SUPPLEMENTAL INFORMATION. Center Hamburg-Eppendorf, Center for Molecular Neurobiology, ZMNH, Hamburg, Germany

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1 SUPPLEMENTAL INFORMATION Title: Inter-motif communication induces hierarchical Ca 2+ -filling of Caldendrin Authors: Uday Kiran 1, Phanindranath Regur 1, Michael R. Kreutz 3,4*, Yogendra Sharma 1,2*, Asima Chakraborty 1*. 1 CSIR-Centre for Cellular and Molecular Biology (CCMB), Uppal Road, Hyderabad , India. 2 Academy of Scientific and Innovative Research (AcSIR), New Delhi, India 3 RG Neuroplasticity, Leibniz Institute for Neurobiology, Brenneckestr. 6, Magdeburg, Germany 4 Leibniz Group 'Dendritic Organelles and Synaptic Function', University Medical Center Hamburg-Eppendorf, Center for Molecular Neurobiology, ZMNH, Hamburg, Germany Supplemental Methods S1

2 Protein purification Briefly, the bacterial pellets were resuspended in 20 mm Tris-HCl, ph 8.5, 500 mm NaCl, 1 mm EDTA and 0.5 mm TCEP (Intein buffer) and lysed under ultrasonication in the presence of 0.1% Triton-X 100 and PMSF, followed by centrifugation at rpm for 1 hour, whereby the protein was retained in the soluble fraction. The supernatant was then loaded on a chitin resin column that was pre-equilibrated with the intein buffer. The protein-bound resin was activated by passing 3 volumes of intein buffer supplemented with 50 mm DTT (elution buffer). The protein was eluted from the column after ~12 hours of incubation in the elution buffer. The protein was rendered Ca 2+ free by 1 mm EDTA to chelate any metal ion in the protein sample, which was then buffer exchanged thoroughly against Chelexpurified buffer (50 mm Tris, ph 7.5, 150 mm NaCl) to obtain the apo protein. Analysis of Red-shifted emission max of EF1W and EF3W Based on the solvent accessible surface area (SASA), one would expect EF1W (Trp165) to show an emission maximum closer to , however, that is not the case in EF1W (emission maximum ~348 nm). It is well known that nearby polar residues and type of bonds with other residues can influence the emission of Trp fluorescence. 1 This might be a possible reason for red shifted emission maxima of EF1W reporter. From the structural model of EF1W reporter, it is apparent that Trp165 could be involved in cation-pi interaction with Lys169 similar to cation-pi interaction between Phe34 and Lys39 in s-cabp1, which can lead to red shifted emission maxima. 2 Best example for this kind of red shifted emission maxima of completely buried Trp residue (Trp16 SASA = Å 2 ) is pobp protein where there is only one cation-pi interaction with Lys120, gives 340 nm emission max. 3 These factors support our experimental observation of red-shifted emission in the case of EF1W reporter. Similarly, in the case of EF3W, Trp is not completely buried in hydrophobic core (SASA = Å 2 ). Trp in this area interacts with the surface-exposed polar residues, like Thr244, SASA= Å 2 ), Arg240, SASA=82.84 Å 2 ). Thus, in the case of EF3W also the red shifted emission maxima (~343 nm) could be because of relatively more solvent accessibility and its interaction with the polar environment created by polar amino acids besides hydrophobic background. S2

3 Supplemental figures Figure S1. Amino acid sequence of Caldendrin, and its alignment with the sequence of s-cabp1, showing the consensus in amino acid composition in the C-terminal domain and the long non-homologous N-terminal stretch in Caldendrin. The Ca 2+ binding loops of the four EF-hands have been underlined. The sites of mutagenesis have been boxed to show the location of the introduced Trp in the primary sequence. EQ3 and EQ4 represent E to Q mutation sites to disable EF3 and EF4 motifs respectively. S3

4 Figure S2. GdmCl unfolding of CDD-WT, reporter mutants EF3W and EF4W at a selected concentration of Ca 2+, plotted by ellipticity at 222 nm. From figure, it is clear that overall unfolding transitions of Trp reporters and wild type proteins are closely similar. S4

Table S1. Thermodynamic parameters of Ca 2+ binding to wild type CDD, its two Trp reporters (EF3W and EF4W) and EF3 binding site disabled mutant (EF3Q_EF4W).

5 Table S1. Thermodynamic parameters of Ca 2+ binding to wild type CDD, its two Trp reporters (EF3W and EF4W) and EF3 binding site disabled mutant (EF3Q_EF4W). N, number of binding sites, H Enthalpy, Ka, association constant, Kd, dissociation constant, global dissociation constant, KD, of 2 high affinity sites (EF-hand1, a weak Ca 2+ binding site was not considered for computing the global affinity as it is physiologically not relevant). From the table, it is clear that there are only marginal changes in affinities of CDD-WT and reporters EF3W and EF4W CDD. In all cases, there are two high affinity sites (corresponds to EF3 and EF4) and the affinity difference between sites is ~10 folds (in agreement with previously report on s-cabp1). With respect to overall enthalpy change, Ca 2+ binding to wild type and EF4W reporter is almost similar, but in case of EF3W reporter, Ca 2+ binding is enthalpically more favorable. S5

6 Supplemental references 1. Reshetnyak, Y. K., Koshevnik, Y., and Burstein, E. A. (2001) Decomposition of protein tryptophan fluorescence spectra into log-normal components. III. Correlation between fluorescence and microenvironment parameters of individual tryptophan residues, Biophys J. 3, Hamm, H. E., Meier, S. M., Liao, G., and Preininger, A. M. (2009) Trp fluorescence reveals an activation-dependent cation-pi interaction in the Switch II region of Galphai proteins, Protein Sci. 11, Stepanenko, O. V., Marabotti, A., Kuznetsova, I. M., Turoverov. K. K., Fini, C., Varriale, A., Staiano, M., Rossi, M., and D'Auria, S. (2008) Hydrophobic interactions and ionic networks play an important role in thermal stability and denaturation mechanism of the porcine odorant-binding protein, Proteins 1, S6

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