Transcript 投影片 1 - Wellesley College

The Effects of Reduction Potential and Number of Disulfide Bonds on the Correct Folding
of Lin-12/Notch Repeats (LNRs) Using Human Notch 1 LNRA as a Model System
Lauren Choi, Wellesley College
Advisor: Dr. Didem Vardar-Ulu
Introduction
Results
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
Negative Regulatory Region of the extra-cellular domain of the Notch Receptor
contains three Lin-12/Notch Repeats (LNR) which protect the cleavage site when the
receptor is in resting conformation prior to ligand-induced cleavage activation.
Mutant LNRA_CG Folding Favors Two of Three Disulfide Bond Configurations
hN1LNRA Wild Type Folding Over Time
hr
hr
hr
hr
folded species
0.018
0.016
0.014
0.012
reduced
AU
0.006
0.005
Most LNRs contain 3 disulfide bonds and coordinate Ca2+ believed to be critical
for LNR structure and function. Exceptions include human Notch4 and PAPP-A which
harbor only four cysteine residues thereby can only form two disulfide bonds. This
finding suggests that only two disulfide bonds are necessary for correct LNR folding,
and that the third disulfide bond is only needed in select LNRs to aide in domain
stabilization. To test the importance of the third disulfide bond, a mutant form of
hN1LNRA was created by replacing the cysteines at positions 4 and 27 with glycine
residues. (mutant LNRA_CG). The importance of the third disulfide bond is elucidated
through tests of folding kinetics, disulfide bonding patterns and calcium coordination
A.
N Ligand Binding Domain
ABC
Intracellular Notch
--Reduced
--without Ca2+
--with Ca2+
reduced
--0 hr
--1 hr
--2 hr
--5hr
two folded species
folded species
0.008
alternatively
folded species
intermediates
0.006
0.004
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
34.00
12.00
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
26.00
27.00
Figure 2. Chromatograms of hN1LNRA_WT (left) and hN1LNRA_CG (right) captured at
different time points of rapid dilution refolding: in its reduced form (black), at 1 hour
(green), 2 hours (blue) and 5 hours (red). For the wild type, the folding process forms
transient intermediates which slowly convert into a single properly folded conformation via
the disulfide shuffling caused by cysteine & cystine in the refolding buffer solution. For the
mutant hN1LNRA_CG, there are two favored folded conformations which form quickly after
rapid dilution in refolding buffer.
reduced
Wild-type hn1_LNRA Ca2+ Coordination Geometry
Electron Donors
Residue Functional group
Distance (Ă) Angle
Asp33 Carboxylate
2.50
159.47
Asp12 Carboxylate
2.41
Identifying the Disulfide Patterns of the
Mutant hN1LNRA_CG’s Two Major Folded Species
C4
B.
C27
N-term
Pre-Cleavage MW: 3621.524 Experimental [3623.7 expected]
Disulfide Bonding
Pattern A (native)
EEAGELPECQE/DAGNKVCSLQCNNHAGGW/DGG/DCS
Fragments DGG EEACELPECQE-DAGNKVCSLQCNNHACGW-DCS
Mass 137.1 3413.1
Disulfide Bonding
Pattern B
EEAGELPECQE/DAGNKVCSLQCNNHAGGW/DGG/DCS
Fragments DGG EEACELPECQE-DAGNKVCSLQCNNHACGW-DCS
Mass 137.1 3413.1
Disulfide Bonding
Pattern C
EEAGELPECQE/DAGNKVCSLQCNNHAGGW/DGG/DCS
Fragments DGG EEACELPECQE–DCS DAGNKVCSLQCNNHACGW
Mass 137.1 1504.6
1910.1
Ca2+
D33
C34
C18
C-term
10
20
30
hN1LNRA_WT: EEACELPECQ EDAGNKVCSL QCNNHACGWD GGDCS
hN1LNRA_CG: EEAGELPECQ EDAGNKVCSL QCNNHAGGWD GGDCS
Figure 1. LNR Construct Sequence and Organization. (A) The Domain Organization of the
Notch receptor. (B) The crystal structure of NRR from Human Notch 2. (C) The NMR solution
structure of LNRA from Human Notch 1 (Orange = three disulfide bonds, Red = Ca2+
coordinating residues) (D) The sequence construct of LNRA_WT and mutant LNRA_CG from
Human notch 1. Our mutant LNRA_CG has cysteines 4 and 27 (orange) removed.
Reduction Potential Effects Amount of Mutant’s Alternatively Folded Species
AU
0.015
--Reduced LNRA_CG
--1:3 cysteine:cystine (-170 mV)
--5:1 cysteine: cystine (-233mV)
--12:1 cysteine: cystine (-241 mV)
alternatively
folded species
0.000
13.00
14.00
15.00
16.00
17.00
18.00
19.00
20.00
21.00
Minutes
67.75
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
During the refolding process, mutant LNRA_CG forms two thermodynamically favored
folded species identified through protease digest as the native disulfide bonding
configuration and a closely related disulfide bonding configuration
•
Reduction potential affects the amount of minor alternatively folded species.
