Transcript 投影片 1 - Wellesley College

IDENTIFYING THE ROLE OF CALCIUM
DURING THE REDUCTION OF THE hN1LNRA PROTEIN
Angie Seo, Wellesley College
Advisor: Dr. Didem Vardar-Ulu
Results:
Abstract:
Notch receptors are transmembrane glycoproteins. Lin12/Notch Repeats are three unique protein
modules that help to regulate the ligand induced proteolytic cleavage of the Notch receptor.1
hN1LNRA is the first of the three tandem Lin12/Notch Repeats in the human Notch1 receptor that
regulates cell fate decisions. Previous works have shown that Ca2+ is required for the correct
folding of this repeat via a unique set of three disulfide bonds.2 The goal of this project is to
investigate the role of Ca2+ in stabilizing these disulfide bonds in the folded protein. Our hypothesis
is that the amount of Ca2+ in the environment will impact the minimum redox potential and/or the
time required to reduce hN1LNRA. To test this hypothesis we have exposed folded hN1LNRA in
varying amounts of Ca2+ to identical predetermined reducing conditions under anaerobic conditions.
We assayed samples at various time points using High Performance Liquid Chromatography
(HPLC) and quantified the ratio of folded vs. reduced protein to determine how the concentration of
Ca2+ in the environment affected this ratio.
Ligand Binding Domain
ABC
Intracellular Notch
Folded LNRA
A.
10µM
Oxidized
DTT
21.4% LNRA-Ca2+ complex
50µM
B.
Reduced LNRA
100µM
77.9% LNRA-Ca2+ complex
C.
500µM
C
95.1% LNRA-Ca2+ complex
N15
C4
C9
C22
C.
Ca2+
C27
D30
C34
1
B.
35
C-term
D33
S19
C18
Figure 1. The Notch Receptor and the LNR. (A) Domain Organization of the Notch receptor.
(B) Sequence of hN1LNRA with cysteine residues highlighted in orange and the characteristic disulfide
pattern indicated above the sequence. (C) NMR solution structure of hN1LNRA, disulfide bonds shown as
orange sticks and Ca2+ coordinating residues represented as yellow sticks.3
Figure 4. Chromatograms of hN1LNRA. (A) Overlay of chromatogram for the fully correctly folded and fully
reduced LNRA monitored at 280 nm. The gray line across the chromatograms show the HPLC gradient used
in the experiments. (B) Full view of chromatogram, which shows the overlay of chromatogram for reduction
experiments carried out for 60 minutes under various free [Ca2+] monitored at 280 nm. The peaks ~ 3 min.
are buffer components that do not bind to the C18 column under the assaying conditions. The peak ~ 10 min.
is from the residual oxidized DTT peak. Peaks at ~ 11 min. and ~ 18 min. correspond to folded and reduced
LNRA, respectively. (C) Close-up view of folded and reduced LNRA peaks after 60 minutes of reduction in 2.5
mM DTT (Redox potential: -320 ± 10 mV) in 0, 25 µM, 100 µM, 1 mM, and 10 mM free [Ca2+].
Reduction of Folded LNRA vs. Time at Redox Potential of -320 ± 10 mV
120
Preliminary Experiments:
Cysteine
-S
HO
OH
Reduced DTT SH
of reduction of
Reduced Cysteine Figure 2. Mechanism
disulfide bonds.5 Disulfide bond
formation is a reversible process
R1
R1
SH R important for many biological processes,
SH
2
such as stabilization of proteins and
HS
regulation of biological activity. The
formation of disulfide bonds in LNRA
R2
S
stabilizes the folded protein.4
HO
Dithiotreitol (DTT), a strong reducing
S agent, is used in this project to reduce
HO
S the disulfide bonds of the folded LNRA.
OH
DTT not only reduces the disulfide
HO
bonds, but also establishes the redox
Oxidized DTT potential of the experiments.
SH
Experimental Set-Up:
• Prepared a set of buffers containing 20mM HEPES pH 8.0, 150mM NaCl, and varying [CaCl2].
• Purged the buffers with N2 gas and placed them in the AtmosBag, which was then filled with N2
gas to provide anaerobic conditions.
• Added 500 µl of 25 mM DTT into each buffer tubes for a final [DTT]
of 2.5 mM. 450µl of the solution was transferred into eppendorf
tubes. All solutions were left open for 2 hours to reach equilibrium.
