We engineered 29 HCCS mutants, changing the conserved residues to (1) an alanine, (2) an amino acid of opposite or like charge, and/or (3) an amino acid with disease relevance (i.e., MLS mutations E159K and R217C).
For the EmrE mutants, changing the loop length leads to relatively small shifts in the EOT ensemble; however, for the CB mutants, the effect of loop length on the EOT ensemble is much larger, with the new short-loop CB mutant exhibiting a much weaker bias towards the Ncyto/Ccyto topology in the EOT ensemble.
It was also evident that the large side chains of Tyr7, Trp32, Tyr140, and His134 were shifted in the mutant, changing the shape and nature of the binding site.
To retain the charge of these surface residues, we made the charge-swapped DG R-X8- DQD R CTD mutant, changing the key Arg residues within the two motifs to Asp, and vice versa.
Our sequence analysis revealed that nucleotide residue 6179 in OsISA1, which was adenine (A) in the wild type, was substituted with guanine (G) in sug-1 and sug-h mutants, thereby changing the amino acid from glycine (Gly) to aspartic acid (Asp).
We found that the nucleotide residue 4732 in wild-type OsBEIIa, which was G, was substituted with A in the sug-h mutant, thereby changing the amino acid from Gly to Asp.
The increased or repressed expression of these Pi transporters was caused, at least in part, by accumulated protein level of Pi transporters in osnla1 mutant, because changing the expression of OsPT4 or OsPT8 affects the expression of Pi transporters in rice (Jia et al., 2011; Zhang et al., 2015).
Alternatively, the lipid composition of cell membranes might be altered in the mutants listed above, thereby changing the properties of the membrane and impacting on the activities of membrane-embedded transporters, sensors or signalling receptors [ 49].
The CD98-pcDNA3.1/V5-His-TOPO S426A-S430A mutant was generated by changing the codon at 1803 1805* and 1815 1817* position from serine 426 and 430, respectively, to alanines using specific primers P2 (forward, 5'-TTC CGG CGG CTA GCT GAC CAG CGG GCT AAG GAG CGC-3'; reverse, 5'-GCG CTC CTT AGC CCG CTG GTC AGC TAG CCG CCG GAA-3').
The CD98-pcDNA3.1/V5-His-TOPO S305A-S307A-S309A mutant was generated by changing the codon at 1440 1442*, 1446 1448* and 1452 1454* position from serine 305, 307 and 309, respectively, to alanines using specific primers P1 (forward, 5'-C TCA TAC CTG GCT GAT GCT GGC GCC ACT GGG GAG C-3'; reverse, 5'-G CTC CCC AGT GGC GCC AGC ATC AGC CAG GTA TGA G-3').
The properties of the PFLs could be altered using LuxR mutants with altered sensitivities without changing the inherent properties of the systems.
