Extended Abstract

Investigation of multiple oligomeric states of Eschecherichia coli

MurE protein

Field: Life Science

Author: Rujipat Permpornpipat, Tanit Yodsirawong, Vachiravit Phitchuanchom
Ratchasima Wittayalai School, SCiUS-Suranaree University of Technology
Adviser: Dr. Sakesit Chamnansilpa, School of Biochemistry, Suranaree University of Technology

Abstract
Currently, the Antibiotic resistance germs are increasing population and becoming more disseminated. In
order to prevent the development of germs reproduction, we have to look for the possibility of eliminating the cell to
prevent the development of drug-resistant ability. MurE protein catalyzes the attachment of meso-diaminopimelic
acid in gram negative bacteria which is composed of the peptidoglycan biosynthesis pathway. Peptidoglycan plays
a critical role in cells' viability, which is the target to annihilate the microbial. This research analyzes the possible
form of MurE in the free transformation condition to demonstrate the conformation of MurE protein, which impacts
the efficiency of antibiotics. A number of the experimental run were carried out in this research. Attempting to
investigate the monomer MurE experiment require EDTA and splitting the series of experiments into two sets. Set I
is an absence of EDTA buffer, set II is the presence of EDTA buffer. For analyzing size exclusion chromatography
(SEC), results of the absence of EDTA series show five peaks of MurE protein, and the presence of EDTA series
show two peaks. Implying the estimated molecular weight of each peak followed by the sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) test, which expresses MurE properties by considering result could
imply purity of MurE protein. By analyzing the accessible conformation condition in the solution of MurE protein
through the Small Angle X-ray Scattering (SAXS), The scattering data was utilized by The ATSAS package program
to analyze the characteristics of MurE protein. The SAXS data processed through the ATSAS program display the
characteristics of each peak of MurE protein from the Native- PAGE test. Considering MurE monomer, the result
display molecular weight is 64.9 kDa, the radius of gyration is 32.4 A, and D-max is 153.3 A.
Keywords: MurE protein, peptidoglycan biosynthesis, and Small Angle X-ray Scattering (SAXS)

