Transcript 投影片 1 - Wellesley College

CHARACTERIZATION OF LIN-12/NOTCH REPEATS (LNRs)
USING HUMAN NOTCH 1 LNRA AS A MODEL SYSTEM
Lauren Choi, Wellesley College
Advisor: Dr. Didem Vardar-Ulu
Results
Introduction
The Folding of hN1LNRA_WT and hN1LNRA_CG is a Dynamic Process
hN1LNRA Wild Type Folding Over Time
hr
hr
hr
hr
folded species
0.018
0.016
0.014
0.012
reduced
0.006
reduced
--0 hr
--1 hr
--2 hr
--5hr
two folded species
0.010
0.005
0.008
0.004
0.006
intermediates
0.003
0.004
0.002
0.001
0.002
0.000
0.000
10.00
12.00
14.00
16.00
18.00
20.00
22.00
Minutes
24.00
26.00
28.00
30.00
32.00
12.00
34.00
13.00
14.00
15.00
16.00
17.00
18.00
19.00
20.00
Minutes
21.00
22.00
23.00
24.00
25.00
--vacuum
--no vacuum
Reduction Potentials of Refolding Buffer (mV)
No Vacuum
Vacuum
1:3
0.015
AU
--0
0.009 --1
0.008 --2
0.007
--5
0.010
0.020
hN1LNRA_CG Mutant Folding Over Time
AU
0.011
AU
Notch receptors are multi-domain trans-membrane proteins that are important for cell-cell
communication and development. Deregulated Notch signaling has been linked to many human
diseases such as sclerosis, artereopathy and leukemia. The extra-cellular domain of the Notch
Receptor contains the Ligand Binding Domain and the Negative Regulatory Region (NRR) which
includes three copies of a Lin-12/Notch Repeat (LNR), a small disulfide-rich sequence of 35
residues. In wild-type Human Notch 1, proper folding of LNR requires the coordination of Ca2+ and
the formation of three specific disulfide bonds. It has been previously shown that the first LNR
from human Notch1, LNRA, can autonomously fold in vitro using a refolding buffer that contains
Ca2+ and a certain reduction potential. In this study, we explored the effect of different reduction
potentials on the folding of wild-type LNRA. We also designed mutant forms of LNRA in which one
pair of cysteine residues was eliminated, and compared their time dependent in vitro folding
pattern with the wild-type’s under varying reduction potentials.
In order to characterize the Ca2+ binding site of LNRA, we carried out an in depth analysis of
the Ca2+ coordination geometry in LNRA and compared it with Ca2+ coordination geometries
observed in other Ca2+ binding proteins. The results of this study will help characterize LNR folding
and elucidate the mechanism of disulfide bond formation during the protein folding process.
LNRA_WT Folding is Faster Under Vacuum Conditions
26.00
27.00
Figure 2. Chromatograms of hN1LNRA_WT (left) and hN1LNRA_CG (right) captured at
different time intervals of rapid dilution refolding: in its reduced form (black), at 1 hour
(green), 2 hours (blue) and 5 hours (red). For either of these constructs, folded
polypeptides will elute earlier due to the surface inaccessibility of hydrophobic residues. For
the wild type, the folding process forms transient intermediates which are slowly converted
into a single properly folded conformation via the disulfide shuffling caused by cysteine :
cystine in the refolding buffer solution. For the mutant hN1LNRA_CG note that there are two
favored folded conformations which form quickly after rapid dilution in refolding buffer.
5:1
12:1
1:3
5:1
12:1
Cystein
e
Cystine
Time
0hr -170 -233 -241 -170 -233 -241 -258
-120
0.010
1hr -169 -226 -230 -169 -231 -238 -250
-111
0.005
3hr -160 -217 -223 -166 -229 -231 -244
-99
5hr -153 -212 -221 -167 -227 -230
0.000
7hr -153 -210 -218 -162 -226 -224
10.00
15.00
20.00
25.00
30.00
Minutes
20hr -97 -213 -219 -96 -214 -220 -224
-87
Figure 4. Chromatograms of hN1LNRA_WT folding after 3 hours in 5:1 cysteine: cystine
under vacuum and air exposed conditions. Anaerobic conditions expedite the folding
process significantly. At three hours, samples under vacuum show primarily folded species
while air exposed samples show primarily reduced species. The table on the right shows the
time dependence of the reduction potential of the refolding buffer solutions without
protein. All values are given in reference to the platinum redox electrode (244mV). The
oxidation of the refolding buffer is slowed under vacuum.
