Transcript 投影片 1 - Wellesley College

LIN-12/Notch Repeat B:
The effects of disulfide bonding and hydrophobic residues on its autonomous folding
Jessica Lin ‘10, Wellesley College
Advisor: Dr. Didem Vardar-Ulu
Results
Abstract
Notch proteins are transmembrane proteins involved in controlling cell differentiation, cell growth, and
cell death during embryonic and adult life. Upon ligand binding, Notch proteins are activated and a proteolytic
cleavage is induced within an extracellular negative regulatory region (NRR) (Gordon et al. 2007). The NRR
contains three contiguous LIN-12/Notch Repeats (LNRs) – LNRA, LNRB, and LNRC – the first two of which are
currently being studied in this lab to understand more thoroughly the important determinants in protein folding.
Previous research has shown that imperative to LNR folding are its calcium ion coordination and correct
disulfide bond formation. Disulfide bonds in proteins are covalent bonds formed by two cysteine residues that
contribute to the tertiary structure of proteins alongside with other global, non-covalent interactions. To study the
effect of each disulfide bond on the independent folding of LNRB, one of its three pairs of disulfide bonds was
eliminated by mutating C45 and C69 to two alanine residues. In this study, the mutant form was folded in vitro
under the same conditions as the wild type, and the folding patterns of the two protein modules were compared.
One other characteristic of much interest in LNRB is its hydrophobic residues and their impact on the
autonomous folding of LNRB. Because of its more central location in relation to the rest of the protein, LNRB
participates in more extensive interactions via its exposed hydrophobic residues such as W29, W42, and W52.
With an indole functional group, tryptophan is one of the most hydrophobic amino acids. Hence, to study the
possible destabilizing effect of hydrophobicity in LNRB, W52 was mutated to an alanine. W52 is especially of
interest because of its location on the outer surface of LNRB right next to C51 and its proximity to W42, which is
located in the LNR-AB linker. It is hypothesized that the hydrophobic interaction between W52 and W42
counteracts the stability imparted by the disulfide bond residing in the core of the LNRB module, formed by C51
and C64. Different lengths of LNRB were tested in conjunction with the above-mentioned mutations in the
hopes of defining the minimal requirements for the autonomous folding of LNRB.
Table 1: LNRB constructs of interests with their molecular weights. The LNR-AB and BC linkers are
highlighted in red. The LNRB_intermediate constructs have part of the AB linker and the LNRB_short constructs do
not have any part of either linker. The cysteines forming disulfide bonds are highlighted in blue, the CA mutations
are highlighted in orange, and the WA mutations are highlighted in purple. The MW’s were calculated using
ExPASy ProtParam and the observed MW’s were found using a MALDI-TOF mass spectrometer. Several observed
MW’s are currently not available due to mechanical issues with the instrument.
Calculated
MW (Da)
Protein construct
LNRB_Original
Electron Donors
Observed
MW (Da)
5368.8
5357.72
5304.7
5291.05
L N F N D P W K N C T Q S L Q C W K Y F S D G H C D S Q C N S A G C L F D G F D C Q R A E G Q
Mut1,5 CA _LNRB_Original
L N F N D P W K N A T Q S L Q C W K Y F S D G H C D S Q C N S A G A L F D G F D C Q R A E G Q
Mut W52A52_LNRB_Original
5253.7
5189.5
N/A
LRNB_Intermediate
4338.7
N/A
Mut 1,5 CA LNRB_Intermediate
Buffers Used:
Buffer A: 50 mM Tris pH 8.0, 25% Sucrose, 200 mM of NaCl
Buffer B: 20 mM Tris pH 8.0, 1% Triton X-100
Buffer C: 20 mM Tris pH 8.0, 200 mM of NaCl
Buffer D: 6 M Guanidine HCl, 50 mM Tris pH 8.0, 10 mM DTT
The protein modules with the leader sequence still attached were dissolved in 70% formic acid then cleaved
with  200 mg cyanogen bromide for 4 hours. The samples were dialyzed overnight in acidic water pH 3.
