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The samples were incubated for 30 min with intermittent mixing, and then duplicate 10-μl aliquots of each sample were spotted onto P-81 phosphocellulose paper. Though we did not find much significant difference in Rg values, however we noticed that at the equilibrium state mutant structure was more compact than the wild-type (Figure 4B). We were also interested to check the ATQ distance from the Qo site during the entire dynamics run. The calculated residuals for 1EZV, 2IBZ, 3CX5, 3H1J and 1BCC templates from our modeled protein were 0.48 Å, 0.44 Å, 0.46 Å, 0.56 Å and 1.02 Å respectively. The supernatant was transferred to a fresh tube and neutralized using K2CO3, and the perchloric acid-precipitated proteins were saved for quantification via Lowry assay. Alderdice (1988) describes the establishment of osmotic regulation during embryogenesis as beginning during gastrulation and being in place by yolk-plug closure or completion of epiboly.

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