Identification of a Critical Amino Acid Residue of YiiP, a Zinc Transporter from Escherichia coli

1
Identification of a Critical Amino Acid Residue
of YiiP, a Zinc Transporter from Escherichia coli
Katherine W. Kao, Yinan Wei, John Trunk and
Dax Fu SUNY at Stony Brook, Stony Brook, NY
11794 Biology Department, Brookhaven National
Laboratory Upton, NY 11973, USA
Purification
Abstract
Figure 1 -FPLC System for Purification of YiiP.
Cell membrane extract was applied to a DEAE
column, which is connected to one of the two
Ni-NTA columns, followed by a desalting column.
Switching valves were used to control solvent
flow in the following sequential order protein
binding, washing, eluting and desalting. The
protein peaks in the flow-through of Ni-NTA and
desalting columns were continuously monitored by
UV detector 1 and 2. The purified YiiP fraction
was fractionated and collected by a
computer-controlled fraction collector.
Homeostasis of metal cations in the cell is
crucial to survival. YiiP is a metal transporter
in E. coli that plays a central role in
regulating cellular concentrations of Zn2, Cd2
and Hg2. Transport of these metals involves a
coupled deprotonation mechanism, however, the
structure and mechanism of function have not yet
been revealed to atomic detail. In this study,
point mutations in wild type YiiP were made and
the stability and secondary structure of YiiP
mutants were determined through CD (circular
dichroism) analysis at 222nm. CD analysis shows
that the protein is 52 alpha helical and reveals
one site in particular, Asp 157 that is critical
to stability suggesting that it may have a role
in the metal transport mechanism.
Figure 3-Instruments at the National Synchrotron
Light Source (NSLS). Experiments were conducted
using a UV beamline at the NSLS. Depicted above
is a 0.2mm path length cuvette containing protein
sample being loaded into test chamber.
Figure 2-Purification and Isolation of YiiP.
Left Panel SDS PAGE showing the purification of
YiiP. Lane 5 corresponds to pure YiiP and Lane 6
corresponds to pure YiiP after cleavage of the
His-tag. Right Panel The sizing HPLC profile of
YiiP, showing the purity and monodispersity of
YiiP. The purified YiiP exhibits a major size
specie with a small bump due to delipidation.

Circular Dichroism
Results of CD of YiiP
Wild Type YiiP
D157A
C287S
a) Chiral molecules are optically active and
absorb polarized light differently. Nature is
full of chiral molecules, for example, amino
acids naturally occur in the L-isoform only.
http//www.sinc.sunysb.edu/Class/che321ff/
Figure C1-CD of YiiP-D157A. CD of mutant D157A
of YiiP taken at 4oC. The curve displays the
characteristics of a protein that is mostly alpha
helical. The percent alpha helicity calculated
based on the absorbance at 208nm is 68.
Figure A1-CD of Wild Type YiiP. CD of wild type
YiiP taken at 4oC. Comparison of the curve with
the standards given in figure-d show that the
protein is mostly alpha helical. The
characteristic double peak at around 205-225 nm
reflects the alpha helicity of the protein.
Calculations based on DA at 208 nm indicate that
alpha helicity is 52.
b) Circular Polarized Light CD is observed when
an optically active sample absorbs right and left
handed polarized light differently.
Figure B1-CD of YiiP-C287S. CD of mutant C287S
taken at 4oC. The curve is characteristic of a
protein consisting mostly of alpha helices.
Calculations based on DA at 208 nm reveal that
the alpha helicity is 45.34.

No EDTA
No EDTA
No EDTA
With EDTA
With EDTA
With EDTA
Aromatics- Phehnylalanine, tryptophan and
tyrosine www.sunysb.edu/chemistry/molecules/aa.htm
l
Disulfide bond http//www.web-books.com/MoBio/Free
/Ch2A3.htm
Alpha Helix http//www.sci.sdsu.edu/TFrey/ProtStru
ctClass/ProtStructClass.html
Beta Sheet
c) Chromophores are optically active groups
within a protein, they include peptide backbones,
disulfide bonds and aromatic side chains such as
Phe, Tyr, and Trp. CD is particularly sensitive
to changes in the alpha helical structure of
proteins at about 222nm making the study of
protein stability possible
Figure A2-Time Scan of Wild Type YiiP at 70oC. A
scan of time vs. change in absorbance was taken
to measure the stability of the protein. Change
in absorbance (DA) was monitored at 222nm to
record the change in secondary structure of alpha
helices. The increase in DA indicates the decay
of alpha helices. The decay for YiiP with EDTA is
more rapid than decay of YiiP without EDTA.
Decay was monitored for 1200s.
Figure B2-Time Scan of C287S at 70oC. A scan of
time vs. DA was taken for mutant C287S. The
curves indicate that the protein denatures more
rapidly when EDTA is added. A comparison to the
wild type time scan indicates that the change in
absorbance when EDTA is added to C287S is not as
pronounced as in wild type YiiP. Decay was
monitored for 1200s.
Figure C2-Time Scan of D157A at 70oC. The time
vs. DA for D157A. The denaturation of D157A in
the presence of EDTA is significantly more rapid
than decay without EDTA. A comparison to wild
type and C287S indicates that at 70oC, D157A
denatures more rapidly in a shorter time frame.
Decay was monitored for 800s.
d) Standard Curves that are characteristic of
each secondary structure of a protein. YiiP is
mostly alpha helical and its CD exhibits the
trend shown for an alpha helical protein.
Figure A3-Denaturation Rate of wild type YiiP.
Graphs monitoring the rate of decay of wild Type
YiiP. Decay remains relatively constant at about
0 s-1.
Figure B3-Denaturation Rate of C287S. Graphs
monitoring decay rate which remained relatively
constant at about 0 s-1.
Figure C3-Denaturation Rate of D157A. Curves
show two major peaks corresponding to maximum
rate of decay. The maximum rate of decay is
greater when EDTA is added.
Conclusions
This study yielded highly purified protein and
identified one point mutation D157A, that
significantly decreased the stability of YiiP.
We also conclude that the presence of EDTA
decreases the stability of the proteins because
it chelates structural metal ions from the ion
channel and thus, the protein denatures more
rapidly at high temperature. Acknowledgements.
Supported by the U.S. Department of
EnergyOffice of Science Education. I would like
to thank my mentors John Trunk, Dr. Yinan Wei and
Dr. Dax Fu for their guidance in this project, as
well as Hong Wang, Denise Monteleone and Michele
Bender for helpful discussions.
http//employees.csbsju.edu/hjakubowski/classes/ch
331/protstructure/cdsecondst.gif