Thermodynamic Stability of Annexin V E17G: Equilibrium Parameters from an Irreversible Unfolding Reaction
详细信息 查看全文
- 作者：Thomas Vogl ; Claudia Jatzke ; Hans-Jü ; rgen Hinz ; Jö ; rg Benz ; and Robert Huber
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：February 18, 1997
- 年：1997
- 卷：36
- 期：7
- 页码：1657 - 1668
- 全文大小：322K
- 年卷期：v.36,no.7(February 18, 1997)
- ISSN：1520-4995
文摘
Conformational stability of the membrane-binding proteinannexin V E17G has been determinedby high-sensitivity differential scanning microcalorimetry (DSC)measurements and by isothermal,guanidinium hydrochloride (GdnHCl)-induced unfolding studies.Wild-type annexin V and the E17Gmutant protein studied here are structurally almost identical.Therefore, it can be expected that the presentresults will not deviate significantly from the stability data of thewild-type molecule. Thermal unfoldingis irreversible, while GdnHCl unfolding shows a high degree ofreversibility. We were able to demonstratethat characteristic features of annexin V E17G unfolding permit us toextract from the kinetically controlledheat capacity curves thermodynamic equilibrium parameters at the highheating rates. The thermodynamicquantities obtained from the DSC studies in phosphate buffer at pH 7.0are as follows: t1/2 = 54.7
C(heating rate of 2.34 K min-1),
H = 690 kJ mol-1, andCp = 10.3 kJmol-1 K-1 whichcorrespondendsto a value of GD (20 C) of 53.4 kJmol-1. When compared on a per grambasis, these thermodynamicparameters classify annexin V E17G as a marginally stable protein.This conclusion is consistent withstructural and functional features of the protein that requireconformational adaptability for hinge-bendingmotions and pore formation on interaction with membranes. Weobserved a large difference between thechange in the Gibbs energy value derived from the heat capacity studiesand that determined from theGdnHCl unfolding curve. The difference appears to stem from aspecific interaction of the protein withthe denaturant that results in both a low half-denaturationconcentration c1/2 of 1.74 M and a smallslope(6.0 kJ L mol-2) of theGapp versus [GdnHCl] plot. Theextraordinary interaction of annexin V withGdnHCl is also manifested in the enormous depression of the transitiontemperature t1/2 (=18 C)whenthe GdnHCl concentration is increased from 0 to 1 M. "Regular"proteins experience an average decreasein the transition temperature of 8 ± 2 C per 1 M change in theconcentration of GdnHCl.
C(heating rate of 2.34 K min-1),H = 690 kJ mol-1, andCp = 10.3 kJmol-1 K-1 whichcorrespondendsto a value of GD (20 C) of 53.4 kJmol-1. When compared on a per grambasis, these thermodynamicparameters classify annexin V E17G as a marginally stable protein.This conclusion is consistent withstructural and functional features of the protein that requireconformational adaptability for hinge-bendingmotions and pore formation on interaction with membranes. Weobserved a large difference between thechange in the Gibbs energy value derived from the heat capacity studiesand that determined from theGdnHCl unfolding curve. The difference appears to stem from aspecific interaction of the protein withthe denaturant that results in both a low half-denaturationconcentration c1/2 of 1.74 M and a smallslope(6.0 kJ L mol-2) of theGapp versus [GdnHCl] plot. Theextraordinary interaction of annexin V withGdnHCl is also manifested in the enormous depression of the transitiontemperature t1/2 (=18 C)whenthe GdnHCl concentration is increased from 0 to 1 M. "Regular"proteins experience an average decreasein the transition temperature of 8 ± 2 C per 1 M change in theconcentration of GdnHCl.