-0 .5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 -8 -6 -4 -2 0 2 -2 -1 0 0 40 80 120 160 200 240 280 320 360 400 T im e (m in) µ cal/sec M ola r R atio kcal/m ole of injectant -0 .5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 -8 -6 -4 -2 0 -2 .0 -1 .5 -1 .0 -0 .5 0.0 0 125 250 375 500 625 T im e (m in) 甥al/sec M olar R atio kcal/m ole ofinjectant -0 .5 0.0 0.5 1.0 1.5 2.0 2.5 3 .0 3.5 4.0 4.5 5.0 0 2 0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 1 .2 0 40 80 120 160 200 240 280 T im e (m in) µ cal/sec M olar R atio kca l/m o le of inje cta nt Tb 3+ Zn 2+ Ca 2+ Metal Ion Selectivity and Affinity of the LIN- Metal Ion Selectivity and Affinity of the LIN- 12/Notch-Repeat 12/Notch-Repeat Christina Hao, Advisor: Didem Vardar-Ulu Wellesley College, Chemistry Department Introduction • Notch receptors are large transmembrane glycoproteins that regulate cell growth, diff erentiation and death in multicellular organisms via a highly conserved signaling pathw ay (Figure A). • Dysregulation of notch signaling pathway in all four identified notch homologs (Notch 1 – Notch4) has been implicated in numerous disease phenotypes. •Three conserved Lin12/Notch Repeat (LNRA, LNRB, and LNRC) modules of about 35 residues each are located in tandem in the extracellular region of the notch receptors. They de corate the heterodimerization (HD) domain of the receptor and conceal the activating cl eavage site in the absence of a ligand. Therefore, they are responsible for maintainin g the receptor in a resting conformation prior to ligand-induced activation. (Figure B). •Each LNR module exhibit highly conserved architecture consisting of three characterist ic disulfide bonds and a group of aspartate/asparagine residue that coordate a Ca2+ ion, which are essential for the correct folding of the module. (Figure C) Objectives • To obtain a molecular understanding for the metal binding affinity and specificity of the LNRs through the determination of thermodynamic parameters associated with the bind ing of different metals to human Notch1 LNRA (hN1LNRA). For this aim we performed calo rimetric titrations of Ca 2+, Zn 2+ , Tb 3+ into hN1LNRA with using isothermal titration calor imetry (ITC). • To test if the binding affinity and specificity of these repeats can be altered throu gh a single amino acid replacement in the Ca 2+ binding pocket of the wild-type hN1LNRA. For this aim we designed a mutant form of hN1LNRA where serine in position 19 is repla ced by an aspartate that is part of the Ca 2+ coordination site in most other LNRs (Figur e D). Material and Methods Protein Expression and Purification: •Wildtype hN1LNRA was expressed in E.coli. as a fusion protein with a modified form of t he trpLE sequence in which the methionine and cysteine residues have been replaced by leucine and alanine ,respectively using the pMML vector (kind gift of S. Blacklow, BW H). The plasmid for hN1LNRA_mt was obtained from the pMML vector using the QuikChang e Site-Directed Mutagenesis protocol (Stratagene) The expressed fusion proteins were p urified from inclusion bodies and cleaved by cyanogen bromide to obtain hN1LNRA or hN 1LNRA_mt. •The protein was refolded in vitro through successive dialysis against a redox buffer (50 mM Tris pH 8.0, 150 mM NaCl, 10 mM CaCl 2 , 2 mM cysteine, 0.5 mM cystine), purified via reversed-phase HPLC, and lyophilized. The identity of the constructs were confirmed u sing MALDI-TOF mass spectrometry. Isothermal titration calorimetry (ITC) Experiments: • Lyophilized protein was solubilized in water at concentration of approximately 0.1mM and then extensively dialyzed against, 35 mM HEPES pH 7, 100 mM NaCl buffer. • To ensure protein samples were completely metal-free, prewashed chelex beads (Sigma chelex 100, were incubated with the sample after dialysis for to remove residual metal s. • Final protein concentration of the sample was determined based on UV absorbance of the sample at 280 nm using a corrected extinction coefficient. •Small aliquot of buffer used to dialyze the protein sample was also chelexed and used to prepare stock metal solution of 1 or 2mM CaCl 2 , Zn(CH3COO) 2 *H2O, and TbCl 3 . •Isothermal titration calorimetry experiments (ITC), were carried out using a high-pre cision 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. •Control experiments of metal solutions titrated into protein free buffer solutions we re performed to correct for the heat of dilution. •Experiments were repeated to ensure reproducibility. •The dissociation constant, the binding stoichiometry, the enthalpy of binding, and th e calculated entropy were determined based on a one-site fitting model