Courtemanche Naomi, Barrick Doug
T.C Jenkins Department of Biophysics, The John Hopkins University, Baltimore, Maryland 21218, USA.
Protein Sci. 2008 Jan;17(1):43-53. doi: 10.1110/ps.073166608.
Although the folding of alpha-helical repeat proteins has been well characterized, much less is known about the folding of repeat proteins containing beta-sheets. Here we investigate the folding thermodynamics and kinetics of the leucine-rich repeat (LRR) domain of Internalin B (InlB), an extracellular virulence factor from the bacterium Lysteria monocytogenes. This domain contains seven tandem leucine-rich repeats, of which each contribute a single beta-strand that forms a continuous beta-sheet with neighboring repeats, and an N-terminal alpha-helical capping motif. Despite its modular structure, InlB folds in an equilibrium two-state manner, as reflected by the identical thermodynamic parameters obtained by monitoring its sigmoidal urea-induced unfolding transition by different spectroscopic probes. Although equilibrium two-state folding is common in alpha-helical repeat proteins, to date, InlB is the only beta-sheet-containing repeat protein for which this behavior is observed. Surprisingly, unlike other repeat proteins exhibiting equilibrium two-state folding, InlB also folds by a simple two-state kinetic mechanism lacking intermediates, aside from the effects of prolyl isomerization on the denatured state. However, like other repeat proteins, InlB also folds significantly more slowly than expected from contact order. When plotted against urea, the rate constants for the fast refolding and single unfolding phases constitute a linear chevron that, when fitted with a kinetic two-state model, yields thermodynamic parameters matching those observed for equilibrium folding. Based on these kinetic parameters, the transition state is estimated to comprise 40% of the total surface area buried upon folding, indicating that a large fraction of the native contacts are formed in the rate-limiting step to folding.
尽管α-螺旋重复蛋白的折叠已得到充分表征，但对于含有β-折叠的重复蛋白的折叠了解较少。在此，我们研究了单核细胞增生李斯特菌的细胞外毒力因子内化素B（InlB）富含亮氨酸重复序列（LRR）结构域的折叠热力学和动力学。该结构域包含七个串联的富含亮氨酸重复序列，每个重复序列贡献一条单一的β-链，与相邻重复序列形成连续的β-折叠，以及一个N端α-螺旋帽基序。尽管其结构具有模块化特点，但InlB以平衡的两态方式折叠，这通过不同光谱探针监测其S形尿素诱导的去折叠转变所获得的相同热力学参数得以体现。虽然平衡两态折叠在α-螺旋重复蛋白中很常见，但迄今为止，InlB是唯一观察到这种行为的含β-折叠的重复蛋白。令人惊讶的是，与其他表现出平衡两态折叠的重复蛋白不同，除了脯氨酰异构化对变性态的影响外，InlB还通过一种缺乏中间体的简单两态动力学机制折叠。然而，与其他重复蛋白一样，InlB的折叠速度也比根据接触序预期的要慢得多。当以尿素为横坐标作图时，快速重折叠和单步去折叠阶段的速率常数构成一条线性的V字形曲线，用动力学两态模型拟合时，得到的热力学参数与平衡折叠时观察到的参数相匹配。基于这些动力学参数，估计过渡态占折叠时埋藏的总表面积的40%，这表明在折叠的限速步骤中形成了很大一部分天然接触。
