Plimstex Results for RasGDP Titrated with Mg2B

In the titration of 1.5 mM ras-GDP with Mg2+, as the [Mg]:[ras-GDP]tot ratio increases, the deuterium uptake decreases as the ternary complex is formed. Fit

Fig. 11.4 PLIMSTEX curve for 1.5 |iM Ras-GDP titrating with Mg2+. Conditions: 90% D2O, 50 mM HEPES buffer, 100 mM KCl, pH 7.4, H/D exchange time = 3 h. EDTA was used to control [Mg2+] in solution. The error bars shown for each data point were based on the deviation from two independent runs.

[Mg2+]free was calculated using the ''Webmaxc Standard'' program on the internet [43]. The solid line was the fitted PLIMSTEX curve for the average data using a 1:1 binding model and three-parameter (b 1, D0, AD1) fitting.

ting the PLIMSTEX curve (Fig. 11.4) gives Ka, ADi, and D0 for ras-GDP as (4.1 G 0.2) x 104 M"1, 25.6 G 0.6 and 105.7 G 0.5, respectively. The root mean square (RMS) of the error between the predicted and the experimentally measured deuterium shifts is 0.7 Da. The results indicate a relatively weak 1/1 interaction between Mg2+ and ras-GDP, causing @ 26 backbone amide protons of the binary complex to become protected with the binding to Mg2+. The binding constant agrees with the literature value (6.9 x 104 M"1) obtained from an equilibrium unfolding stability study using circular dichroism [41]. The protection quantified by PLIMSTEX (AD1 = 26) is also consistent with the difference between the number of the slow exchanging amide protons of ras in its ternary complex ras-GDP-Mg2+ (@76) and that in the binary complex ras-GDP (@48).

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