Ca2+ Coordination Geometry
Electron Donors
hN1LNRA_wt Folding Under Different Reduction Potentials
Asp12
Carboxylate
Distance (Ă) Angle
2.50
159.47
2.41
0.007
Asp12
Backbone carbonyl
2.66
67.75
0.006
Asn15
Side chain carbonyl
2.49
88.37
Val17
Backbone carbonyl
2.00
82.96
Asp30
Carboxylate_a
2.18
48.55
Asp30
Carboxylate_b
2.78
71.98
0.010
0.009
AU
0.008
Figure 1. LNR Contruct and Organization. (A) The Domain Organization of the Notch receptor. The
negative regulatory region (NRR) is circled. (B) The crystal structure of NRR from Human Notch 2.
Note that the LNR modules surround the S2 cleavage site in the resting conformation (1). (C) The
NMR solution structure of LNRA from Human Notch 1. The three disulfide bonds are highlighted in
orange and Ca2+ coordinating residues are marked in red (aspartate) and green (asparagine) (2). The
disulfide bond formed between the cysteine residues at position 4 and 27 are being removed in our
mutant strains. (D) The sequence construct of LNRA_WT, LNRA_CG and LNRA_CS and mutant
from Human notch 1. The mutated cysteine residues are highlighted in orange.
Material and Methods
Protein Expression and Purification:
• The pMML-LNRA vector contains the wild-type hN1 LNRA gene fused to modified gene that codes for
the TrpLE sequence in which the Met and Cys residues have been replaced by Leu and Ala,
respectively.
•The mutant plasmids for hN1LNRA_CG and hN1LNRA_CS were made from the kanamyacin resistant
pMML vector for hN1LNRA_WT via QuikChange© Site Directed Mutagenesis (Strategene) and verified
via DNA sequencing.
• Both wildtype and mutant plasmids were transformed into BL21DE3PlysS E.Coli cell line
(chloramphenicol resistant)
•hN1LNRA_WT and hN1LNRA_CG were expressed by growing transformed cells to A600 ~0.6 in Luria
Broth Miller with kanamyacin (50 g/ml) and chloramphenicol (34 g/ml). at 37 °C in a shaking
incubator at 220 rpm and inducing protein production with 0.25 mM IPTG.
•The proteins were purified from inclusion bodies through successive resuspension/ centrifugation
steps and cleaved by cyanogen bromide to obtain the protein of interest.
• A reduced form of the target protein was purified via reversed-phase HPLC, and lyophilized.
• The identity of the constructs were confirmed using MALDI-TOF mass spectrometry.
Reduction Potential Refolding Assays:
• Lyophilized protein was resuspended in water to a concentration of approximately 225 μM.
• 15 μof the protein stock solution in a rapid dilution refolding buffer of
50mM Tris pH 8.0
100 mM NaCl
10mM CaCl2
+ plus one of the following:
Ratio of
Cysteine :
Cystine
Effective
Concentration
5:1
2.5mM : 0.5mM
12:1
3.0mM : 0.25mM
1:3
0.5mM : 1.5mM
• Refolding results analyzed using an analytical HPLC (C18 column) running a gradient of .25% a
minute from 12-20% Buffer B (Buffer A: 90% H20, 10% Acetonitrile, 0.1% TFA; Buffer B: 10% H20, 90%
Acetonitrile, 0.1% TFA)
--reduced
--1:3
--5:1
--12:1
Residue Functional group
Asp33 Carboxylate
folded species
reduced
A.
0.005
0.004
B.
intermediates
0.003
0.002
0.001
0.000
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
Minutes
28.00
30.00
32.00
34.00
36.00
38.00
40.00
Figure 3. Chromatograms of hN1LNRA_WT folded in 1:3, 5:1 and 12:1 cysteine: cystine after
one hour. The difference in proportions of cysteine: cystine effectively changes the reduction
potential of the refolding buffer solution (Table in Fig. 4). At one hour, protein folding in 12:1
cysteine:cystine has little progress with the majority of the species still in the reduced form. 1:3
cysteine: cystine has had the most progress, as most of its peaks are correctly folded. Folding in
5:1 cysteine: cystine has proceeded somewhere in between the other two concentrations with
about the same amount of the species in the reduced and folded forms.