• At the start of the experiment, folded LNRA, which was dialyzed
against respective Ca2+ concentrations, was aliquoted to a final
concentration of ~ 15 µM.
• At predetermined time points, ~150 µl of this sample was placed in
~100 µl of 0.5 M Ammonium Acetate pH 5.3 to quench the reduction
of proteins at that point.
• Each sample was assayed using analytical high performance liquid Figure 3. Picture of the
chromatography (HPLC), using a C-18 column running a gradient experimental set-up. In order
to ensure anaerobic conditions
of 12 to 13.4% Buffer B at 0.1%/min
that would support constant
(Buffer A: 95% 5 mM Ammonium Acetate pH 5.8, 5% Acetonitrile;
Buffer B: 10% 5 mM Ammonium Acetate pH 5.8, 90% Acetonitrile). redox potentials over the course
of the experiments, experiments
• The amount of folded, reduced, and misfolded proteins were
were carried out in an
quantitated by integrating the area under each corresponding
AtmosBag filled with N2 gas.
peak on the chromatogram.
100
Reduced Proteins (%)
• To obtain a range of redox potentials between -180 and -425 mV, varying concentrations of DTT
(between 0.10 and 2.5 mM) were used in different buffers.
• To determine how long it takes to reach equilibrium, a redox electrode was used to monitor the
redox potentials over time. It was determined that redox reaches equilibrium in about 2 to 3 hours
and remains constant for 3 more hours under anaerobic conditions.
H2
H
COOH
C
C
NH2
HOOC
S S
C
H2N
C
H
H2
Oxidized
70.0% LNRA-Ca2+ complex
EDTA
80
25µM
Ca2+
60
40
100µM
Ca2+
20
1mM
Ca2+
0
10mM
Ca2+
0
15
-20
30
45
60
Time (min.)
0 µM Ca2+(0.5 mM EDTA)
0% LNRA-Ca2+ complex
25 µM
42.7% LNRA-Ca2+ complex
100 µM
77.9% LNRA-Ca2+ complex
1 mM
97.5% LNRA-Ca2+ complex
% folded
% reduced
% misfolded
% folded
% reduced
% misfolded
% folded
% reduced
% misfolded
% folded
% reduced
% misfolded
% folded
% reduced
% misfolded
0 min.
86.27
4.46
9.27
84.77
1.03
14.2
75.66
1.31
23.03
99.20
0
0.8
99.33
0
0.67
15 min.
3.52
89.30
7.18
58.38
20.52
21.1
66.89
6.11
27.00
98.38
0
1.62
99.67
0
0.33
Figure 5. Reduction of folded
LNRA as a function of time at
-320 ± 10 mV in 20 mM HEPES
pH 8.0, 150 mM NaCl, 2.5 mM
DTT and various free [Ca2+].
Percent of reduced LNRA for each
experiment was determined by
dividing the integrated peak area
under the reduced peak by the total
area calculated as the sum of all
LNRA peaks in the corresponding
individual chromatograms. These
percentages were plotted as a
function of time in 15 minute
intervals for each experimental
conditions.
Time
30 min.
0.73
93.28
5.99
41.71
39.74
18.55
57.05
17.10
25.94
97.80
0
2.2
99.34
0
0.66
45 min.
0.72
95.17
4.11
27.79
50.39
21.82
43.87
20.09
36.04
94.69
1.44
5.31
99.71
0
0.29
60 min.
0.87
94.46
4.67
17.72
60.92
21.36
43.24
30.00
26.76
94.02
1.98
5.98
99.72
0
0.28
10 mM
99.8% LNRA-Ca2+ complex
Table 1. Quantification of LNRA reduction over time in 20 mM HEPES pH 8.0, 150 mM NaCl, 2.5 mM
DTT, and varying concentrations of Ca2+ at -320 ± 10 mV. Percent of folded, reduced, and misfolded LNRA
at 15 minute time intervals were calculated by dividing the integrated peak area of the corresponding peaks
by the total area of all LNRA peaks from individual chromatograms. The percent of protein complexed with
Ca2+ at each [Ca2+] was calculated using the formula ML = {(Lo+Mo+Kd)–((Lo+Mo+Kd)2 – 4MoLo)1/2 }/2; ML
represents the percent bound complex, Mo is the initial [LNRA] = ~ 15 µM, Lo is the initial [Ca2+] of the
experiments included in the table, and Kd is the dissociation constant 25 µM.6
1 mM
% folded
% reduced
% misfolded
% folded
% reduced
% misfolded
% folded
% reduced
% misfolded
% folded
% reduced
% misfolded
% folded
% reduced
% misfolded
0 min. 15 min.
