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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?
BACKGROUND: Escherichia coli is known to have a ferric citrate
transport system involving outer membrane permease, periplasmic
transporters and an inner transmembrane protein encoded by the gene
fepD. The fepD gene can also be found in other gram negative bacteria
such as Pseudomonas aeruginosa PAO1.
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 charge makeup to the corresponding
regions of FepD in E. coli, P. aeruginosa, etc.
http://hugroup.cems.umn.edu/Research/Images_research/Ecoli.jpg
These segments would
be the same in the
three species
SALTY
ENVIRONMENT,
For
Chromohalobacter
These segments in
Chromohalobacter
would be more acidic
than in the other two
species
METHODS
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 (NP415122) 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.
Using Biology Workbench, we uploaded these
three aa sequences and used GREASE to
reveal the hydrophobicity profile (graph) for
each protein chain.
Using PI, we obtained the overall isoelectric
point at each pH, with our interest being pH7 to
compare the three proteins overall. Our
hypothesis predicts that Chromohalobacter
would have the lowest isoelectric point (more
acidic residues).
However, stronger evidence would be obtained
by comparing each hydrophilic region in a
pairwise fashion. TMAP allowed us to obtain a
more precise prediction of which segments
were hydrophobic vs. hydrophilic. There were 9
predicted transmembrane regions. Finally, each
hydrophilic segment was submitted for PI
analysis and the isoelectric point for each
segment was compared among the three
species.
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.
E. coli Data
Sequence:
MSGSVAVTRAIAVPGLLLLL
IIATALSLLI
GAKSLPASVV
LEAFSGTCQS ADCTIVLDAR LPRTLAGLLA GGALGLAGAL
MQTLTRNPLA DPGLLGVNAG ASFAIVLGAALFGYSSAQEQ
LAMAFAGALV ASLIVAFTGS QGGGQLSPVR LTLAGVALAA
VLEGLTSGIA LLNPDVYDQL RFWQAGSLDI RNLHTLKVVL
IPVLIAGATA
LLLSRALNSL SLGSDTATAL GSRVARTQLI
GLLAITVLCG SATAIVGPIA FIGLMMPHMA RWLVGADHRW
SLPVTLLATP ALLLFADIIG
RVIVPGELRV SVVSAFIGAP
VLIFLVRRKT RGGA
Isoelectric Point:
10.476999
Kyte-Doolittle Hydropathy Profile
Predicted Transmembrane
Segments for E. coli:
•
•
•
•
•
•
•
•
•
TM
TM
TM
TM
TM
TM
TM
TM
TM
1:
2:
3:
4:
5:
6:
7:
8:
9:
11 - 39 (29)
66 - 86 (21)
94 - 114 (21)
121 - 143 (23)
152 - 173 (22)
193 - 221 (29)
243 - 271 (29)
283 - 303 (21)
310 - 330 (21)
pI of Non-Transmembrane
Segments
Segment
1
2
3
4
5
6
7
8
9
Residues
1 – 10
40 – 65
87 – 93
115 – 120
144 – 151
174 – 192
222 – 242
272 – 282
304 - 334
pI
11.045
6.265
3.099
3.294
11.045
4.171
10.888
10.888
12.406
P. aeruginosa Data
Sequence:
MQASPMRRRR
AVTLDALQAV
VAGALMQALT
SMGQYLGCAF
