1. Characterization of protein folding determinants for LIN-12/Notch-Repeats (LNRs) using Human Notch1 LNRB as a model system Sharline Madera Advisor: Dr. Didem Vardar-Ulu Wellesley College

2. 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

3. Domain Architecture of Notch Have 3 LNRs in Negative Regulatory Region (NRR) –maintain protein in resting position Intracellualr Notch Cell LNR Domain B A EGF C HD Domain NRR S1S2S3 Gordon, W. R. et al. (2007) Nature.

4. Characteristics of an LNR Structurally independent Small Disulfide Rich Folds –6 cysteines out of 32-36 total –Require Ca 2+ 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)

5. 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 BAC 1:52:4 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

6. Defining LNRB Recall LNRs: Tandem C A CC B CC C C SLNFNDPWKN Linker_AB 10 conserved residues Linker_BC 6 residues QRAEGQ What Next?

7. C A CC B CC C C SLNFNDPWKNQRAEGQ 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_ABLinker_BC LNRALNRBLNRC

8. Protein Production All constructs expressed as inclusion bodies using BL21(DE3) PlysS E. coli cell line system C A CC B CC C C S LNFNDPWKNLFDGFD START END pMML Vector Sequence GROW Cleave & Separate Met

9. Protein Folding Dialyze: Refolding Buffer Cysteine Cystine cysteine & cystine –Important for folding –Provide proper environment –LNRA 5:1 ratio red:ox LNRB 10mM CaCl 2 100mM NaCl 20mM Tris pH8

10. 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

11. LNRB_orig

12. Folding Verification Reverse Phase HPLC –C18 column –RP chromatography: Separates on hydrophobicity Run sample on RP-HPLC –Incubate in DTT for ~2hrs

13. LNRB_orig Post 2hr DTT Incubation

14. 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 DTT26 DTT28 Calc. MWMass Spec MW 5368.85368.7

15. LNRB_orig, LNRB_delBC & LNRB_int fold Post DTT incubation LNRB_orig LNRB_int LNRB_delBC

16. 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

17. Optimize Folding Cysteine/Cystine ratio –redox potential Eukaryotic Protein Disulfide Isomerase (PDI) –endoplasmic reticulum –strong redox potential: -110mV 5:1 cysteine/cystine:-4.5mV

18. Experimental Redox Ratios Cysteine/Cystine RatioRedox Potential (mV) 30:1-51.5 15:1-35.6 10:1-31.6 5:1-4.5 2:137.2

19. Redox Chromatograms 2:1 grey 5:1 orange 10:1 black 15:1 red 30:1 purple 5:1 Best

20. 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

21. Future Directions Test relevance of 3 disulfide bonds to folding –LNRB variants containing differing number of disulfide bonds. Metal specificity of LNR folding –Mg 2+, Mn 2+ & Zn 2+ Metal affinity –Isothermal Calorimetry Ca 2+, Mg 2+, Mn 2+ & Zn 2+

22. 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

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24. Metal Specificity: LNRB_orig CaCl 2 ZnCl 2

25. Metal Affinity: LNRB_int 10 mMCaCl 2 1 mMCaCl 2

26. Table 1. Construct Sequences 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 Ca 2+ ions Orange Residues: Disulfide forming cysteines 2:41:53:6 ABC LNR Domain HD Domain

27. 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

28. Notch Proteins Have 3 LNRs in Negative Regulatory Region (NRR) – maintain protein in resting position