98.49 96.26
0
0
1.51
3.74
99.49 69.38
0
29.77
0.51
0.85
98.94 98.90
0
0
1.06
1.1
-
30 min.
100
0
0
42.14
57.86
0
79.88
18.96
1.16
-
1 hr.
0
100
0
17.26
81.57
1.17
10.13
89.87
0
62.39
36.79
0.82
67.76
29.16
3.08
2hr.
0
99.41
0.59
4.12
95.33
0.55
0.72
98.62
0.66
36.79
63.21
0
44.45
52.94
2.61
3 hr.
0
98.41
1.58
0.80
96.94
2.26
1.09
98.91
0
24.77
75.23
0
30.85
67.80
1.35
97.5% LNRA-Ca2+ complex
Table 2. Quantification of LNRA reduction over time in 20 mM HEPES pH 8.0, 150 mM NaCl, 2.5 mM
DTT, and varying concentrations of Ca2+ at -360 ± 10 mV. Percent of folded, reduced, and misfolded
LNRA at 15 minute time intervals were calculated by dividing the integrated peak area of the corresponding
peaks by the total area of all LNRA peaks from individual chromatograms. The percent of protein complexed
with Ca2+ at each [Ca2+] was calculated using the formula ML = {(Lo+Mo+Kd)–((Lo+Mo+Kd)2 – 4MoLo)1/2 }/2; ML
represents the percent bound complex, Mo is the initial [LNRA] = ~ 15 µM, Lo is the initial [Ca2+] of the
experiments included in the table, and Kd is the dissociation constant 25 µM.6
Reduced LNRA vs. Redox Potential
Reduced LNRA (%)
A. N
Overlay of Chromatograms for Reduction of LNRA at Redox Potential of -320 ± 10 mV
Time
45 min.
3.25
96.75
0
23.46
76.54
0
69.08
29.55
1.37
-
89.87%
100
100 µM
Ca2+
80
60
40
30%
20
0
29.16%
1 mM
Ca2+
1.98%
-320 ± 10
-360 ± 10
Redox Potential (mV)
Figure 6. Comparison of
reduced LNRA at two different
redox potentials and two
different [Ca2+]. Folded LNRA
was reduced in buffers
containing 100 µM and 1 mM
[Ca2+] with redox potentials of
-320 ± 10 and -360 ± 10 mV.
Conclusions:
• Stability of the folded LNRA against reduction is dependent on the free [Ca2+] in the
environment.
• Reduction of the folded LNRA is slower at higher free [Ca2+].
• In the absence of Ca2+, folded LNRA was reduced quickly.
• In more reduced environments (lower redox potentials), folded LNRA is reduced faster than in
more oxidized (higher redox potentials), for the same free [Ca2+].
Future Directions:
• Collect more data for additional [Ca2+] for the same time points and same redox potentials
• Conduct additional experiments under different redox potentials
• Extend the time of experiment to determine the time needed for complete reduction of LNRA at
each [Ca2+] and redox potential.
References:
1. NIH AREA Grant Proposal_DVU_2009
2. Vardar, D. North, C.L., Sanchez-Irizarry, C., Aster, J. C., Blacklow, S.C. “Nuclear Magnetic Resonance
Structure of a Prototype Lin12-Notch Repeat Module from Human Notch1.” Biochemistry. 2003, 42,
7061-7067.
3. Gordon, W.R., Vardar-Ulu, D., Histen, G., Sanchez-Irizarry, C., Aster, J. C., Blacklow, S. C. “Structural
basis for autoinhibition of Notch.” Nature Structural & Molecular Biology. 2007, 1-6.
4. Gilbert, Hiram F. “Thiol/Disulfide Exchange Equilibria and Disulfide Bond Stability.” (1995)
http://www.ncbi.nlm.nih.gov/pubmed/7651233.
5. Dithiothreitol. (2010) http://en.wikipedia.org/wiki/Dithiothreitol.
6. Jakubowski. “Chapter 5 – Binding. A: Reversible Binding 1 Equations and Curves.” (2010)
http://employees.csbsju.edu/hjakubowski/classes/ch331/bind/olbindderveq.html.
Acknowledgements:
• Research Supported by the Sophomore Early Research Program
• Dr. Didem Vardar Ulu