using the Origi Results Conclusions • Ca 2+ exhibits exothermic binding to wildtype Human Notch1 LNRA (hN1L NRA) with a dissociation constant of 22.05 +/- 3.27 µM and a stoichio metry of 1:1 at pH 7.0. • No quantifiable binding is observed for hN1LNRA with zinc at pH 7.0. However, preliminary displacement experiments indicate that Ca 2+ b inding affinity of hN1LNRA is slightly decreased after it has been pr e-saturated with Zn 2+. This finding may imply some very weak binding of Zn 2+ to the Ca 2+ site or an indirect effect of nonspecific binding of Z n 2+ on the native conformation of hN1LNRA altering the molecular detai ls of the Ca 2+ binding site. • Although Tb 3+ clearly exhibits endothermic binding to (hN1LNRA), pre liminary results indicate that a single binding site model does not f it the experimental data. However, displacement experiments indicate that Ca 2+ binding affinity of hN1LNRA is decreased dramatically after it has been pre-saturated with Tb 3+ . Taken together, these data sugge st strongly that Tb 3+ most likely binds to the Ca 2+ coordination site a s well as to other specific or non-specific sites on the protein. • Preliminary data suggest mutant human Notch 1 LNR_A where a serine in position 19 is replaced by an aspartate (nN1LNRA_mt) binds to calc ium approximated 2.5 fold more tightly than the wildtype counterpart. However, the stoichiometry of binding is drastically altered. Zn 2+ a nd Tb 3+ binding to hN1LNRA_mt follows the same trends as the wild-type protein 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.; Bl acklow, S. C. “NMR Structure of a Prototype LNR Module from Human Notch1” Biochemistry 2003, 42, 7061–7067. Future Directions • Repeat the preliminary results on hNLNRA_mt to determine reproducib le thermodynamic profile for Ca 2+ binding. • Determine a reliable model to quantify Tb 3+ binding to hN1LNRA. • Test additional metals on both wild-type and mutant hN1LNRA. • Design other mutants that alter binding specificity of hN1LNRA. • Conduct additional ITC experiments to test the effect of pH and tem perature on the binding affinity of the different metals to both the wild-type and mutant hN1LNRA. Representative ITC data on the calorimetric titrations of hN1LNRA with Ca 2+ ,Zn 2+ ,Tb 3+ Ca 2+ N15 D33 S19 D30 N-term C-term Ca 2+ N-term C-term D30 D33 N15 D19 Ca 2+ coordination in hN1LNRA based on the NMR structure 2 Ca 2+ coordination in hN1LNRA_mt modeled on the hN1LNRA structure N K d (µM) H(kcal/mol) S (kcal/mol) hN1LNRA 0.9600.005 22.053.27 -9.140.25 -9.870.73 hN1LNRA_mt 0.0940.039 9.374.99 -36.446.83 -100.823.96 Tb 3+ Zn 2+ Ca 2+ Representative ITC data on the calorimetric titrations of hN1LNRA_mt with Ca 2+ ,Zn 2+ ,Tb 3+ Summary of thermodynamic parameters associated with the binding of Ca 2+ to hN1LNRA and hN1LNRA_mt Representative ITC data on the calorimetric titrations of hN1LNRA pre-saturated with Zn 2+ or Tb 3+ with Ca 2+ -0 .5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 -1 .6 -1 .4 -1 .2 -1 .0 -0 .8 -0 .6 -0 .7 -0 .6 -0 .5 -0 .4 -0 .3 -0 .2 -0 .1 0.0 0.1 0 40 80 120 160 200 T im e (m in) 甥al/sec M o lar R atio kcal/m ole ofinjectant -0.50.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 -6 -4 -2 0 -1.6 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0 40 80 120 160 200 240 280 D ata:w tlnCAintoZ_N D H M odel:OneSites C hi^2/D oF = 1071 N 0.659 ±0.0316 K 1.36E4 ±446 H -1.282E 4 ±695.1 S -24.8 T im e (m in) µ cal/se c M o lar R atio kca l/m ole of injectant N K d (µM) H(kcal/mol) S (kcal/mol) hN1LNRA presat with Zn2+ 0.660.03 73.532.24 -12.820.70 -24.8 hN1LNRA presat with Tb3+ Too weak to quantify Summary of thermodynamic parameters associated with the binding of Ca 2+ to Zn 2+ and Tb 3+ presaturated hN1LNRA Domain Organization of the Notch Receptors and the Notch Signaling Pathway Figure A Crystal Structure of LNR and HD Domain of human Notch2 1 Figure B N- term C-term Ca 2+ N15 D33 S19 C22 C18 C9 C27 C4 D30 C34 NMR Structure of hN1LNRA 2 Figure C Green: wildtype human Notch1 LNRA Blue: mutant human Notch 1 LNR A Brown: LNRA from other human Notch hom ologs Orange: Cysteines (disulfide bonding p attern indicated on the top) Highlighted in yellow: Ca 2+ -coordinati ng residues Sequence Alignment of LNRAs from human Notch homologs: hN1LNRA : EEAC--ELPECQ-EDAGNKVCSLQCNNHACGWDGGDC hN1LNRA_mt: EEAC--ELPECQ-EDAGNKVCDLQCNNHACGWDGGDC hN2LNRA : PATC--LSQYCA-DKARDGVCDEACNSHACQWDGGDC hN3LNRA : P-RC--PRAACQ-AKRGDQRCDRECNSPGCGWDGGDC hN4LNRA : P-----GAKGCE-GRSGDGACDAGCSGPGGNWDGGDC 1 35 19
Tb 3 + Zn 2+ Ca 2+ Metal Ion Selectivity and Affinity of the LIN-12/Notch-Repeat Christina Hao, Advisor: Didem Vardar-Ulu Wellesley College, Chemistry.
