Misselwitz R, Hausdorf G, Welfle K, Höhne W E, Welfle H
Institute of Biochemistry, Medical Faculty (Charité), Humboldt University, Berlin, Germany.
Biochim Biophys Acta. 1995 Jul 3;1250(1):9-18. doi: 10.1016/0167-4838(95)00041-r.
Conformation and stability of the recombinant protein HIV-1 rp24 were analyzed by circular dichroism, fluorescence spectroscopy and differential scanning calorimetry under different solvent conditions. From circular dichroism measurements, HIV-1 rp24 at pH 5.8 can be classified as an all alpha-helical protein. A fluorescence maximum of about 330 nm indicates a predominantly hydrophobic environment of the five tryptophan residues. The GdnHCl-induced unfolding curves monitored by CD and fluorescence are sigmoidal and single phasic and the midpoints of transitions are independent on the protein concentration. For the calculation of free energy of unfolding delta GuH2O a 'two-state' model was applied. The calculated values are between 18 and 24 kJ/mol and thus on the lower limit of the conformational stability of globular proteins. Melting experiments at pH 5.8 are impaired by a strong irreversible aggregation at higher temperatures. However, at pH 3.0 and in the presence of 0.1% (w/v) ocytl beta-glucopyranoside the melting curves show a large degree of reversibility with a Tm value of 38 degrees C and a molar enthalpy change delta Hm of 218 kJ/mol. At pH < 2.5 HIV-1 rp24 can adopt a new conformation which is characterized by a high alpha-helical content, a strongly decreased CD in the aromatic region, a red-shift of the fluorescence spectrum and a strong binding of ANS. These spectral features of the acid-induced conformational state are similar to those obtained for molten globule-like folding states. HIV-1 rp24 unfolds cooperatively at pH 2.0 in the concentration range of about 1.5-3.0 M GdnHCl. The calculated values delta GuH2O at pH 2.0 of about 12 kJ/mol are significantly decreased in comparison to the delta GuH2O values of the protein at pH 5.8.
在不同溶剂条件下，通过圆二色性、荧光光谱和差示扫描量热法对重组蛋白HIV-1 rp24的构象和稳定性进行了分析。从圆二色性测量结果来看，pH 5.8时的HIV-1 rp24可归类为全α螺旋蛋白。约330 nm的荧光最大值表明五个色氨酸残基主要处于疏水环境中。通过圆二色性和荧光监测的盐酸胍诱导的解折叠曲线呈S形且单相，转变中点与蛋白质浓度无关。为了计算解折叠自由能ΔGuH2O，应用了“两态”模型。计算值在18至24 kJ/mol之间，因此处于球状蛋白构象稳定性的下限。pH 5.8时的熔解实验在较高温度下受到强烈不可逆聚集的影响。然而，在pH 3.0且存在0.1%（w/v）辛基β-D-吡喃葡萄糖苷的情况下，熔解曲线显示出高度的可逆性，熔解温度（Tm）值为38℃，摩尔焓变ΔHm为218 kJ/mol。在pH < 2.5时，HIV-1 rp24可采用一种新的构象，其特征是α螺旋含量高、芳香区的圆二色性强烈降低、荧光光谱红移以及与8-苯胺基-1-萘磺酸（ANS）强烈结合。酸诱导构象状态的这些光谱特征与熔球样折叠状态所获得的特征相似。HIV-1 rp24在pH 2.0时，在约1.5 - 3.0 M盐酸胍浓度范围内协同解折叠。与pH 5.8时蛋白质的ΔGuH2O值相比，pH 2.0时计算得到的约12 kJ/mol的ΔGuH2O值显著降低。
