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concentration ratios (0, 15, 500) which represent, for a titration with Mg2+: the initial (no ligand), an intermediate point, and the end. The data were fitted using a three-group, pseudo-first-order kinetics model [25] to give, for 162 amide hydrogens in C-terminal-truncated ras, the distribution of the fast-, intermediate-, and slow-exchanging amide hydrogens under these three [Mg2+]:[ras-GDP]tot ratios (Table 11.2). As the ratio of [Mg2+]:[ras-GDP]totaj increases, more binary ras-GDP complex is transformed to the ras-GDP-Mg2+ ternary complex, and the number of fast-exchanging amide hydrogens decreases monotonically from 79 to 65, whereas the number of slow-exchanging hydrogens increases from 48 to 76. This distribution is a "signature" of ras protein's conformation and should, in general, be a useful indicator for an unknown protein to guide theoretical calculations of folding. Fast-exchanging hydrogens are likely to be involved in minimal hydrogen bonding, whereas slow ones are protected by high-order structure and hydrogen bonding. That more hydrogens shift from fast to intermediate and slow groups upon Mg2+-binding is consistent with a global folding of ras into a more compact and stable, less solvent-accessible form. After 3 h of exchange, the deuterium uptake levels off for each of the three concentration ratios, indicating that the H/D exchange of the fast and intermediate amides had arrived at a near steady state. The small experimental errors in exchange time do not contribute significantly to the measurement of deuterium uptakes. Pointing to a successful application of PLIMSTEX are detectable differences between deuterium levels in ras-GDP (no Mg2+) and ras-GDP-Mg2+ complex at this time. Therefore, we chose 3 h as the exchange time for the PLIMSTEX titration (i.e., the time of the quench in exchange).

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