28 ± 1
Binding expected to be hindered at allosteric site	SosHD-DH-PH-Cat	H-Ras·GTPγS	24 ± 2
Binding expected to be hindered at both sites of Sos	SosCatW729E	H-RasY64A·GTPγS	32 ± 3
Binding expected to be hindered at allosteric site	SosCatW729E	H-Ras·GDP	67 ± 2
H-RasY64A·GTPγS expected to partially saturate allosteric site	SosCat (preloaded with 30 μm H-RasY64A·GTPγS)	H-Ras·GTPγS	21 ± 2b
None	SosCat	K-Ras·GTPγS	10 ± 1
Oncogenic variant	SosCat	K-RasG12V·GTPγS	31 ± 2

^a Unless stated otherwise, the WT version of protein was used.

^b Lower limit estimate.