Fig. 3.

Kinetics and unfolding properties of G protein α subunit chimeras. (A) Cartoon representations of the chimeras used in this study. (B) Temperature-induced unfolding of the indicated G protein α subunits as monitored by CD spectroscopy at 208 nm. (C and D) GTP-γ-S binding rates were measured from intrinsic fluorescence changes as described in Fig. 1 with GTP-γ-S (2 μM). (E) Single-turnover GTP hydrolysis. Purified His-tagged G protein α subunits (900 nM) were loaded with [γ-³²P]GTP before the reaction was started by addition of Mg²⁺. Hydrolyzed ³²PO₄ was extracted with charcoal and quantified. (F) Fluorescence-based GTP binding and hydrolysis were measured as described in Fig. 1 with GTP (400 nM). (G) Steady-state GTP hydrolysis. Purified G protein α subunits (400 nM) were incubated with [γ-³²P]GTP (10 μM) for the indicated times before hydrolyzed ³²PO₄ was extracted with charcoal and quantified. For all of the panels, data are representative of at least two experiments. Error bars indicate SEM.