Jaswal Sheila S, Truhlar Stephanie M E, Dill Ken A, Agard David A
Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94143, USA.
J Mol Biol. 2005 Mar 25;347(2):355-66. doi: 10.1016/j.jmb.2005.01.032. Epub 2005 Jan 28.
Alpha-lytic protease (alpha LP) and Streptomyces griseus protease B (SGPB) are two extracellular serine proteases whose folding is absolutely dependent on the existence of their companion pro regions. Moreover, the native states of these proteins are, at best, marginally stable, with the apparent stability resulting from being kinetically trapped in the native state by large barriers to unfolding. Here, in an effort to understand the physical properties that distinguish kinetically and thermodynamically stable proteins, we study the temperature-dependences of the folding and unfolding kinetics of alpha LP and SGPB without their pro regions, and compare their behavior to a comprehensive set of other proteins. For the folding activation thermodynamics, we find some remarkable universal behaviors in the thermodynamically stable proteins that are violated dramatically by alpha LP. Despite significant variations in deltaC(P,F)++, the maximal folding speed occurs within the narrow biological temperature range for all proteins, except for alpha LP, with its maximal folding speed shifted lower by 200 K. This implies evolutionary pressures on folding speed for typical proteins, but not for alpha LP. In addition, the folding free energy barrier in the biological temperature range for most proteins is predominantly enthalpic, but purely entropic for alpha LP. The unfolding of alpha LP and SGPB is distinguished by three properties: a remarkably large deltaC(P,U)++, a very high deltaG(U)++, and a maximum deltaG(u)++ at the optimal growth temperature for the organism. While other proteins display each of these traits to some approximation, the simultaneous optimization of all three occurs only in the kinetically stable proteins, and appears to be required to maximize their unfolding cooperativity, by suppressing local unfolding events, and slowing the rate of global unfolding. Together, these properties extend the lifetime of these enzymes in the highly proteolytic extracellular environment. Attaining such functional properties seems possible only through the gross perturbation of the folding thermodynamics, which in turn has required the co-evolution of pro regions as folding catalysts.
α-裂解蛋白酶（α-LP）和灰色链霉菌蛋白酶B（SGPB）是两种细胞外丝氨酸蛋白酶，它们的折叠绝对依赖于其伴侣前肽区域的存在。此外，这些蛋白质的天然状态充其量只是勉强稳定，其表观稳定性是由于在展开过程中存在巨大障碍而在动力学上被困在天然状态所致。在这里，为了理解区分动力学稳定和热力学稳定蛋白质的物理性质，我们研究了去除前肽区域的α-LP和SGPB的折叠和展开动力学的温度依赖性，并将它们的行为与其他一系列蛋白质进行比较。对于折叠活化热力学，我们在热力学稳定的蛋白质中发现了一些显著的普遍行为，而α-LP则显著违背了这些行为。尽管ΔC(P,F)++存在显著差异，但除α-LP外，所有蛋白质的最大折叠速度都出现在狭窄的生物温度范围内，α-LP的最大折叠速度下移了200 K。这意味着典型蛋白质在折叠速度上受到进化压力，但α-LP不受此影响。此外，大多数蛋白质在生物温度范围内的折叠自由能障碍主要是焓性的，而α-LP则完全是熵性的。α-LP和SGPB的展开具有三个特点：显著大的ΔC(P,U)++、非常高的ΔG(U)++以及在生物体最佳生长温度下的最大ΔG(u)++。虽然其他蛋白质在某种程度上也表现出这些特征，但只有在动力学稳定的蛋白质中才同时优化了这三个特征，并且似乎需要通过抑制局部展开事件和减缓全局展开速率来最大化它们的展开协同性。这些特性共同延长了这些酶在高度蛋白水解的细胞外环境中的寿命。只有通过折叠热力学的严重扰动才有可能获得这些功能特性，而这反过来又需要前肽区域作为折叠催化剂共同进化。
