protein in the presence of EGTA, unless indicated otherwise. For investigating the effect of free Ca2+, the GST-C2B was buffered with 1mM EGTA and Ca2+ added to yield the indicated free Ca2+ concentrations using a pKd value of 10.995 for EGTA[51]. Statistical analysis was performed using Microsoft Excel. Signif- icant differences were determined using Student’s T-test on two populations of the same variance. P values of less than 0.05 were considered significant. Liposome pretreatment of GST-C2B Liposomes were prepared as described by Zhang et al. [52]. GST-C2B was preincubated with liposomes (2 mg/ml) for 30 min at room temperature in 20 mM HEPES containing 1 mM CaCl2 and then 45 min at 4uC. Composition of liposomes were prepared as described previously, consisted of 60%PC (phosphatidylcho- line), 20%PE (phosphatidylethanolamine) and either 20%PS (phosphatidylserine) or a further 20%PC [20]. The purified MBP-Stoned proteins were added and allowed to bind for 2 h at 4uC, followed by washing and analyzed as described above. Peptide competition experiments The peptide pC2B1 (Biotin-KICLVVTVVDYDRIGTSE) which contains the aspartate residues (D3,4) at loop 3 of the SYT-1-C2B domain was added at the same time as addition of MBP-tagged Stoned proteins to the GST-C2B domain as described above in the protein-protein interactions assay. Besides pC2B1, pC2B1.M (Biotin-KICLVVTVVDADRIGTSE) and p- CTRL (Biotin-SINPHLGPLQISLGR) were also tested. The binding assays were performed as described above in Ca2+ binding buffer. Supporting Information Figure S1 STNA and STNB proteins bind to the GST- C2B of SYT-1. The MBP-STNA and MBP-STNB bind significantly stronger to GST-C2B in comparison to GST C2A; 3and 4 fold respectively (n = 4, p,0.05). Independent experiments were done in the presence of 1 mM Ca2+. The bar graph References 1. Littleton JT, Stern M, Schulze K, Perin M, Bellen HJ (1993) Mutational analysis of Drosophila Synaptotagmin demonstrates its essential role in Ca2+-activated neurotransmitter release Cell 74: 1125–1134. 2. Nonet ML, Grundahl K, Meyer BJ, Rand JB (1993) Synaptic function is impaired but not eliminated in C. elegans mutants lacking synaptotagmin. Cell 73: 1291–1305. 3. DiAntonio A, Schwarz TL (1994) The effect on synaptic physiology of synaptotagmin mutations in Drosophila. Neuron 12: 909–920. 4. Geppert M, Goda Y, Hammer RE, Li C, Rosahl TW, et al. (1994) Synaptotagmin 1: a major Ca2+ sensor for transmitter release at a central synapse. Cell 79: 717–727. 5. Littleton JT, Stern M, Perin M, Bellen HJ (1994) Calcium-dependence of neurotransmitter release and rate of spontaneous vesicle fusions are altered in Drosophila Synaptotagmin mutants Proceedings of the National Academy of Sciences of the United States of America 91: 10888–10892. 6. Fernandez-Chacon R, Konigstorfer A, Gerber SH, Garcia J, Matos MF, et al. (2001) Synaptotagmin I functions as a calcium regulator of release probability. Nature 410: 41–49. 7. Littleton JT, Bai JH, Vyas B, Desai R, Baltus AE, et al. (2001) synaptotagmin mutants reveal essential functions for the C2B domain in Ca2+-triggered fusion and recycling of synaptic vesicles in vivo. Journal of Neuroscience 21: 1421– 1433. 8. Li C, Ullrich B, Zhang JZ, Anderson RGW, Brose N, et al. (1995) Ca2+- dependent and Ca2+-independent activities of neural and nonneural synapto- tagmins Nature 375: 594–599. 9. Davis AF, Bai JH, Fasshauer D, Wolowick MJ, Lewis JL, et al. (1999) Kinetics of synaptotagmin responses to Ca2+ and assembly with the core SNARE complex onto membranes. Neuron 24: 363–376. 