After the overnight dialysis, the leader sequence was crashed out by raising the pH of the samples to ~8.5.
The protein modules were further purified using reversed-phase (RP) prep HPLC. After purification, the
identities of the protein modules were confirmed using MALDI-TOF mass spectrometry.
4274.6
N/A
4223.6
N/A
Mut 1,5 CA_Mut W52A52 LNRB_Intermediate
4159.4
N/A
15 l of 200 M protein was immersed in 285 l of refolding buffer for different lengths of time.
Time points used: 0 hr, 1 hr, 1.5 hrs, 3 hrs, 7 hrs, overnight (16 hours)
Folding was quenched using 50 l of 4% TFA. The “folded” samples were run on the RP - analytical HPLC.
2.23
Y54
2.35
D57
2.65
H59
2.39
D75
2.44
3940.3
N/A
0.006
3825.1
N/A
0.004
D P W K N A T Q S L Q C A K Y F S D G H C D S Q C N S A G A L F D G F D C Q
LNRB_Short
--- Short _ 0hr
---Short_overnight
---mut W52A52_Short_0hr
---mut W52A52_Short_overnight
K N C T Q S L Q C W K Y F S D G H C D S Q C N S A G C L F D G F D C Q
Mut W52A52 LNRB_Short
AU
K N C T Q S L Q C A K Y F S D G H C D S Q C N S A G C L F D G F D C Q
0.002
0.000
Mut 1,5 CA
Original_overnight
Original_overnight
Mut 1,5 CA
Original_reduced
Original_reduced
Figure 3: Observing the effects of
eliminating the hydrophobic residue
W52 on the folding of LNRB_original.
A comparison was made between the
folding abilities of LNRB original and mut
W52A52_ original. This
chromatogram shows that although
there was one preferred folded
conformation reached for samples of the
two constructs allowed to fold overnight,
there were several additional
conformations reached for the mut
W52A52_original construct.
Figure 2: Observing the effects of eliminating
a pair of disulfide bond on folding. A
comparison was made between the folding
abilities of LNRB original and mut 1,5
CA_original. The samples in this figure were
refolded overnight and overlaid with their
reduced forms. The chromatogram shows a
single thermodynamically preferred folded
conformation for LNRB_original and three main
folded conformations for mut 1,5 CA_original.
Mut W52A52
Original_reduced
Mut W52A52
Original_overnight
Original_overnight
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
55.00
Minutes
60.00
Discussion and Future Direction:
• eliminating two cysteines (resulting in four cysteines) in LNRB_original leads to three thermodynamically
preferred folded conformations instead of just one preferred conformation, which is seen in the wild type
LNRB_original with six cysteines
• in LNRB_original, changing W52 to a less hydrophobic residue, alanine, does not increase the stability of the
autonomous folding of LNRB as was initially hypothesized; after analyzing the results, it is now hypothesized
that eliminating W52 actually destabilizes LNRB_original because the stabilizing hydrophobic interactions
between W52 and W42 are eliminated as well
•in LNRB_short, no conclusive effects of eliminating W52 on folding were observed; at the 3-hour refolding time
point for mutW52A52_short, a more defined peak was observed later in the gradient (a more hydrophobic
conformation), but the peak was no longer visible in the sample that refolded overnight
Mut 1,5 CA Mut W52A52 Original
Mut W52A52 Original
-0.002
Figure 6: Observing the effects of
eliminating the hydrophobic residue
W52 on the folding of LNRB_short. A
comparison made between
LNRB_short_0hr and mut
W52A52_short_0hr shows that the
elimination of W52 did have some effects
on the module. However, a comparison
between LNRB_short_overnight and mut
W52A52_short_overnight shows that
similar conformations were reached
regardless of the W52 mutation.
