THE PUZZLING PROPERTIES OF THE PERMEASE (PPP) Kim Finer, Jennifer Galovich, Ruth Gyure, Dave Westenberg March 4, 2006. IS THERE AN E.COLI -TYPE IRON PERMEASE GENE (FepD) IN CHROMOHALOBACTER SP . AND IF SO, IS THE PERIPLASMIC-FACING RESIDUE SEQUENCE SIGNIFICANTLY DIFFERENT?.
Chromohalobacter salexigens is a newly described species of bacterium able to survive in moderately saline environments. The species includes the previously described species, Halomonas elongata. One of its survival adaptations is the use of higher percentages of acidic amino acid residues in proteins that come into contact with the high salt environment. This modification protects the proteins from ‘salting out.’
HYPOTHESIS: We predict that transmembrane proteins such as FepD would also demonstrate this characteristic enrichment with acidic residues, but only in the hydrophilic regions facing the salty periplasm. Regions of protein hydrophilic regions facing the cytoplasm would be similar in chargemakeup to the corresponding regions of FepD in E. coli, P. aeruginosa, etc.
These segments would be the same in the three species
These segments in Chromohalobacter would be more acidic than in the other two species
We began by accessing an Excel spreadsheet of annotated C. salexigens sequences available at JGI. The spreadsheet was searched for ferric citrate transporter. The sequence reference was found (NP-415122) and the homologous sequences from E. coli and P. aeruginosa were identified.
The protein sequence for this accession was obtained from the JGI website.
Protein sequences for FepD from E. coli K12 and P. aeruginosa (PAO1) were obtained from the NCBI website.
A third approach was to analyze the sequences using the Excel based analysis program, Protein Analysis. Protein analysis was used to plot the location of acidic amino acid residues along the entire peptide sequence.
MSGSVAVTRA IAVPGLLLLL IIATALSLLI GAKSLPASVV LEAFSGTCQS ADCTIVLDAR LPRTLAGLLA GGALGLAGAL MQTLTRNPLA DPGLLGVNAG ASFAIVLGAA LFGYSSAQEQ LAMAFAGALV ASLIVAFTGS QGGGQLSPVR LTLAGVALAA VLEGLTSGIA LLNPDVYDQL RFWQAGSLDI RNLHTLKVVL IPVLIAGATA LLLSRALNSL SLGSDTATAL GSRVARTQLI GLLAITVLCG SATAIVGPIA FIGLMMPHMA RWLVGADHRW SLPVTLLATP ALLLFADIIG RVIVPGELRV SVVSAFIGAP VLIFLVRRKT RGGA
MQASPMRRRR LRAWGLLAGA LLLALAALAS LALGSRPVPL AVTLDALQAV DPHDDRHLVV RELRLPRTLV ALLAGAALG VAGALMQALT RNPLAEPGLL GINAGAALAV IVGVALFDLA SMGQYLGCAF LGAGLAGIAV FLLGQARETG TNPVRLVLAG AGLSVMLASL TGIIVLNAPP EVFDRFRHWA AGSLSGSGFA
LLGWPGLAIG AGLAAAFALAA RLNALALGQE I GQALGVDLRL
TWLLACLAVM LLAGAATALAG PIAFVGLVAP HLARLLAGPD
QRWILPFSAL IAAGLLLGAD ILGRLLAAPT EIAAGIVALL LGGPAFIVLV RRFRLSRL
MLTRRTTRLA GLLAGLVLMA TTFAASVMLG TTELPPSTFI ATLLHYDPSR VAHIIIVKER LPRAVIAVLV GASLAIAGTL MQTLTRNPLA SPGILGINAG AMCFVVIAVA LLPLHAPADY
VWAALLGALV AACLVLMLSR GGGRAGPSSL RVVLAGVAVT AMFVSFSQGL LIIDHQSFES VLYWLAGSVS GRELSLVVPL LPLFGIALLL CMLLVRHANA LMLGDDMVTS LGMHAGTIKL LLGLVVILLA GSSVALTGMI GFVGLIVPHM ARGLFGFDHR WLLPACALLG ACLLLLADVA SRFLMPPQDV PVGVMTALIG TPFFIYLARR QQARP
Hydropathy plots illustrate a similar topology for the FepD proteins of C. salexigens, E.coli and P. aeruginosa. We predicted that the loop domains or the C. salexigens FepD protein, exposed to the exterior of the cell would have a lower pI than the exterior domains of E. coli and P. aeruginosa. The calculated pI of the entire C. salexigens FepD protein is lower than the pI of the other two organisms. However, the calculated pI of individual loop domains and a plot of acidic residues show that the overall hypothesis is not supported. Several loop domains of the C. salexigens FepD protein have a lower calculated pI than the corresponding segments of E. coli, However, comparison to the corresponding domains of the P. aeruginosa FepP protein do not show the same trend. In fact, P. aeruginosa seems to have more acidic loop domains than C. salexigens.