Jan 11, 2016
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Metal Ion Selectivity and Affinity of the LIN-12/Notch-RepeatMetal Ion Selectivity and Affinity of the LIN-12/Notch-RepeatChristina Hao, Advisor: Didem Vardar-Ulu
Wellesley College, Chemistry Department
Introduction• Notch receptors are large transmembrane glycoproteins that regulate cell growth, differentiation and death in multicellular organisms via a highly conserved signaling pathway (Figure A).
• Dysregulation of notch signaling pathway in all four identified notch homologs (Notch1 – Notch4) has been implicated in numerous disease phenotypes.
•Three conserved Lin12/Notch Repeat (LNRA, LNRB, and LNRC) modules of about 35 residues each are located in tandem in the extracellular region of the notch receptors. They decorate the heterodimerization (HD) domain of the receptor and conceal the activating cleavage site in the absence of a ligand. Therefore, they are responsible for maintaining the receptor in a resting conformation prior to ligand-induced activation. (Figure B).
•Each LNR module exhibit highly conserved architecture consisting of three characteristic disulfide bonds and a group of aspartate/asparagine residue that coordate a Ca2+ ion, which are essential for the correct folding of the module. (Figure C)
Objectives• To obtain a molecular understanding for the metal binding affinity and specificity of the LNRs through the determination of thermodynamic parameters associated with the binding of different metals to human Notch1 LNRA (hN1LNRA). For this aim we performed calorimetric titrations of Ca2+, Zn2+, Tb3+ into hN1LNRA with using isothermal titration calorimetry (ITC).
• To test if the binding affinity and specificity of these repeats can be altered through a single amino acid replacement in the Ca 2+ binding pocket of the wild-type hN1LNRA. For this aim we designed a mutant form of hN1LNRA where serine in position 19 is replaced by an aspartate that is part of the Ca2+ coordination site in most other LNRs (Figure D).
Material and MethodsProtein Expression and Purification:•Wildtype hN1LNRA was expressed in E.coli. as a fusion protein with a modified form of the trpLE sequence in which the methionine and cysteine residues have been replaced by leucine and alanine ,respectively using the pMML vector (kind gift of S. Blacklow, BWH). The plasmid for hN1LNRA_mt was obtained from the pMML vector using the QuikChange Site-Directed Mutagenesis protocol (Stratagene) The expressed fusion proteins were purified from inclusion bodies and cleaved by cyanogen bromide to obtain hN1LNRA or hN1LNRA_mt.
•The protein was refolded in vitro through successive dialysis against a redox buffer (50 mM Tris pH 8.0, 150 mM NaCl, 10 mM CaCl2, 2 mM cysteine, 0.5 mM cystine), purified via reversed-phase HPLC, and lyophilized. The identity of the constructs were confirmed using MALDI-TOF mass spectrometry.
Isothermal titration calorimetry (ITC) Experiments:• Lyophilized protein was solubilized in water at concentration of approximately 0.1mM and then extensively dialyzed against, 35 mM HEPES pH 7, 100 mM NaCl buffer.
• To ensure protein samples were completely metal-free, prewashed chelex beads (Sigma chelex 100, were incubated with the sample after dialysis for to remove residual metals.
• Final protein concentration of the sample was determined based on UV absorbance of the sample at 280 nm using a corrected extinction coefficient.
•Small aliquot of buffer used to dialyze the protein sample was also chelexed and used to prepare stock metal solution of 1 or 2mM CaCl2, Zn(CH3COO)2*H2O, and TbCl3.
•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.
•Control experiments of metal solutions titrated into protein free buffer solutions were performed to correct for the heat of dilution.
•Experiments were repeated to ensure reproducibility.
•The dissociation constant, the binding stoichiometry, the enthalpy of binding, and the calculated entropy were determined based on a one-site fitting model using the Origin software, version 7.0. after correcting for heat of dilution.