Original_reduced
Original
Mut 1,5 CA Original
HPLC Buffers Used: Buffer A: 10% Acetonitrile, 90% H2O, 0.1% TFA
Buffer B: 90% Acetonitrile, 10% H2O, 0.1% TFA
Refolding Tests:
Refolding Buffer: 20 mM Tris pH 8.0-8.5,10mM CaCl2, 100 mM NaCl, 2.5 mM cysteine, 0.5 mM cystine
D61
D P W K N C T Q S L Q C A K Y F S D G H C D S Q C N S A G C L F D G F D C Q
Cell Growth and Protein Expression:
The protein constructs of interest were expressed by transforming BL21(DE3)-pLysS E. coli cells with the
kanamycin-resistant pMML-LNRB vector that contained the DNA sequences for the constructs of interest with
an N-terminal hydrophobic leader sequence. These mutations were made via QuikChange© Site Directed
Mutagenesis and confirmed with DNA sequencing.
Inclusion Body Purification and Identification:
To dispose of undesired cellular parts, the samples were suspended in different buffers via sonication. After
suspension in each buffer, the samples were centrifuged in SS34-rotor tubes at 19K for 30 minutes to
separate the proteins from the undesired cellular parts.
2.68
Figure 5 and Table 2: Distances between
coordinating ligands and the Ca2+. Measurements
were made on PyMol and a criterion of < 3 Å was used
to determine the Ca2+ coordinating ligands in LNRB.
The distances are listed in Table 2 on the right with
their respective coordinating ligands.
D P W K N C T Q S L Q C W K Y F S D G H C D S Q C N S A G C L F D G F D C Q
Mut W52A52 LNRB_Intermediate
Material and Methods
D72
L N F N D P W K N A T Q S L Q C A K Y F S D G H C D S Q C N S A G A L F D G F D C Q R A E G Q
D P W K N A T Q S L Q C W K Y F S D G H C D S Q C N S A G A L F D G F D C Q
Figure 1: The 3-D structure of LNRB in
relationship to LNRA and the α- helix
of the heterodimerization (HD) domain.
The light purple structure on the left is
LNRB and the greenish - yellow
structure on the right is LNRA. The αhelix of the HD domain is shown in the
bottom center in red. W52, which is
mutated to an alanine in some constructs,
is shown in cyan blue. W42, which is
located in the LNR-AB linker, is shown in
navy blue. Located in LNRA is W29,
which is shown in violet. The Ca2+ ion is
shown as a blue sphere.
Distance to Ca2+ (Å)
N/A
L N F N D P W K N C T Q S L Q C A K Y F S D G H C D S Q C N S A G C L F D G F D C Q R A E G Q
Mut 1,5 CA_Mut W52A52 LNRB_Original
Residue
• Future Direction
• testing the four LNRB_intermediate constructs to see whether the same effects of eliminating two
cysteines and mutating W52 to an alanine on folding will be observed
• optimizing the conditions in which to refold and analyze the LNRB_short constructs in order to obtain more
conclusive results
Reference
Gordon, W.R., Vardar-Ulu, D., Histen, G., Sanchez-Irizarry, C., Aster, J.C., Blacklow, S.C. (2007)
Structural basis for autoinhibition of notch. Nature Structural &Molecular Biology.
Figure 4: Comparison of the folding patterns for the four “original” constructs at 1.5 hours. The four different
constructs of LNRB_original were refolded for 1.5 hours. The two constructs with only four cysteines (mut 1,5
CA_original and mut 1,5 CA mut W52A52 original) both showed three major folded conformations after 1.5 hours
whereas the wild type and the mut W52A52_original showed one main folded conformation after 1.5 hours. However,
the refolded mut W52A52_original displayed additional conformations similar to the ones shown in Figure 3.
Acknowledgments
Dr. Didem Vardar-Ulu
Other members of the lab: Sharline Madera ’08, Christina Hao ’09, Lauren Choi ’10
Roberta Day Staley and Karl A. Staley Fund for Cancer-Related Research