Introduction  the production route could preclude the cell division

More than 2.8 million antibiotic-resistant
infections occur in the U.S. each year, and more than
35,000 people die as a result. According to CDC's
2019 Antibiotic Resistance (AR) Threats Report [1].
Multiple drug-resistant (MDR) bacteria are a
significant problem in public health. These bacteria
develop antibiotic resistance ability appears to
increase over time unfortunate that the rate of
developing the new antibiotic medicine is slower than
the appearance of MDR bacteria. By considering that
gram-negative bacteria have more probability of
surviving than gram-positive bacteria, gram-negative
bacteria have one more layer of a cell membrane
surrounding the peptidoglycan protecting cell.
Preventing the development of the antibiotic
ability of bacteria by finding a solution to eliminating
bacteria could be a new significant method. By
experiment based on the E. coli, which E. coli is an
archetype of gram-negative bacteria. Based upon the
fact that peptidoglycans play a significant role in cell
viability by targeting the peptidoglycan synthesis
pathway in the cell division sequence, interrupting
by accomplishing that method could control
bacteria's reproduction. The Mur complex is a set of
enzymes related to Peptidoglycan synthesis. MurE is
one of the Mur complexes. During the cytoplasmic
step of peptidoglycan precursor synthesis, the
EcMurE from gram-negative bacteria begins the
reaction by adding the meso-diaminopimelic acid to
the nucleotide precursor, which is known as UDP-N-
acetylmuramoyl-L-D-glutamate. (Fig.1) Thus,
understanding the structural detail of this enzyme
might significantly provide the information for the
development of new medicine
Fig.1 Peptidoglycan synthesis. MurE is responsible for attaching
meso-diaminopimelic acid to UDP-MurNAc-L-Ala-D-Glu and
transferring it to UDP-MurNAc-L-Ala-D-Glu-meso-DAP.
Objective  wash, and elution fractions. Following SDS-PAGE
The purpose of this research is to
characterize the oligomeric state of Escherichia coli
MurE, in addition, to elucidate the MurE proteins
characteristic in solution by size exclusion
chromatography and Small Angle X-ray Scattering.
Method and Experimental Detail
Cloning and Expression MurE
The MurE gene was amplified in a strain of
E.coli ATCC25922 (EcMurE) by utilizing the
Polymerase Chain Reaction (PCR) method and
cloning it into a modified pET21d(+) vector (pSY5),
followed by overnight pre-culture at 37 °C. 5 ml of
LB medium were inoculated with a 1:30 dilution of
pre-cultures and incubated at 37 °C with 200 rpm
shaking. Once the optical density (OD) reaches 0.6 -
0.8, afterward kept on ice. After cooling the sample,
1 mM isopropyl-d-1-thiogalactopyranoside (IPTG)
was added to induce expression, and the culture was
incubated at the temperature to be evaluated with
shaking. Following that, 200 μl of overnight
induction cell culture was centrifuged at 5,000 rpm
for 30 minutes at 4 °C and resuspended in 50 l of
blinding buffer containing 50 mM tris-HCI pH 7.5
and 150 mM NaCl.
Purification of Protein
IPTG-induced E. coli BL21 (DE3) cells
expressing EcMurE (Novagen) were resuspended in a
binding buffer containing 50 mM tris pH7.5, 150 mM
NaCl, and 100 mM NaCl. Following that, the cell
suspension was divided into several conical tubes,
each holding 50 mL. (40 mL each). Sonication was
used to lyse the cells in each tube containing cell
suspension. Sonication was utilized for 10 minutes
(20 cycles of 30 sec of sonication at 50 percent
amplitude). Sonication of the cell debris was
followed by pelleting at 15,000 x g for 30 minutes at
4 °C. Chromatography with Immobilized Metal
Affinity (IMAC), After centrifugation, the clarified
lysate was put onto a 1 ml Ni-NTA column (GE
Healthcare), which had been pre-equilibrated with
binding buffer. Following that, the column was
washed with 5 mL (5 CV) of wash buffer (20 mM
imidazole, 500 mM NaCl, 50 mM Tris-HCI pH 8.0).
The protein was then eluted with 5CV, or until the
OD280 value reached zero, using an elution buffer
comprising 20 mM imidazole, 500 mM sodium
chloride, and 50 mM Tris-HCI pH 8 0. Wash and
eluted fractions were collected at a concentration of 1
mL each fraction, respectively. SDS-PAGE was used
to analyze the insoluble fraction (cell pellet), the
soluble fraction (clarified lysate), the flow-through,
analysis, EcMurE-containing fractions were pooled.
Size Exclusion Chromatography (SEC)
The sample of pooled fraction from a
previous purification was loaded the concentrated
Mur protein sample (0.5 mL) onto Superdex Increase
S200 10/300 column (GE Healthcare). The
experiment is conducted at the temperature of 25 °C
in protein buffer pre-equilibrated with this buffer
before. Elution was carried out using 26 mL (1.2 CV)
of the same buffer used for column pre-equilibration.
A flow rate of 0.5 mL per minute was maintained
throughout the procedure. Following SDS- PAGE
analysis, fractions containing Murs proteins were
pooled concentrated of about 20 mg mL- and kept for
working stock. The purification of MurE oligomer
forms was purified in buffer containing 50 mM Tris-
HCI pH 7.5 150 mM NaCl. The purification of MurE
monomer form was pooled and further purified by
size exclusion chromatography in protein buffer
containing 50 mM Tris, pH 7.5, 150 mM NaCl, and 1
mM EDTA. The SEC result of absence EDTA buffer
shows 5 peaks, The SEC result of presence EDTA
buffer shows 2 peaks
Small Angle X-Ray Scattering (SAXS)
SAXS is used for the determination of
microscale or nanoscale structure of particle systems
in terms of such parameters as averaged particle
sizes, shapes, distribution, and surface-to-volume
ratio. SAXS data from the solution of EcMurE were
collected at the Synchrotron Light Research
Institute's (SLRI) beamline 1.3W: SAXS/WAXS in
Nakhon Ratchasima, Thailand. Using a MarCCD
detector at a distance of 1.7 m from the sample
detector with a wavelength of Y jo 1.38 A. The
momentum transfer range was 0.13q 3.76nm1 (g =
4rsin0/2, where 20 is the scattering angle). A sample
volume of at least 60 uL was utilized. At 16 °C and
10-minute exposure of 10 minutes, the protein
concentrations of 5 and 10 mg/mL were determined.
The scattering data were adjusted to the transmitted
beam's intensity and averaged radially.
Result And Discussion
Scattering Data
The data from SAXS (Fig.2) is used to
calculate the Kratky plot (Fig.3) by PRIMUS
program[4], Kratky plot, I(q)*q2 vs. q plot, is thus
informative to check globularity and flexibility of
your protein. In the case of a well-folded globular
protein, the Kratky plot will exhibit a "bell-shape"
peak at low q and converges to the q axis at high q.
The Kratky plot will not converge to the q axis if the
protein has pronounced flexibility. Then the
CRYSOL program[3] was used to evaluate the
solution scattering from macromolecules with known
atomic structure and fit it to experimental scattering
curves from Small-Angle X-ray Scattering (SAXS)
(Fig.4) as the result show MurE peak I proportion
with 2wtz PDB(Fig.4A), peak II proportion with
2wtz PDB(Fig.4B), peak III proportion with 1gg4
PDB(Fig.4C), peak IV proportion with 1gg4
PDB(Fig.4D), peak V 5mg proportion with 2wtz
PDB(Fig.4E), peak V 10mg proportion with 2wtz
PDB(Fig.4F), MurE monomer 5mg proportion with
2y1o PDB(Fig.4G).
Fig.2 Small angle x-ray scattering plot of EcMurE
Then using PRIMUS program to plot Pair-
wise distance distribution function (P(r) Function)
(Fig.5) result show D-max of peak I = 190.42
Å(Fig.5A) , D-max of peak II = 155.21 Å(Fig.5B),
D-max of peak III = 143.23 Å(Fig.5C), D-max of
peak IV = 116.30 Å(Fig.5D), D-max of peak V 5mg
= 178.46 Å(Fig.5E), D-max of peak V 10mg =
188.28 Å(Fig.5F), D-max of peak monomer EcMurE
= 153.28 Å(Fig.5G).

