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UA BIOC 585 - BIOC 585 Lecture Notes

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Figure 2: Fluorescence emission spectra of CaM34/110-AEDANS/DAB. The doubly labeled CaM mutant, CaM34/110-AEDANS/DAB (1.0 µM) was mixed with 0.5 mM EGTA (+EGTA), which promotes a rigid, extended conformation, 1 mM CaCl2 (+Ca2+), which promotes a more flexible conformation, 1mM CaCl2 and 18 µM helix H (+helix H), or with 1 mM CaCl2 and 2 µM CyaA1-412 (+CyaA). In a control experiment using CaM34/110 labeled only with the donor, minimal change in fluorescence emission was observed indicating that the changes in the fluorescent signal reflect changes in the efficiency of energy transfer from the donor (AEDANS) to the acceptor (DAB)7. Methods: The expression and purification of recombinant CaM were performed as described previously1. To express the adenylyl cyclase domain (amino acids 1-412) of CyaA, pET-CyaA1-412 (kindly provided by Dr. Emanuel Hanski at Hebrew Univ.-Hadassah Medical School) were transformed into E. coli B834(DE3) cells harboring pREP4. The resulting cells were cultured at the optimized condition for CyaA expression (19-hour induction at 24 oC with 30 µM IPTG) and the high-speed supernatant of E. coli lysate was prepared without denaturant. Recombinant CyaA1-412 with greater than 95% purity was obtained by fractionation through a Ni-chelating column followed by a Q-sepharose column, concentrated to greater than 10 mg/ml, and stored at -80 oC. The identity of CyaA1-412 was confirmed by its molecular weight (46,216 daltons based on mass spectrometry, 32 daltons higher than its ideal mass) and its CaM-activated adenylyl cyclase activity. The yield was around 12 mg from each liter of E. coli culture. To perform the FRET experiment, CaM34/110, a human CaM mutant in which T34 and T110 were both changed to cysteine was randomly labeled with a fluorescence donor, N-iodoacetyl-N’-(5-sulfo-1-naphtyl)ethylenediamine (1,5-IAEDANS) and a non-fluorescence acceptor, 4-dimethylaminophenylazophenyl-4’-maleimide (DAB) and purified by HPLC. The steady-state fluorescence emission of CaM34/110-AEDANS/DAB was recorded as described1. The dissociation constant between CaM and CyaA and between CaM and the EF helix H peptide was determined by titration of donor-acceptor labeled CaM34/110 with CyaA1-412 protein and EF H peptide, respectively1.1. Drum, C. L. et al. An extended conformation of calmodulin induces interactions between the structural domains of adenylyl cyclase from Bacillus anthracis to promote catalysis. J Biol Chem 275, 36334-40 (2000). Figure 3: Effect of YbCl3 on the adenylyl cyclase activity of EF. The adenylyl cyclase assay was performed under conditions that mimicked the solution in EF-CaM crystals (1 mM MgCl2, 1:1.25 molar ratio of EF to CaM, and low pH (6.5)). The experiment is done in triplicate. At 0.3 mM YbCl3 concentration which was used to generate EF-CaM crystal, YbCl3 had a marginal effect on the activity of EF. At higher than 2.5 mM YbCl3 concentration, the assay solution formed macroscopic precipitates, resulting in drastically reduced activity. Supplemental table Crystallographic and refinement statistics A. Crystallographic data Crystal EF-CH6-CaM-3'dATP* H6-EF EF-CH6-CaM Space group I222 P21212 I222 a (Å) 117.60 50.47 116.73 b (Å) 167.44 203.60 167.31 c (Å) 343.48 74.03 344.30 X-Ray source APS, 14-BM-C APS, 14-BM-C NSLS X-25 Resolution (Å) 2.75 2.6 2.95 completeness (%) (last shell) 91.6 (57.2) 98.5 (95.4) 98.8 (98.1) R-sym (last shell) 9.7 (26.4) 8.7 (27.3) 6.1 (26.6) I/σ (last shell) 4.6 (2.8) 5.6 (1.7) 18.5 (4.1) Redundancy (last shell) 10.9 (3.3) 6.5 (4.8) 16.0 (5.5) B. Refinement Resolution (Å) 20-2.75 30-2.6 30-2.95 R-factor/free (%) 21.5/28.6 22.8/27.6 27.8/31.5 Bond length (Å) 0.005 0.011 0.011 Bond angle (degree) 1.8 1.55 1.6 * H6-EF and EF-CH6 are tagged with hexahistidine at the N- and C-terminal end,


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