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Most single mutations did not affect the critical shape of the molecule, and multiple mutations tended to compensate for those that did.
As expected, most single mutations (D332A, G348A, D349A, S394A, N395A and V442A) lead to complete or nearly complete loss of binding (Fig. 8D, 8I J and 8L), indicating that these residues are strictly required for the structural and functional integrity of the HBGA binding interface.
On an individual basis, it was striking that most single mutations led to only modest and non-significant decreases in the amplitude of the T cell response.
In this context one might expect that most single mutations that finally become stabilized in coevolving pairs are neutral in the context of the protein in which they occur, but become beneficial (even at a much later time) in the presence of the partner in the pair [63].
Most single mutations in the bulges are critical.
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Conversely, most single point mutations throughout key conserved features in peripheral tertiary architecture supporting the SAM binding pocket have relatively little impact on riboswitch activity.
Moreover, the anti-Bax function of PrP is situated within helix 3 of PrP, the area with the most single point mutations in PrP diseases [30].
Several thousand variants were screened per round, theoretically covering most single point mutations in the ∼1000 bp PTE gene.
In each round, 2000 10,000 colonies were screened in this fashion, theoretically covering most single point mutations in the 330 amino acid PTE gene.
We have also made a double mutant, combining two of the most beneficial single mutations.
Of these, most contained single mutations: eight had either H3 A7V or H3 A7T mutations, seven had an H3 T6I mutation, and one bore an H3 G33V mutation.
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