Characterization of the Ca Binding Affinity and Coordination Site of the LIN-12/Notch-Repeat 2+ Christina Hao, Didem Vardar-Ulu Wellesley College Chemistry Department Wellesley, Massachusetts Introduction Results • Notch receptors are transmembrane glycoproteins that regulate cell fate in multicellular organisms via a highly conserved signaling pathway (Figure A). Ca2+ dependency during folding of hN1 LNRA, glucose transferase and hN4LNRA Representations of overall structures and calcium binding sites for (A) hN1LNRA (B) Glucose Transferase LNRA (C) hN4LNRA (A)NMR Structure. (B) and (C) homology Modeling • Deregulation of notch signaling pathway in all four identified notch homologues (Notch1 – Notch4) has been implicated in numerous disease phenotypes. A. hN1 LNRA • Three conserved Lin12/Notch Repeat (LNRA, LNRB, and LNRC) modules are located in tandem in the extracellular region of the notch receptors. They maintain the receptor in a resting conformation prior to ligand-induced activation. (Figure B). B. Glucose Transferase LNRA C. hN4 LNRA • Each LNR module in the Notch receptor consists of three characteristic disulfide bonds and a Ca 2+ ion, essential for structural integrity (Figure C). Domain Organization of the Notch Receptors and the Notch Signaling Pathway Crystal Structure of LNR and HD Domain of human Notch21 Figure B NMR Structure of hN1LNRA2 Figure C C 4 N15 C9 C27 Figure A Nterm Key: C22 D30 • Calcium binding sites (7 angstroms from the Ca2+): red and green ribbons Red sticks: Aspartates - Green sticks: other residues besides aspartates - Blue sphere: calcium ion Ca2+ C34 • Non-binding sites: silver ribbons D33 S19 C18 C-term Representative ITC data on the calorimetric titrations of hN1LNRA with Ca2+,Zn2+ ,Tb3+ Distances and distribution of coordinating residues from Ca 2+ Objectives • Quantify calcium binding affinity and specificity of LNR homologues across different proteins using ITC • Determine calcium dependency of different LNR homologues for autonomous folding • Understand the molecular basis of calcium binding in LNR using computer modeling Tb Zn2 Ca 3+ + 2+ Key: Red residues: residues that coordinate calcium with both side chain and backbone oxygen moiety Sequence Alignment of LNR homologues investigated in this study: Distances ranges highlighted yellow: aspartate is present in this distance range Conclusions • HN1 LNRA exclusively binds to Ca2+ in an exothermic reaction with a dissociation constant of 22.05 +/3.27 µM and a stoichiometry of 1:1 at pH 7.0. Representative ITC data on the calorimetric titrations of glucose transferase and hN4LNRA with Ca2+ Material and Methods Glucose transferase LNRA hN1 LNR A hN4 LNR A Protein Acquisition: • Human N1LNRA recombinantly expressed in Escherichia coli. Human Notch 4 LNRA and glucose transferase LNRA were synthesized by EZ Biolabs. • All proteins were folded and purified as follows: • Folding: 6-8 hours dialysis against refolding buffer: 2.5mM cysteine, 0.5mM cystine, 100mM NaCl, 200mM sucrose, 10mM CaCl2 and 20mM Tris pH 8. • Purification: Elution through reverse phase high pressure liquid chromatography (RPHPLC) using 0.1% formic acid in acetonitrile based buffer systems. Peaks corresponding to the folded species were collected and lyophilized. Folding Experiments: • Denatured proteins were refolded in redox solution containing 5:1 cysteine/cystine ratio, 100mM NaCl and 20mM Tris at pH 8 and with/without 10mM CaCl2. Proteins were folded under partial nitrogen atmosphere for six hours and promptly analyzed on RP-HPLC. Isothermal titration calorimetry (ITC) Experiments: • Lyophilized protein of appropriate concentration was demetalized with sigma chelex beads and suspended in 35mM Hepes pH7, 100mM NaCl buffer . • Stock metal solution of 0.2 – 1mM CaCl2 was used • Isothermal titration calorimetry experiments (ITC), were carried out using a high-precision VP-ITC titration calorimetry instrument (Microcal Inc., Northampton, MA) where the metal solution was titrated in 5µL increments into the protein solution at 20°C. Computer Modeling Software used: • Clustal W: sequence alignment • Modeller: homology modeling • Pymol: visualization • Glucose transferase LNRA display strong binding to calcium in a non-stoichiometric manner • HN4 LNRA does not bind to calcium • Calcium is necessary for the folding of HN1 LNRA and glucose transferase LNRA but not for HN4 LNRA • Homology modeling suggests differences in distribution of aspartic acids lead to distinct calcium binding behaviors of the LNR repeats. Future Directions • Determine precise roles of disulphide bonds and aspartic acids in calcium binding affinity mutational studies. through • Correlate calcium binding affinity and specificity with structural stability to gain insight into the biological significance of calcium binding by the LNRs in vivo. • Design of calcium binding peptides through de novo experiments based on understanding of the LNRs Summary of thermodynamic parameters associated with the binding of Ca2+ to Zn2+ and Tb3+ presaturated hN1LNRA Constructs hN1LNRA Metal Calcium N 0.9600.005 Kd (µM) 22.053.27 Zinc Calcium hN4LNRA Calcium -9.140.25 No binding Terbium GlucTran H (kcal/mol) Undefined mode of binding 0.07 9 -4.64 E4  1.36E4 No binding References 1) Gordon, W. R.;* Vardar-Ulu, D.;* Histen, G.; Sanchez-Irizarry, C.; Aster, J. C.; Blacklow, S. C. “Structural basis for autoinhibition of Notch” Nat Struct Mol Biol. 2007, 14, 295–300.2. 