Staniforth R A, Bigotti M G, Cutruzzolà F, Allocatelli C T, Brunori M
Dipartimento di Scienze Biochimiche, A. Rossi Fanelli, Istituto Pasteur-Fondazione Cenci Bolognetti, Roma, Italy.
J Mol Biol. 1998 Jan 9;275(1):133-48. doi: 10.1006/jmbi.1997.1409.
The equilibrium unfolding pathway of Aplysia apomyoglobin has been studied under various solvent conditions. The protein exhibits a single unfolding transition in acid in contrast to the two transitions observed for the mammalian apomyoglobins with which it shares a common fold but a low level of sequence identity (24%). This acid-unfolded species has considerable residual structure as evidenced by both tryptophan fluorescence and far-UV CD spectroscopy. It remains 40% alpha-helical under low salt conditions (2 mM citrate, 4 degrees C); the folded form is 65% helical. A similar species is observed for the mammalian globins in mild acid conditions. Titration with GdnHCl at pH 7 reveals two unfolding transitions, the first having common features with that observed in acid and the second resulting in a completely unfolded state. Under the same conditions, urea unfolds the protein completely in an apparently single cooperative transition. Assuming a simple three-state model (F<-->I<-->U), data from GdnHCl and urea titrations over a range of pH conditions were used to derive values for the apparent stability (delta Gw(app) and solvent accessibility (n(app)) of the folded (F) and intermediate (I) forms of the protein. Urea titrations were then repeated over a range of KCl concentrations in order to understand the contribution of Cl- to the different unfolding activity of GdnHCl. A three-state scheme is justified when changes in delta G(w(app)) occur without changes in n(app). The change in free energy of folding of I<-->F (delta Gw(F/I)) decreases to 0 at pH 4 as expected from the acid unfolding curve. delta Gw(I/U) reaches its maximum at pH 4.5, the isoelectric point of the protein. Variations of this value with pH and chloride are as much as 3 kcal mol-1 and correlate closely with changes in n(app) although there is no change in the alpha-helical content of I across the pH range. This observation is interpreted here as a deviation of the unfolding of the I state of Aplysia apomyoglobin from a cooperative behaviour.
在不同溶剂条件下研究了海兔脱辅基肌红蛋白的平衡去折叠途径。与哺乳动物脱辅基肌红蛋白观察到的两个转变不同，该蛋白质在酸性条件下呈现单一的去折叠转变，尽管它们具有共同的折叠结构，但序列同一性较低（24%）。色氨酸荧光和远紫外圆二色光谱均表明，这种酸性去折叠物种具有相当多的残余结构。在低盐条件下（2 mM柠檬酸盐，4℃），它仍保持40%的α-螺旋结构；折叠形式为65%螺旋结构。在温和酸性条件下，哺乳动物球蛋白也观察到类似的物种。在pH 7下用盐酸胍滴定显示两个去折叠转变，第一个与在酸性条件下观察到的有共同特征，第二个导致完全去折叠状态。在相同条件下，尿素以明显的单一协同转变使蛋白质完全去折叠。假设一个简单的三态模型（F<-->I<-->U），在一系列pH条件下盐酸胍和尿素滴定的数据用于推导蛋白质折叠态（F）和中间态（I）的表观稳定性（ΔGw(app)）和溶剂可及性（n(app)）值。然后在一系列氯化钾浓度范围内重复尿素滴定，以了解氯离子对盐酸胍不同去折叠活性的贡献。当ΔG(w(app))发生变化而n(app)不变时，三态模型是合理的。如酸性去折叠曲线所预期的，I<-->F的折叠自由能变化（ΔGw(F/I)）在pH 4时降至0。ΔGw(I/U)在pH 4.5（蛋白质的等电点）达到最大值。该值随pH和氯离子的变化高达3 kcal mol-1，并且与n(app)的变化密切相关，尽管在整个pH范围内I的α-螺旋含量没有变化。在此，这一观察结果被解释为海兔脱辅基肌红蛋白I态的去折叠偏离了协同行为。
