Actitudes de conducta 1 Definición de actitud

From amino acid alignments of 118 orthologous proteins from eukaryotes and prokaryotes as performed in Chapter Three, Section 3.2.1, T83 was shown to be conserved in just 25 % of eukaryotic and 29 % of prokaryotic NrtA homologues studied. This site is generally occupied by serine or alanine in eukaryotes (respectively 55 and 17 % of the time) whereas glycine is

more frequent in prokaryotes (68 % occurrence), alanine is rare (3 % occurrence). Thus, it seems that a small residue is required at this site to provide flexibility to the arginine residue as it interacts with the substrate. Here T83 was targeted for mutagenesis to analyse its function. T83 was altered to alanine, valine, serine, glutamine and arginine with varying degrees of complementation. Phenotypic data presented in Figure 4.1 suggest that changes to alanine and serine are permitted by the protein. Alanine and serine residues are common in this position in prokaryotic homologues. Further, serine seems to be tolerated more than alanine, though nitrate uptake studies showed alanine to transport more nitrate than serine (Table 4.1). However, accounting for experimental error, uptake in these mutant strains can probably be considered similar. Tracer uptake studies were carried out on T83S to determine if an alteration in this position altered the Km or Vmax of NrtA. However, uptake kinetics were unaffected (Figure 4.5), implying that this residue is unlikely to interact with nitrate. Valine is of comparable size to threonine, and it would be expected that mutants to valine would complement on nitrate containing agar. Unexpectedly, this was not the case (Figure 4.2). Valine is commonly found in β-pleated sheets and it is possible that properties that favour valine in these regions are unfavourable in the α-helix, potentially the split C side- chain could affect available space. As stated Western analysis of these mutants produced an apparently cleaved protein, though the reason for this is currently under investigation. However, as activity is observed in both T83A and T83S it is apparent that nitrate was transported by NrtA. However, whether the reduction in nitrate uptake is as a result of the point mutation or protein cleavage is unclear from these results.

The importance of the relevant position of the arginine residues (R87 and R368) in relation to the polar residues T83 and N364 was investigated, initially by the construction of a double mutant which effectively swapped the positions of the T83 and R87. This mutant did not show growth on nitrate media, indicating that the positioning of these residues is critical for NrtA function. Further to this it was thought that if the position of the arginine was elevated in the helix then the interactions with nitrate might not occur on the same plane of the bilayer while the opposite arginine remained in a lower position (for residue positioning see Figure 4.1). All amino acids involved with substrate binding in LacY are located on the same plane in the transporter (Guan & Kaback, 2006) and therefore it is likely that this will be the case for NrtA. To test this, a quadruple mutation was created which moved the threonine and arginine in Tm 2 in addition to exchanging the positions of N364 and R368 in Tm 8. This mutant did not show growth on nitrate, suggesting that the positioning of these residues is critical for the efficient functioning of the substrate binding site. Perhaps as the substrate is moved towards or out of the binding site, there are additional interactions that are required for

the transit of nitrate which are unavailable in this mutant. As a final note of interest, mutagenesis work in parallel, on the polar residue (N364) above second interactive arginine (R368), preliminary results show that mutations to alanine and glutamine are tolerated in this position while cysteine, lysine and arginine are not, this too fits in with the theory that drastic size alterations are not permitted at these positions above the arginines.

To study the binding site, parallels are best drawn to previous studies on NrtA as have been done here. While LacY and GlpT are models for MFS proteins it is notable that the substrates in these permeases are structurally very different from nitrate as discussed in Chapter Three. While modelling of GlpT has provided clues as to the orientation of specified amino acids and their relationship to the binding site this model has to be backed up by experimental evidence on NrtA. For example in LacY the key residues known to be involved directly with the substrate are located in helices 4, 5, 8, 9 and 10 (i.e. substrate binding residues E126, R144 located at the intracellular edge of Tms 4 and 5, and residues involved with proton coupling E269, R302, H322 and E325, located on Tm 8, 9 and 10) (Frillingos et al., 1998; Kaback et al., 2001; Vazquez-Ibar et al., 2004; Kaback, 2005; Mirza et al., 2006). While charged residues in NrtA are known to reside on Tms 2 and 8 (R87 and R368 respectively), residues involved in proton coupling have yet to be identified in NrtA. This shows that although it is possible to employ these models as a guide for the targeting of residues caution should be exerted when drawing parallels.

The significance of results for growth on chlorate was as discussed in Section 3.3.