R in only one relevant amino acidde Francisco et al. BMC

Experiments carried out on the green fluorescent protein (GFP), after changing lysine for arginine on the protein surface and retaining protein activity, have shown that the GFP variant was relatively more stable compared to the biological activity control PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27385778 GFP (in the presence of urea, basic pH or ionic detergents), but the thermal stability of the protein was similar to the control [45]. On the other hand, proteins with elevated solubility, a higher expression and abundant intracellular levels have an increased ratio of Lys content to Arg content [46]. In general, MTs have large number of Lys residues and null or very few Arg residues per molecule, preferring higher solubility and the avoidance of protein aggregates to higher stability by forming a larger number of electrostatic interactions (salt-bridges or hydrogen bonds). This could therefore mean that the asymmetry (Lys > > Arg) reported in all MTs is a basic requirement for forming proteins with an elevated solubility and high intracellular level to respond to metal stress.Analysis of Tetrahymena MT cDNAsAfter comparing all new isolated cDNAs of putative MT genes with the genomic DNA from the corresponding Tetrahymena species, we confirmed that none of these genes have cancer introns in their open reading frames, which is also the case in all previously reported Tetrahymena MT genes [13]. The absence of introns could be related to a faster gene response to different environmental stressors. The presence of introns can delay regulatory responses and they are selected against in genes with transcripts requiring rapid adjustment in order to survive environmental changes [47].In general, the gene expression of Tetrahymena MT genes is very fast, as reported in a CdMT gene (MT-1) from T. pigmentosa, in which an approximately tenfold increase of this transcript was detected after 30 min Cd treatment [15]. An exception was reported in the gene isoform MTT5 of T. thermophila, in which PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28192408 an intron was located in the 3'UTR of the corresponding cDNA.R in only one relevant amino acidde Francisco et al. BMC Genomics (2016) 17:Page 12 ofposition (Asn89/Lys89), and it is interesting how this amino acid position change increases the CuMT character (higher affinity for Cu ions) of TtheMTT2 (Asn) in relation to TtheMTT4 (Lys) [25]. This special feature has also been reported in snail MTs, balancing in favour of Zn/ CdMTs (Lys) or CuMTs (Asn) [44]. Another peculiar characteristic of MTs (including those from Tetrahymena) is the large asymmetry in the ratio of specific amino acids, such as the positively charged amino acids Lys and Arg. There is a considerably higher use of Lys residues with respect to Arg residues, and this asymmetry (Lys > > Arg) appears in all reported MTs. In ciliate MTs the Arg residues are almost absent of the CdMTs, only two (TtheMTT3 and ThegMT2) present one Arg residue (Fig. 3), and only two CuMTs (TborMTT3 and ImMTT2) have Arg residues, with one and two residues, respectively (Fig. 2). The location of these Arg residues is the same as those of Lys residues in other ciliate MT sequences. These two positively charged amino acids (Lys and Arg) are mostly exposed to the protein surface and play important roles in protein stability by forming electrostatic interactions. Arginine forms a higher number of electrostatic interactions compared to lysine.