1. --0 hr --1 hr --2 hr --5hr two folded species reduced Material and Methods Protein Expression and Purification: The pMML-LNRA vector contains the wild-type hN1 LNRA gene fused to modified gene that codes for the TrpLE sequence in which the Met and Cys residues have been replaced by Leu and Ala, respectively. The mutant plasmids for hN1LNRA_CG and hN1LNRA_CS were made from the kanamyacin resistant pMML vector for hN1LNRA_WT via QuikChange© Site Directed Mutagenesis (Strategene) and verified via DNA sequencing. Both wildtype and mutant plasmids were transformed into BL21DE3PlysS E.Coli cell line (chloramphenicol resistant) hN1LNRA_WT and hN1LNRA_CG were expressed by growing transformed cells to A 600 ~0.6 in Luria Broth Miller with kanamyacin (50  g/ml) and chloramphenicol (34  g/ml). at 37 °C in a shaking incubator at 220 rpm and inducing protein production with 0.25 mM IPTG. The proteins were purified from inclusion bodies through successive resuspension/ centrifugation steps and cleaved by cyanogen bromide to obtain the protein of interest. A reduced form of the target protein was purified via reversed-phase HPLC, and lyophilized. The identity of the constructs were confirmed using MALDI-TOF mass spectrometry. Reduction Potential Refolding Assays: Lyophilized protein was resuspended in water to a concentration of approximately 225 μM. 15 μof the protein stock solution in a rapid dilution refolding buffer of Refolding results analyzed using an analytical HPLC (C18 column) running a gradient of.25% a minute from 12-20% Buffer B (Buffer A: 90% H 2 0, 10% Acetonitrile, 0.1% TFA; Buffer B: 10% H 2 0, 90% Acetonitrile, 0.1% TFA) CHARACTERIZATION OF LIN-12/NOTCH REPEATS (LNRs) USING HUMAN NOTCH 1 LNRA AS A MODEL SYSTEM Lauren Choi, Wellesley College Advisor: Dr. Didem Vardar-Ulu Results 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)Dokmanić, I., M. Šikić, and S. Tomić. 2008. Metals in proteins. Acta Crystallogr. D. 64:257-263. 4)DeLano, WL. The PyMOL Molecular Graphics System (2002) http://www.pymol.org LNRA_WT Folding is Faster Under Vacuum Conditions Conclusions During the refolding process, LNRA has folding intermediates which can be captured through acidification and resolved using an analytical reverse-phase liquid chromatograph. The folding of LNRA Wild Type is faster under vacuum conditions which correlates with the slower oxidation of cysteine/cystine in the refolding buffer. The Ca 2+ binding site of hN1LNRA_WT has seven electron donors and coordination geometry of a pentagonal bipyrimid characteristic of most Ca 2+ binding proteins (3). hN1LNRA folding is favored in a more oxidizing environment. The Folding of hN1LNRA_WT and hN1LNRA_CG is a Dynamic Process Figure 2. Chromatograms of hN1LNRA_WT (left) and hN1LNRA_CG (right) captured at different time intervals of rapid dilution refolding: in its reduced form (black), at 1 hour (green), 2 hours (blue) and 5 hours (red). For either of these constructs, folded polypeptides will elute earlier due to the surface inaccessibility of hydrophobic residues. For the wild type, the folding process forms transient intermediates which are slowly converted into a single properly folded conformation via the disulfide shuffling caused by cysteine : cystine in the refolding buffer solution. For the mutant hN1LNRA_CG note that there are two favored folded conformations which form quickly after rapid dilution in refolding buffer. Figure 4. Chromatograms of hN1LNRA_WT folding after 3 hours in 5:1 cysteine: cystine under vacuum and air exposed conditions. Anaerobic conditions expedite the folding process significantly. At three hours, samples under vacuum show primarily folded species while air exposed samples show primarily reduced species. The table on the right shows the time dependence of the reduction potential of the refolding buffer solutions without protein. All values are given in reference to the platinum redox electrode (244mV). The oxidation of the refolding buffer is slowed under vacuum. Introduction Notch receptors are multi-domain trans-membrane proteins that are important for cell-cell communication and development. Deregulated Notch signaling has been linked to many human diseases such as sclerosis, artereopathy and leukemia. The extra-cellular domain of the Notch Receptor contains the Ligand Binding Domain and the Negative Regulatory Region (NRR) which includes three copies of a Lin-12/Notch Repeat (LNR), a small disulfide-rich sequence of 35 residues. In wild-type Human Notch 1, proper folding of LNR requires the coordination of Ca2+ and the formation of three specific disulfide bonds. It has been previously shown that the first LNR from human Notch1, LNRA, can autonomously fold in vitro using a refolding buffer that contains Ca2+ and a certain reduction potential. In this study, we explored the effect of different reduction potentials on the folding of wild-type LNRA. We also designed mutant forms of LNRA in which one pair of cysteine residues was eliminated, and compared their time dependent in vitro folding pattern with the wild-type’s under varying reduction potentials. In order to characterize the Ca2+ binding site of LNRA, we carried out an in depth analysis of the Ca2+ coordination geometry in LNRA and compared it with Ca2+ coordination geometries observed in other Ca2+ binding proteins. The results of this study will help characterize LNR folding and elucidate the mechanism of disulfide bond formation during the protein folding process. Figure 1. LNR Contruct and Organization. (A) The Domain Organization of the Notch receptor. The negative regulatory region (NRR) is circled. (B) The crystal structure of NRR from Human Notch 2. Note that the LNR modules surround the S2 cleavage site in the resting conformation (1). (C) The NMR solution structure of LNRA from Human Notch 1. The three disulfide bonds are highlighted in orange and Ca2+ coordinating residues are marked in red (aspartate) and green (asparagine) (2). The disulfide bond formed between the cysteine residues at position 4 and 27 are being removed in our mutant strains. (D) The sequence construct of LNRA_WT, LNRA_CG and LNRA_CS and mutant from Human notch 1. The mutated cysteine residues are highlighted in orange. Ratio of Cysteine : Cystine Effective Concentration 5:12.5mM : 0.5mM 12:13.0mM : 0.25mM 1:30.5mM : 1.5mM 50mM Tris pH 8.0 100 mM NaCl 10mM CaCl 2 + plus one of the following: Electron Donors ResidueFunctional groupDistance (Ă)Angle Asp33Carboxylate2.50 159.47 Asp12Carboxylate2.41 Asp12Backbone carbonyl2.6667.75 Asn15Side chain carbonyl2.4988.37 Val17Backbone carbonyl2.0082.96 Asp30Carboxylate_a2.1848.55 Asp30Carboxylate_b2.7871.98 Future Directions Characterize the other mutant LNRA_CS by comparing its time dependent in vitro folding pattern with the wild type’s under varying reduction potentials. Mutate the electron donating residues in the Ca 2+ binding site to change the binding affinity of Ca 2+ and ion selectivity of the binding site. Perform the folding experiments under complete vacuum conditions where no oxidation occurs. hN1LNRA_CG Mutant Folding Over Time Figure 3. Chromatograms of hN1LNRA_WT folded in 1:3, 5:1 and 12:1 cysteine: cystine after one hour. The difference in proportions of cysteine: cystine effectively changes the reduction potential of the refolding buffer solution (Table in Fig. 4). At one hour, protein folding in 12:1 cysteine:cystine has little progress with the majority of the species still in the reduced form. 1:3 cysteine: cystine has had the most progress, as most of its peaks are correctly folded. Folding in 5:1 cysteine: cystine has proceeded somewhere in between the other two concentrations with about the same amount of the species in the reduced and folded forms. --vacuum --no vacuum --0 hr --1 hr --2 hr --5 hr reduced intermediates folded species hN1LNRA Wild Type Folding Over Time Quantified Folding of hN1LNRA Wild Type Table 1. Quantified folding of hN1LNRA_wt showing the percent reduced, misfolded and folded at each hourly interval after initial rapid dilution in refolding buffer dilution. Chromatograms of hN1LNRA_WT folded in 1:3, 5:1 and 12:1 cysteine: cystine were processed. The percent of each species was determined by integration of corresponding peaks of the reverse- phase high performance liquid chromatograms. The reduced species elutes at 12.5% Buffer B, and fully folded species elute at 10% Buffer B. Misfolded species are composed of peaks which elute between 11-12% Buffer B. The table shows that the progression of folding under more oxidizing conditions (1:3) is faster than under more reducing conditions (12:1) hN1LNRA_wt Folding Under Different Reduction Potentials reduced folded species intermediates --reduced --1:3 --5:1 --12:1 Reduction Potentials of Refolding Buffer (mV) Time No VacuumVacuum 1:35:112:11:35:112:1Cystein e Cystine 0hr-170-233-241-170-233-241-258-120 1hr-169-226-230-169-231-238-250-111 3hr-160-217-223-166-229-231-244-99 5hr-153-212-221-167-227-230 7hr-153-210-218-162-226-224 20hr-97-213-219-96-214-220-224-87 A. B. Acknowledgements Research Supported by the Sherman Fairchild Foundation Summer Research Award Dr. Didem Vardar Ulu, Jessica Lin, Christina Hao Ca 2+ Coordination Geometry Figure 5. Geometry of the Ca 2+ binding site of hN1LNRA_WT. (a) Pymol(4) analysis of the crystal structure of LNRA from hN1NRR reveals the identity of seven electron donating atoms within 3 Ă of the Ca 2+ atom. Electron donors are either carbonyls in the side chain or backbone of the protein, or the carboxylate in the side chain of an acidic residue, such as aspartate. If the electron donor is a carboxylate, one or both of the oxygen atoms can participate in Ca 2+ coordination, monodentate or bidentate, respectively. These seven atoms are arranged in a pentagonal bipyramid (b) which has two axial donors and five equatorial donors. The table (right) shows the distance between the electron donors and the Ca 2+ atom, as well as the angles between significant atoms at the Ca 2+ atom. time0hr1hr2hr4hr5hr 1:3 % reduced100 14.14.72.02.9 % misfolded0 34.225.510.32.2 % folded0 51.769.987.794.9 5:1 % reduced100 39.211.35.43.8 % misfolded0 38.155.680.312.9 % folded0 22.733.114.383.3 12:1 % reduced100 85.576.654.616.0 % misfolded0 10.218.229.963.4 % folded0 4.35.215.620.6