Results
Conclusions • Ca2+ exhibits exothermic binding to wildtype Human Notch1 LNRA (hN1LNRA) with a dissociation constant of 22.05 +/- 3.27 µM and a stoichiometry of 1:1 at pH 7.0.
• No quantifiable binding is observed for hN1LNRA with zinc at pH 7.0. However, preliminary displacement experiments indicate that Ca2+ binding affinity of hN1LNRA is slightly decreased after it has been pre-saturated with Zn2+. This finding may imply some very weak binding of Zn2+ to the Ca2+ site or an indirect effect of nonspecific binding of Zn2+ on the native conformation of hN1LNRA altering the molecular details of the Ca2+ binding site.
• Although Tb3+ clearly exhibits endothermic binding to (hN1LNRA), preliminary results indicate that a single binding site model does not fit the experimental data. However, displacement experiments indicate that Ca2+ binding affinity of hN1LNRA is decreased dramatically after it has been pre-saturated with Tb3+. Taken together, these data suggest strongly that Tb3+ most likely binds to the Ca2+ coordination site as well as to other specific or non-specific sites on the protein.
• Preliminary data suggest mutant human Notch 1 LNR_A where a serine in position 19 is replaced by an aspartate (nN1LNRA_mt) binds to calcium approximated 2.5 fold more tightly than the wildtype counterpart. However, the stoichiometry of binding is drastically altered. Zn2+ and Tb3+ binding to hN1LNRA_mt follows the same trends as the wild-type protein
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.
Future Directions • Repeat the preliminary results on hNLNRA_mt to determine reproducible thermodynamic profile for Ca2+ binding.
• Determine a reliable model to quantify Tb3+ binding to hN1LNRA.
• Test additional metals on both wild-type and mutant hN1LNRA.
• Design other mutants that alter binding specificity of hN1LNRA. • Conduct additional ITC experiments to test the effect of pH and temperature on the binding affinity of the different metals to both the wild-type and mutant hN1LNRA.
Representative ITC data on the calorimetric titrations of hN1LNRA with Ca2+,Zn2+ ,Tb3+
Ca2+
N15
D33
S19
D30
N-term
C-term
Ca2+
N-term
C-term
D30
D33
N15
D19
Ca2+ coordination in hN1LNRA based on the NMR structure2
Ca2+ coordination in hN1LNRA_mt modeled on the hN1LNRA structure
N Kd (µM) H(kcal/mol) S (kcal/mol)
hN1LNRA 0.9600.005 22.053.27 -9.140.25 -9.870.73
hN1LNRA_mt 0.0940.039 9.374.99 -36.446.83 -100.823.96
Tb3+Zn2+
Ca2+
Representative ITC data on the calorimetric titrations of hN1LNRA_mt with Ca2+,Zn2+ ,Tb3+
Summary of thermodynamic parameters associated with the binding of Ca2+ to hN1LNRA and hN1LNRA_mt
Representative ITC data on the calorimetric titrations of hN1LNRA pre-saturated with Zn2+ or Tb3+ with Ca2+
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Data: wtlnCAintoZ_NDHModel: OneSitesChi^2/DoF = 1071N 0.659 ±0.0316K 1.36E4 ±446H -1.282E4 ±695.1S -24.8
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hN1LNRA presat with Zn2+
0.660.03 73.532.24 -12.820.70 -24.8
hN1LNRA presat with Tb3+
Too weak to quantify
Summary of thermodynamic parameters associated with the binding of Ca2+ to Zn2+ and Tb3+ presaturated hN1LNRA
Domain Organization of the Notch Receptors and the Notch Signaling Pathway
Figure A
Crystal Structure of LNR and HD Domain of human Notch21
Figure B
N-term
C-term
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N15
D33
S19
C22
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C9
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NMR Structure of hN1LNRA2
Figure C
Green: wildtype human Notch1 LNRABlue: mutant human Notch 1 LNR A Brown: LNRA from other human Notch homologs Orange: Cysteines (disulfide bonding pattern indicated on the top) Highlighted in yellow: Ca 2+-coordinating residues
Sequence Alignment of LNRAs from human Notch homologs:
hN1LNRA : EEAC--ELPECQ-EDAGNKVCSLQCNNHACGWDGGDC hN1LNRA_mt: EEAC--ELPECQ-EDAGNKVCDLQCNNHACGWDGGDC hN2LNRA : PATC--LSQYCA-DKARDGVCDEACNSHACQWDGGDC hN3LNRA : P-RC--PRAACQ-AKRGDQRCDRECNSPGCGWDGGDC hN4LNRA : P-----GAKGCE-GRSGDGACDAGCSGPGGNWDGGDC
1 3519
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