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We also saw that, even when lysines of Ngn3 have been mutated, further mutation of cysteines has little effect on half-life.
However, mutation of cysteines that coordinate a structural zinc decreased the levels of 5 and 14 kDa forms, suggesting importance of protein structure for biosynthesis and/stability of these forms.
Evidently, the species of origin, the mutation of cysteines in the catalytic domain, as well as the sequence and length of the NL, can individually affect kinesin motility.
To illustrate this, in the absence of internal lysines, Ngn3KO half-life is 29.3±2.8 minutes, while the further mutation of cysteines to alanines results in an increased half-life for Ngn3KOCO of 50.3±0.5 minutes.
The mutation of cysteines to alanines does not result in significant stabilisation of the otherwise native Ngn3 protein in mitotic extract (Ngn3 10.6±1.1 minutes, Ngn3CO 11.5±0.2 minutes).
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In addition, mutation of cysteine 95 to alanine in barley α-amylase was found to increase the recombinant strain's starch hydrolyzing ability.
The report from Pradhan et al. also showed that mutation of cysteine 667 to glycine within the CXXC domain of human DNMT1 disrupts DNA binding and enzymatic activity.
MS/MS analysis of mutated peptide (aa. 400 407) obtained from trypsin and chymotrypsin digested protein showed the mutation of cysteine to serine (data not shown).
This abrogation was not seen with the mutation of cysteine 176 on the inner side of the LRR5, demonstrating that inter-receptor disulfide bonding was not involved in ectodomain multimer formation.
Mutation of cysteine to serine is unlikely to have greatly affected protein structure.
Mutation of cysteine residues 113 and 118 to alanine generated a version of the RecX protein that yielded crystals suitable for high-resolution X-ray analysis.
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