We therefore mutated the equivalent residue in GluN2A, K80, and its possible interacting partner, to cysteine.
It has previously been shown that a large hydrophobic residue in the headgroup is essential for the N N interaction in several species (Kitagawa et al., 2011; van Breugel et al., 2011), so we mutated the equivalent residue, F143, to Asp.
In this regard SUB appears to differ from BRI1, where mutating the equivalent two cysteines resulted in a functional protein [63], but behaves similarly to Cf-9, where equivalent mutations caused absence of Cf-9 activity [47].
In contrast to these observations, mutating the equivalent sequences in the MC4R poly(A) site had the opposite effects.
Mutating the equivalent residue in Lam4S2 (G1119R) completely inhibited sterol binding in vitro, showing that this residue is indeed critical.
Using quantitative actin-binding assays, we show that mutating the equivalent residue abolishes F-actin binding in Coronin 1A, but not Coronin 1C.
Mutating the equivalent methionine to smaller residues in EAAT3 also resulted in less steep dependence of the ionic currents on Na+ concentration (Rosental and Kanner, 2010).
For both HucR and PecS, substitution of residues corresponding to MftR residues W11, D56, and R63 generally ablates the response to urate, while mutating the equivalent of R89 in the DNA recognition helix severely compromises DNA binding.
For example, mutation of the acidic residue, E288, in the extended Walker-B motif of NOD1 results in an inactive response, whereas in NOD2 mutating the equivalent residue, E383, results in a hyperactive receptor.
Similar mean ΔΔG energy values were obtained upon mutating the equivalent Tyrosine residues in each of the three templates used for modelling RIN3-VPS9, corroborathis this result (Supplementary Material, Table S3).
In this work, the two cofactor-binding loops (residues 185 192 and 382 392) were progressively mutated towards the equivalent sequence from the thermostable Thermus thermophilus TK and variants assessed for their impact on both thermostability and activity.