Quantified Folding of hN1LNRA Wild Type
1:3
5:1
time 0hr
% reduced 100
% misfolded 0
1hr
14.1
34.2
2hr
4.7
25.5
4hr
2.0
10.3
5hr
2.9
2.2
%
%
%
%
%
51.7
39.2
38.1
22.7
69.9
11.3
55.6
33.1
87.7
5.4
80.3
14.3
94.9
3.8
12.9
83.3
85.5
10.2
4.3
76.6
18.2
5.2
54.6
29.9
15.6
16.0
63.4
20.6
folded
reduced
misfolded
folded
reduced
0
100
0
0
100
12:1 % misfolded 0
% folded
0
Table 1. Quantified folding of hN1LNRA_wt showing the percent reduced, misfolded and
folded at each hourly interval after initial rapid dilution in refolding buffer dilution.
Chromatograms of hN1LNRA_WT folded in 1:3, 5:1 and 12:1 cysteine: cystine were processed. The
percent of each species was determined by integration of corresponding peaks of the reversephase high performance liquid chromatograms. The reduced species elutes at 12.5% Buffer B, and
fully folded species elute at 10% Buffer B. Misfolded species are composed of peaks which elute
between 11-12% Buffer B. The table shows that the progression of folding under more oxidizing
conditions (1:3) is faster than under more reducing conditions (12:1)
Figure 5. Geometry of the Ca2+ binding site of hN1LNRA_WT. (a) Pymol(4) analysis of the
crystal structure of LNRA from hN1NRR reveals the identity of seven electron donating atoms
within 3 Ă of the Ca2+ atom. Electron donors are either carbonyls in the side chain or backbone
of the protein, or the carboxylate in the side chain of an acidic residue, such as aspartate. If the
electron donor is a carboxylate, one or both of the oxygen atoms can participate in Ca2+
coordination, monodentate or bidentate, respectively. These seven atoms are arranged in a
pentagonal bipyramid (b) which has two axial donors and five equatorial donors. The table
(right) shows the distance between the electron donors and the Ca2+ atom, as well as the angles
between significant atoms at the Ca2+ atom.
Conclusions
• During the refolding process, LNRA has folding intermediates which can be captured through
acidification and resolved using an analytical reverse-phase liquid chromatograph.
The folding of LNRA Wild Type is faster under vacuum conditions which correlates with the slower
oxidation of cysteine/cystine in the refolding buffer.
• The Ca2+ binding site of hN1LNRA_WT has seven electron donors and coordination geometry of a
pentagonal bipyrimid characteristic of most Ca2+ binding proteins (3).
• hN1LNRA folding is favored in a more oxidizing environment.
Future Directions
• Characterize the other mutant LNRA_CS by comparing its time dependent in vitro folding pattern
with the wild type’s under varying reduction potentials.
• Mutate the electron donating residues in the Ca2+ binding site to change the binding affinity of Ca2+
and ion selectivity of the binding site.
• Perform the folding experiments under complete vacuum conditions where no oxidation occurs.
References
1) Gordon, W. R.; Vardar-Ulu, D.; Histen, G.; Sanchez-Irizarry, C.; Aster, J. C.; Blacklow, S. C.
“Structural basis for autoinhibition of Notch” Nat Struct Mol Biol. 2007, 14, 295–300.2.
2) Vardar, D.; North, C. L.; Sanchez-Irizarry, C.; Aster, J. C.; Blacklow, S. C. “NMR Structure of a
Prototype LNR Module from Human Notch1” Biochemistry 2003, 42, 7061–7067.
3) Dokmanić, I., M. Šikić, and S. Tomić. 2008. Metals in proteins. Acta Crystallogr. D. 64:257-263.
4) DeLano, WL. The PyMOL Molecular Graphics System (2002) http://www.pymol.org
Acknowledgements
• Research Supported by the Sherman Fairchild Foundation Summer Research Award
• Dr. Didem Vardar Ulu, Jessica Lin, Christina Hao