10. Gerona RRL, Larsen EC, Kowalchyk JA, Martin TFJ (2000) The C terminus of SNAP-25 is essential for Ca2+-dependent binding of synaptotagmin to SNARE complexes. Journal of Biological Chemistry 275: 6328–6336. Interaction between Stoned and Synaptotagmin-1 represents the amount of quantified MBP-Stoned proteins normalized against the amount of eluted GST-C2A or GST- C2B proteins. Each value represents mean 6 S.E.M. (TIF) Figure S2 Molecular Dynamics (MD) simulation of C- terminal truncations and D3,4N of C2B. The RMSD of the structures during 100 ps MD simulation at 300 K suggested all of the C-terminal truncations and D3,4N of C2B examined have similar structural integrity compared to the full-length C2B. The MD simulation data suggested that the deletion/mutation did not affect the protein structure and indicated that all constructs tested are as stable as the wild type. Therefore, the changes in biochemical properties reflect the importance of each region deleted or changed by the mutation. (TIF) Figure S3 The effect of pC2B.M peptide on STNB binding. Increasing concentration of random peptide pCTRL has no effect on MBP-STNB binding to GST-C2B. Similar to pC2B1, addition of pC2B.M reduced the MBP-STNB binding. The experiment was performed in the presence of 1 mM CaCl2. The upper panel of Western Blot image shows the MBP-STNB bound to GST-C2B and detected by anti-MBP antibody while the lower panel is the Ponceau Red staining of GST-C2B protein of the respective blots. (TIF) Acknowledgments We would like to thank Dr A. Marie Phillips (Department of Genetics, The University of Melbourne), Dr Elizabeth Coulson (QBI, University of Queensland), Dr Peter Thorn (SBMS, University of Queensland) for various help and critical reading of the manuscript. Author Contributions Conceived and designed the experiments: CS CA LEK. Performed the experiments: CS CA. Analyzed the data: CS CA. Contributed reagents/ materials/analysis tools: LEK. Wrote the paper: CS LEK. 11. Chapman ER, An S, Edwardson JM, Jahn R (1996) A novel function for the second C2 domain of synaptotagmin – Ca2+-triggered dimerization. Journal of Biological Chemistry 271: 5844–5849. 12. Schiavo G, Stenbeck G, Rothman JE, Sollner TH (1997) Binding of the synaptic vesicle v-SNARE, synaptotagmin, to the plasma membrane t-SNARE, SNAP- 25, can explain docked vesicles at neurotoxin-treated synapses. Proceedings of the National Academy of Sciences of the United States of America 94: 997– 1001. 13. Zhang JZ, Davletov BA, Sudhof TC, Anderson RG (1994) Synaptotagmin I is a high affinity receptor for clathrin AP-2: implications for membrane recycling. Cell 78: 751–760. 14. Grass I, Thiel S, Honing S, Haucke V (2004) Recognition of a basic AP-2 binding motif within the C2B domain of synaptotagmin is dependent on multimerization. Journal of Biological Chemistry 279: 54872–54880. 15. Haucke V, Wenk MR, Chapman ER, Farsad K, De Camilli P (2000) Dual interaction of synaptotagmin with mu 2-and alpha-adaptin facilitates clathrin- coated pit nucleation. Embo Journal 19: 6011–6019. 16. Martens S, Kozlov MM, McMahon HT (2007) How synaptotagmin promotes membrane fusion. Science 316: 1205–1208. 17. Phillips AM, Smith M, Ramaswami M, Kelly LE (2000) The products of the Drosophila stoned locus interact with synaptic vesicles via synaptotagmin. Journal of Neuroscience 20: 8254–8261. 18. Zhang JZ, Davletov BA, Sudhof TC, Anderson RGW (1994) Synaptotagmin-I is a high-affinity receptor for clathrin-AP-2 – Implications for membrane recycling. Cell 78: 751–760. 19. Chapman ER, Desai RC, Davis AF, Tornehl CK (1998) Delineation of the oligomerization, AP-2 binding, and synprint binding region of the C2B domain of synaptotagmin. Journal of Biological Chemistry 273: 32966–32972. 20. Grass I, Thiel S, Honig S, Haucke V (2004) Recognition of a basic AP-2 binding motif within the C2B domain of Synaptotagmin is dependent on multi- merization. Journal of Biological Chemistry 279: 54872–54880. PLoS ONE | www.plosone.org 10 June 2012 | Volume 7 | Issue 6 | e38822