AGLSVMLASL
LLGWPGLAIG
TWLLACLAVM
QRWILPFSAL
LGGPAFIVLV
LRAWGLLAGA
DPHDDRHLVV
RNPLAEPGLL
LGAGLAGIAV
TGIIVLNAPP
AGLAAAFALAA
LLAGAATALAG
IAAGLLLGAD
RRFRLSRL
Isoelectric Point:
11.822999
LLLALAALAS LALGSRPVPL
RELRLPRTLV ALLAGAALG
GINAGAALAV IVGVALFDLA
FLLGQARETG TNPVRLVLAG
EVFDRFRHWA AGSLSGSGFA
RLNALALGQE I GQALGVDLRL
PIAFVGLVAP HLARLLAGPD
ILGRLLAAPT EIAAGIVALL
Predicted Transmembrane
Segments for P. aeruginosa:
•
•
•
•
•
•
•
•
•
TM
TM
TM
TM
TM
TM
TM
TM
TM
1: 11 - 39
2: 66 - 94
3: 100 - 120
4: 126 - 146
5: 156 - 184
6: 202 - 226
7: 246 - 274
8: 289 - 309
9: 316 - 336
(29)
(29)
(21)
(21)
(29)
(25)
(29)
(21)
(21)
Kyte-Doolittle Hydropathy Profile
pI of Non-Transmembrane
Segments
Segment
1
2
3
4
5
6
7
8
9
Residues
1-10
40-65
95-99
121-125
147-155
185-201
227-245
275-288
310-316
pI
11.04
5.24
3.29
5.92
6.01
11.04
4.07
11.04
3.29
C. salexigens Data
Sequence:
MLTRRTTRLA GLLAGLVLMA TTFAASVMLG
ATLLHYDPSR VAHIIIVKER
LPRAVIAVLV
MQTLTRNPLA SPGILGINAG
AMCFVVIAVA
VWAALLGALV AACLVLMLSR GGGRAGPSSL
AMFVSFSQGL LIIDHQSFES
VLYWLAGSVS
LPLFGIALLL
CMLLVRHANA LMLGDDMVTS
LLGLVVILLA
GSSVALTGMI GFVGLIVPHM
WLLPACALLG ACLLLLADVA SRFLMPPQDV
TPFFIYLARR
QQARP
Isoelectric Point:
9.970999
TTELPPSTFI
GASLAIAGTL
LLPLHAPADY
RVVLAGVAVT
GRELSLVVPL
LGMHAGTIKL
ARGLFGFDHR
PVGVMTALIG
Predicted Transmembrane
Segments for C. salexigens:
•
•
•
•
•
•
•
TM
TM
TM
TM
TM
TM
TM
1:
2:
3:
4:
5:
6:
7:
5 - 33 (29)
62 - 90 (29)
96 - 116 (21)
122 - 142 (21)
150 - 177 (28)
194 - 222 (29)
242 - 270 (29)
• TM 8: 281 - 308 (28)
Kyte-Doolittle Hydropathy Profile
pI of Non-Transmembrane
Segments
Segment
Residues
pI
1
2
3
4
5
6
7
8
9
1–4
34 – 69
91 – 95
117 – 121
143 – 149
178 – 193
223 – 241
271 – 280
309 – 335
11.045
7.891
6.015
3.099
11.045
4.256
5.097
10.888
11.042
Comparison of pI Data
Segment E.
coli
1
11.045
2
6.265
3
3.099
4
3.294
5
11.045
6
4.171
7
10.888
8
10.888
9
12.406
P.
C.
aeruginosa salexigens
11.04
5.24
3.29
5.92
6.01
11.04
4.07
11.04
3.29
11.045
7.891
6.015
3.099
11.045
4.256
5.097
10.888
11.042
Protein Analysis Program plot of
acidic amino acids - E. coli
Charge / 28 Amio Acids
Charge Along Protein Sequence
6
5
4
3
2
1
0
-1
-2
-3
-4
Total per
28
28 per.
Mov. Avg.
(Total per
28)
0
100
200
Amino Acid Residue Number
300
400
Protein Analysis Program plot of
acidic amino acids - P. aeruginosa
Charge Along Protein Sequence
Charge / 28 Amio Acids
6
Total per
28
28 per.
Mov. Avg.
(Total per
28)
5
4
3
2
1
0
-1
-2
-3
0
100
200
Amino Acid Residue Number
300
400
Protein Analysis Program plot of
acidic amino acids - C. salexigens
Charge Along Protein Sequence
Charge / 28 Amio Acids
4
Total per
28
28 per.
Mov. Avg.
(Total per
28)
3
2
1
0
-1
-2
0
100
200
Amino Acid Residue Number
300
400
CONCLUSIONS
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.