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Characterization of protein folding Characterization of protein folding determinants for LIN-12/Notch-Repeats determinants for LIN-12/Notch-Repeats (LNRs) using Human Notch1 LNRB as (LNRs) using Human Notch1 LNRB as
a model systema model system
Sharline Madera
Advisor: Dr. Didem Vardar-Ulu
Wellesley College
NOTCH PROTEINS• Transmembrane receptor protein
• Found in many different animals– from worms to humans.
– mammals have four Notch homologs: Notch1-4
• Function through highly conserved Notch signaling pathway
– 3 cleavages at sites: S1, S2 & S3• S1: Processed on way to cell surface• S2, S3: Upon ligand binding
Domain Architecture of Notch• Have 3 LNRs in Negative Regulatory Region (NRR)
– maintain protein in resting position
Intracellualr Notch
Cell
LNR Domain
BA
EGF
C HD Domain
NRR
S1 S2 S3
Gordon, W. R. et al. (2007) Nature.
Characteristics of an LNR• Structurally independent Small Disulfide Rich Folds
–6 cysteines out of 32-36 total
–Require Ca2+ for folding
• Currently defined based on hN1 LNRA E E A C E L P E C Q E D A G N K V C S L Q C N N H A C G W D G G D C S
Vardar, et.al (2003)
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
Target LNR: LNRB of hNotch 1
LNR Domain
BA C
1:5 2:4 3:6
HD Domain
• Not in literature• Important
– LNRs structurally independent– LNRB has key interactions with neighbors
• Folding of LNRB– LNR function: Maintain Notch Receptor at rest
• Goal: Define LNRB specifics & optimize folding– How?
• Define the minimum LNRB amino acid sequence that can achieve the correct fold autonomously
• Determine the optimum redox potential that ensures correct folding
Defining LNRB
• Recall LNRs: Tandem
C A C C B C C C CSLNFNDPWKN
Linker_AB 10 conserved
residues
Linker_BC 6 residues
QRAEGQ
What Next?
C A C C B C C C CSLNFNDPWKN QRAEGQ
LNRB_orig
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
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
LNRB_short
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
LNRB_delBC
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
LNRB_int
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
LNRB_delAB
5 LNRB Constructs
Linker_AB Linker_BC
LNRA LNRB LNRC
Protein Production• All constructs expressed as inclusion bodies using
BL21(DE3) PlysS E. coli cell line system
C A C C B C C C CS LNFNDPWKN LFDGFD
START END
pMML Vector
Sequence
GROW
Cleave & Separate
Met
Protein Folding
• Dialyze: Refolding Buffer
Cysteine
Cystine
cysteine & cystine
– Important for folding– Provide proper
environment– LNRA
5:1 ratio red:ox
LNRB
10mM CaCl2
100mM NaCl
20mM Tris pH8
Folding Verification
• Reverse Phase HPLC– C18 column– RP chromatography:
• Separates on hydrophobicity
• Run sample on RP-HPLC– Slow Gradient: 0.25% Buffer B/min
• Buffer A: 90% Water 10% Acetonitrile• Buffer B: 90% Acetonitrile 10% Water
Folding Verification• Reverse Phase HPLC
– C18 column– RP chromatography:
• Separates on hydrophobicity
• Run sample on RP-HPLC– Incubate in DTT for ~2hrs
Folding Verification• Reverse Phase HPLC
– C18 column– RP chromatography:
• Separates on hydrophobicity
• Run sample on RP-HPLC– Incubate in DTT for ~2hrs
– Expected Shift– Mass Spec.
Condition %Buffer B Elution
No DTT 26
DTT 28
Calc. MW Mass Spec MW
5368.8 5368.7
LNRB_delAB & LNRB_short do not fold
Post DTT incubation
LNRB_short
LNRB_delAB
• Conclusion: Minimum folding requirements• Structurally important residues located in linker _AB
Optimize Folding
• Cysteine/Cystine ratio– redox potential
• Eukaryotic Protein Disulfide Isomerase (PDI) – endoplasmic reticulum– strong redox potential: -110mV
• 5:1 cysteine/cystine:-4.5mV
Experimental Redox Ratios
Cysteine/Cystine Ratio Redox Potential (mV)
30:1 -51.5
15:1 -35.6
10:1 -31.6
5:1 -4.5
2:1 37.2
Conclusion
• Consensus of defining and predicting LNRs
– Linker_AB is important• Certain residues are crucial for folding
– Protein folding is sensitive to redox potential• Target potential:
– ~-4.5mV– Obtained 5:1 cysteine:cystine
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 L
Future Directions
• Test relevance of 3 disulfide bonds to folding – LNRB variants containing differing number of
disulfide bonds.
• Metal specificity of LNR folding– Mg2+, Mn2+ & Zn2+
• Metal affinity – Isothermal Calorimetry
• Ca2+, Mg2+, Mn2+ & Zn2+
Acknowledgements
• Vardar-Ulu Lab– Christina Hao– Ursela Siddiqui– Fathima F. Jahufar– Dr. Didem Vardar-Ulu
• NSF-REU
• Wellesley College Faculty Award
• Wellesley College– Chemistry
Department
• Protein Society Review Committee
• FASEB MARC Travel Award
• All of you
References1) Aster, J. et al. (1999) Biochemistry. 38: 4736-4742. 2) Brou, C. et al. (2000) Mol. Cell. 2: 207-216.3) Ellisen, L. W. et al. (1991) Cell. 66:649–661.4) Gordon, W. R. et al. (2007) Nature.5) Joutel, A. et al. (1996) Nature. 383: 707–710. 6) Kopan, R. et al. (2000) Genes Dev. 14: 2799-2806. 7) Lawrence, N. (2000) Development. 127: 3185-3195.8) Li, L. et al. (1997) Nat. Genet. 16: 243–251.9) Logeat, F. et al. (1998) Proc. Natl. Acad. Sci. USA. 95: 8108-8112.10)Oda, T. et al. (1997) Nat. Genet. 16: 235–242.11)Rand, M. et al. (2000) Molec. and Cell. Biol. 20: 1825-1835.12)Sanchez-Irizarry, C. et al. (2004) Molec. and Cell. Biol. 24: 9265-
9273.13)Vardar, D. et al. (2003) Biochemistry. 42: 7061-7067.14)Weng, A.P. et al. (2004) Science. 306: 269–271.
5 Constructs
LNRB_orig: 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
LNRB_short: 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
LNRB_delAB: 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
LNRB_delBC: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
LNRB_int: 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
Red Residues: Coordinate Ca2+ ions Orange Residues: Disulfide forming cysteines
2:41:5 3:6
A B C
LNR Domain
HD Domain
5 Constructs
LNRB_Orig: 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
LNRB_short: 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
LNRB_delAB: 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
LNRB_delBC: 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
LNRB_int: 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