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

LNRB_orig

Folding Verification• Reverse Phase HPLC

– C18 column– RP chromatography:

• Separates on hydrophobicity

• Run sample on RP-HPLC– Incubate in DTT for ~2hrs

LNRB_orig Post 2hr DTT Incubation

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_orig, LNRB_delBC & LNRB_int fold

Post DTT incubation

LNRB_orig

LNRB_int

LNRB_delBC

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

Redox Chromatograms2:1 grey 5:1 orange 10:1 black 15:1 red 30:1 purple

5:1 Best

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.

Metal Specificity: LNRB_orig

CaCl2

ZnCl2

Metal Affinity: LNRB_int

10 mMCaCl2

1 mMCaCl2

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

Notch Proteins• Have 3 LNRs in Negative Regulatory Region (NRR)

– maintain protein in resting position