Then calculated the molecular weight of

EcMurE by using PRIMUS result show molecular
weight of EcMurE peak I = 146.8 kDa, peak II =
242.6 kDa, peak III = 136.7 kDa, peak IV = 125.6
kDa, peak V 5mg = 67.5 kDa, peak V 10 mg = 194.6 Fig.3 The Kratky Plot of each peak of EcMurE
kDa, peak monomer EcMurE 64.9 kDa

The molecular model calculated by

DAMMIF are being superimposition with the crytal
structure of EcMurR (Fig.6)
Fig.4A Crysol plot of MurE peak I Fig.4E Crysol plot of MurE peak V 5mg

Fig.4B Crysol plot of MurE peak II

Fig.4F Crysol plot of MurE peak V 10mg

Fig.4C Crysol plot of MurE peak III Fig.4G Crysol plot of monomer MurE

Fig.4D Crysol plot of MurE peak IV

Fig.5A Pair-wise distance distribution function of MurE Fig.5E Pair-wise distance distribution function of MurE
peak I peak V 5mg

Fig.5B Pair-wise distance distribution function of MurE Fig.5F Pair-wise distance distribution function of MurE
peak II peak V 10mg

Fig.5C Pair-wise distance distribution function of MurE

peak III Fig.5G Pair-wise distance distribution function of MurE
peak of monomer MurE 5mg