2) Vardar, D.; North, C. L.; Sanchez-Irizarry, C.; Aster, J. C.; Blacklow, S. C. “NMR Structure of a Prototype LNR Module from Human Notch1” Biochemistry 2003, 42, 7061–7067. 3) N. Eswar, M.A. Marti-Renom, b. Webb, m.S. Madhusudhan, D. Eramian, M. Shen, U. Pieper, A. Sali, Comparative Protein Structure Modeling with MODELLER. Current Protocols in Bioinformatics, John Wilery & Sons, Inc., Supplement 15, 5.6. 1-5.6.30, 2000 4) Cheng G, Baker D and Samudrala R. A Novel Small Molecule Crystal Structure Derived Potential Function To Predict Protein Metal Ion Binding Site, Affinity and Specificity From Structure. xxxx.YYYY,aa-bb,2007 http://protinfo.compbio.washington.edu/soak/
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Opposite and Adjacent 1. What side is the hypotenuse? x Note: The other two sides are called the “legs” 2. What angle is opposite the hypotenuse? 2 3. What is the measure of the angle  90 opposite the hypotenuse? 4. What leg is opposite angle 1? z 5. What leg is adjacent to angle 1? y 6. What leg is opposite angle 3? y 7. What leg is adjacent to angle 3? z y 1 x 2 z 3 8. Describe the relationship between the legs that are opposite and adjacent to angles 1 and 3. 3 The leg that is opposite angle 1 is adjacent to angle ____ 1 The leg that is opposite angle 3 is adjacent to angle ____
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What is Notch?  Transmembrane protein receptors of 300-350kDa  Highly conserved  Regulates cell growth, differentiation, and cell death in a vast array of tissues through Notch signaling pathway  Deregulation of Notch signaling pathway is associated wi th diseases, eg. Cancer  Four mammalian Notch homologs identified (Notch 1-4)
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6. Trajectory Generation 150 150 100 100 50  1 0  2  3 Cartesian Angle in  Joint Angle in  Cartesian Space TG: Joint Angle Time History 200 -50 50  1 0  2  3 -50   4 4  5 -100  5 -100   6 -150 0 0.2 0.4 0.6 0.8 1 1.2 Time in sec 1.4 1.6 1.8 6 -150 2 0 0.2 px -0.5 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 Normalized Units 1 0 kx -0.5 -1 0.4 0.6 0.8 1 1.2 1.4 0.8 1 1.2 Time in sec 1.4 1.6 1.8 2 0 px py -0.5 0 py 0.5 0.2 0.6 0.5 0.2 0.4 pz Joint Space TG: Axis of Rotation 0 Tool Position in m 0 1.6 1.8 ky 2 k z 0.6 0.8 1 1.2 1.4 1.6 1.8 2 Cartesian Space TG: Axis of Rotation Normalized Units Tool Position in m 0.5 0 0.4 Cartesian Space TG: Positional Time History Joint Space TG: Positional Time History 1 0.5 0 kx -0.5 1.6 1.8 ky 2k z 1.6 1.8 2 -1 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Cartesian Space TG: Angle of Rotation Joint Space TG: Angle of Rotation 200 Angle in  200 Angle in  Joint Space Trajectory Generation Joint Space TG: Joint Angle Time History 200 150 150 100 100 0 0.2 0.4 © 2011, Dr. Stephen Bruder 0.6 0.8 1 1.2 Time in sec 1.4 1.6 1.8 2 ME 482/582: Robotics Engineering 0 0.2 0.4 0.6 0.8 1 1.2 Time in sec 1.4 Thursday 25th Oct 2012 pz Cartesian Space Trajectory Generation 6.4 An Example: Joint vs Cartesian Slide 12 / 12
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Part I Problems:  Multiple choice  Using protractor measure angles.  Properly line up protractor to vertex of angle  Properly read measurement of angle on protractor  Mathematically, if an angle is 30 degrees, it is 60 degrees complementary to what angle?  Is the angle acute or obtuse?  If an angle is 70 degrees, it is supplementary to what angle?110 degrees  Is the angle acute or obtuse?
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UNCLASSIFIED Impact Angle Correlation 3-D Model Impact Angle Versus Composite Experiment Data Impact Angle 180 150 120 Regression Equation y = 0.7899x + 16.765 R 2 = 0.2099 Model Impact Angle (degree) 90 60 30 Impact Angle Composite Data Comparison 0 -180 -150 -120 -90 -60 -30 One to one correlation 0 30 60 -30 90 120 150 180 Linear Regression (Impact Angle) -60 -90 -120 -150 -180 Experiment Impact Angle (degrees) UNCLASSIFIED
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Q33.2 Light passes from vacuum (index of refraction n = 1) into water (n = 1.333). If the incident angle a is in the range 0° < a < 90°, A. the refracted angle is greater than the incident angle. B. the refracted angle is equal to the incident angle. C. the refracted angle is less than the incident angle. D. the answer depends on the specific value of a . © 2012 Pearson Education, Inc.
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A33.2 Light passes from vacuum (index of refraction n = 1) into water (n = 1.333). If the incident angle a is in the range 0° < a < 90°, A. the refracted angle is greater than the incident angle. B. the refracted angle is equal to the incident angle. C. the refracted angle is less than the incident angle. D. the answer depends on the specific value of a . © 2012 Pearson Education, Inc.
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