•
Ca2+ addition does not significantly change thermodynamics of LNRA_CG folding
•
The third disulfide bond requires Ca2+ coordination and stabilizes the protein to favor
a single species
Future Directions
•
Find the disulfide bonding pattern of naturally occurring homologs of hN1_LNRA that
have only four cysteines.
Identify the disulfide bonding pattern of the two thermodynamically favored species
of mutant LNRA_CG
References
0.010
0.005
2.66
•
reduced
two folded species
Backbone carbonyl
Conclusions
•
0.020
B.
Asp12
Figure 6. Geometry of the Ca2+ binding site of hN1LNRA_WT. (A) Pymol2 analysis of the
known crystal structure of LNRA reveals the identity of seven electron donating atoms within 3
Ă of the Ca2+ atom. 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 and angles
between the electron donors and the Ca2+ atom.
Figure 3. Identification of the two major folded species of Mutant LNRA_CG. There are three
possible disulfide bonding patterns (A, B & C, see table) for fully oxidized (folded) mutant
LNRA_CG, however only two are thermodynamically favored. After digestion of the two major
folded species of mutant LNRA_CG with Asp-N Endopeptidase, cleaved protein’s mass (circled)
matched the expected fragment mass from the native disulfide bonding pattern (Pattern A)
and a closely related disulfide bonding pattern (Pattern B).
Protein Expression and Purification:
• The mutant plasmids for hN1LNRA_CG were made from hN1LNRA_WT pMML vector.
• Both wildtype and mutant plasmids were transformed into BL21DE3PlysS E.Coli cell line.
• Transformed cells were cultured, induced for protein expression.
• Proteins were purified through successive resuspension, centrifugation, cyanogen
bromine cleavage and final purification using C18 reversed-phase reversed-phase HPLC.
Identifying Disulfide Bonding Pattern:
• Pure hn1LNRA_CG was digested with .02 g/L Asp-N cleavage in 50 mM sodium
phosphate digestion buffer, pH 7.5.
• The digested fragments were identified using MALDI-TOF mass spectroscopy.
Pre-cleavage
Asp-N Endopeptidase Cleaved Fragments
Material and Methods
Protein Folding Experiments:
• Pure proteins were folded in a rapid dilution refolding buffer of 50mM Tris pH 8.0,100
mM NaCl, 10mM CaCl2 and 5:1 cysteine:cystine.
• Folded proteins were analyzed via HPLC (C18 column) using 0.1% TFA system with
varying ratios of H20 and Acetonitrile
• For “stop-and-go”1 time dependent experiments (Figure 2), acid (TFA) mediated
disulfide quenching was employed to capture folding intermediates
A.
AspN Endoproteinase Cleavage pattern for hN1_LNRA_CG:
EEAGELPECQE/DAGNKVCSLQCNNHAGGW/DGG/DCS
D30
C.
D.
C9 C22
reduced
alternatively
folded species
intermediates
C
N15
--Reduced
--without Ca2+
two folded species
--with Ca2+
Figure 5. Chromatograms of hN1LNRA_CG folding after 7 hours in 10 mM Ca2+ or no Ca2+.
Mutant folding is nearly identical in folding conditions with and without Ca2+suggesting that
the loss of the third disulfide bond affects calcium coordination. This trend is seen in other
LNRs with four disulfide bonds.
0.002
0.001
hN1LNRA_CG Mutant
0.010
0.004
0.003
hN1LNRA Wild Type
hN1LNRA_CG Mutant Folding Over Time
AU
--0
0.010
0.009 --1
0.008 --2
0.007
--5
0.011
Ca2+ Addition Does Not Significantly Change Thermodynamics of
Mutant LNRA_CG Folding
22.00
23.00
24.00
25.00
26.00
27.00
28.00
Figure 4. Folding of Mutant LNRA_CG in various reduction potentials effected the amount of
alternatively folded species. The overall ratios of the two major thermodynamically favored
folded species remains constant under different reduction potentials. More reducing
environments favor the minor folded species (denoted by arrows).
1.
2.
3.
4.
5.
Chatrenet,B.,Chang, J.Y., J. Biol. Chem 1993. 268,20998-20996
DeLano, WL. The PyMOL Molecular Graphics System (2002)
Dokmanić, I., M. Šikić, Acta Crystallogr. D. 64:257-263.
Gordon, W. R.; Vardar-Ulu, D, Nature. 2007, 14, 295–300.2.
Vardar, D.; North, C. L.; Blacklow, S. C. Biochemistry 2003, 42, 7061–7067
Acknowledgements
•
•
Research Supported by the Sherman Fairchild Foundation Summer Research Award
Wellesley College Chemistry Department, Dr. Didem Vardar Ulu’s Research Lab