Fig.5D Pair-wise distance distribution function of MurE

peak IV
Fig.6A Averaged and Fig.6E Averaged and
filtered DAMMIF filtered DAMMIF model Table 1: The analyzed information of each peak table.
model of MurE peak I of MurE peak II (White
(White surface) surface) overlaid with Conclusion
overlaid with the the crystal structure MurE of E.coli was amplified by PCR into
crystal structure (PDB (PDB 2wtz.: blue). the expression vector and being purified by using size
2wtz: blue).
exclusion chromatography. Furthermore, EDTA adds
to test the monomer form of the MurE. The absence
Series Molecula Radius of Dmax(Å) Fitted EDTA buffer showed 5 peaks of protein and presence
r weight gyration(Å PDB EDTA buffer showed 2 peaks, SDS PAGE used to
(kDa) ) calculate the molecular weight, and Scattering data
Peak I 146.8 29.3 190.42 2wtz collected from the SAXS were analyzed by utilizing
the ATSAS software Package.
.CRYSOL program to evaluate the fitted PDB from
Peak II 242.6 49.7 155.21 2wtz
SAXS data show different oligomer states of absence
EDTA buffer, and in presence EDTA buffer was
Peak III 136.7 45.8 143.23 1gg4 being estimated radius of gyration was 32.4 Å D-max
was 153.28 Å, Molecular weight was 64.9 kDa which
is reasonable agreement with the theoretical mass of
Fig.6B
Peak IVAveraged and
125.6 Fig.6F Averaged
40.8 116.30and 1gg4 EcMurE monomer (59.2 kDa) by this result suggest
filtered DAMMIF filtered DAMMIF model that EcMurE has different oligomeric states.
model of MurE peak III of MurE peak IV (White
(White
Peak v surface)67.5 surface) overlaid
26.5 178.46with 2wtz References
overlaid with the
5mg the crystal structure
crystal structure (PDB (PDB 2wtz: blue). [1] CDC. Antibiotic Resistance Threats in the
2wtz:vblue). 194.6
Peak 52.3 188.28 2wtz United States, 2019. Atlanta, GA: U.S.
10mg Department of Health and Human Services,
CDC; 2019.
Peak 64.9 32.4 153.28 2y1o
monomer [2] Franke, D., et al. (2017). ATSAS 2.8: a
MurE
comprehensive data analysis suite for small-
angle scattering from macromolecular solutions,
Journal of Applied Crystallography 50(4): 1212-
1225.

[3] Svergun, D., Barberato, C. & Koch, MHJ

(1995) CRYSOL - a Program to Evaluate X-ray
Fig.6C Averaged and Fig.6G Averaged and Solution.
filtered DAMMIF filtered DAMMIF model
model of MurE peak V of MurE peak V 10mg
5mg (White surface) (White surface) overlaid
[4] Konarev, PV, Volkov, VV, Sokolova, AV,
overlaid with the with the crystal structure Koch, MHJ Svergun, DI (2003). PRIMUS: a
crystal structure (PDB (PDB 2y1o: blue). Windows PC- based system for small-angle
1gg4: blue). scattering Journal of applied crystallography. 36,
1277-1282. data analysis.

Acknowledgements
The project is supported by Science
Classroom in University Affiliated School (SCIUS)
under the Suranaree University of Technology and
Rajsima Wittayalai School. The funding of SCIUS
provided by the Ministry of Science and Technology
is highly appreciated. Center for Biomolecular
Structure, Function, and Application, Suranaree
University of Technology, Nakhon Ratchasima,
Yutaekool as trainer and supporter. Dr. Nuntaporn
Fig.6D Averaged and
filtered DAMMIF
model of monomer
MurE (White surface)
overlaid with the
crystal structure (PDB
1gg4: blue).
Kamonsuttipaijit at BL1.3W: SAXS/WAXS,
Synchrotron Light Research Institute (SLRI), Nakhon
Ratchasima, Thailand.