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Isothermal titration calorimetry (ITC) of nucelotide binding
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The titration experiments were performed using the MSC system. In each experiment, 16 aliquots of 15 μL of Geldanamycin (300 μM in 1% DMSO) were injected into 1.3 mL of protein (31 μM in 20 mM Tris-HCl, pH 7.5, 1 mM EDTA) at 25°C, and the resulting data were fit after subtracting the heats of dilution. Heats of dilution were determined in separate experiments from addition of Geldanamycin into buffer and buffer into protein. No evidence for binding of DMSO in the nucleotide binding site was observed. Titration data were fit using a nonlinear least-squares curve-fitting algorithm with three floating variables: stoichiometry, binding constant (Kb = 1/Kd), and change of enthalpy of interaction (ΔH°). The dissociation constant estimated for Geldanamycin binding to intact yeast Hsp90 was 1.22 μM, and for binding to Hsp90 N-terminal domain was 0.78 μM. No meaningful heat was observed with binding to the C-terminal fragment.
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References:
[1]. Roe SM, Prodromou C, O'Brien R, Ladbury JE, Piper PW, Pearl LH. Structural basis for inhibition of the Hsp90 molecular chaperone by the antitumor antibiotics radicicol and geldanamycin. J Med Chem. 1999 Jan 28;42(2):260-6.
[2]. McIlwrath AJ, Brunton VG, Brown R. Cell-cycle arrest and p53 accumulation induced by geldanamycin in human ovarian tumour cells. Cancer Chemother Pharmacol. 1996;37(5):423-8.
[3]. Schnur RC, Corman ML, Gallaschun RJ, Cooper BA, Dee MF, Doty JL, Muzzi ML, Moyer JD, DiOrio CI, Barbacci EG, et al. Inhibition of the oncogene product p185erbB-2 in vitro and in vivo by geldanamycin and dihydrogeldanamycin derivatives. J Med Chem. 1995 Sep 15;38(19):3806-12.
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