PDF(Pubmed)
Abstract：
Kinetic stability is thought to be an attribute of proteins that require a long lifetime, such as the transporter of thyroxine and holo retinol-binding protein or transthyretin (TTR) functioning in the bloodstream, cerebrospinal fluid, and vitreous humor. TTR evolved from ancestral enzymes known as TTR-related proteins (TRPs). Here, we develop a rate-expansion approach that allows unfolding rates to be measured directly at low denaturant concentration, revealing that kinetic stability exists in the Escherichia coli TRP (EcTRP), even though the enzyme structure is more energetically frustrated and has a more mutation-sensitive folding mechanism than human TTR. Thus, the ancient tetrameric enzyme may already have been poised to mutate into a kinetically stable human transporter. An extensive mutational study that exchanges residues at key sites within the TTR and EcTRP dimer-dimer interface shows that tyrosine 111, replaced by a threonine in TTR, is the gatekeeper of frustration in EcTRP because it is critical for function. Frustration, virtually absent in TTR, occurs at multiple sites in EcTRP and even cooperatively for certain pairs of mutations. We present evidence that evolution at the C terminus of TTR was a compensatory event to maintain the preexisting kinetic stability while reducing frustration and sensitivity to mutation. We propose an \"overcompensation\" pathway from EcTRPs to functional hybrids to modern TTRs that is consistent with the biophysics discussed here. An alternative plausible pathway is also presented.
摘要：
动力学稳定性被认为是需要长寿命的蛋白质的属性，如甲状腺素和全视黄醇结合蛋白的转运蛋白或运甲状腺素蛋白（TTR）在血流中起作用，脑脊液,和玻璃体幽默。TTR从称为TTR相关蛋白(TRPs)的祖先酶进化而来。这里,我们开发了一种速率膨胀方法，允许在低变性剂浓度下直接测量展开速率，揭示了大肠杆菌TRP（EcTRP）中存在动力学稳定性，即使酶结构比人类TTR更有活力地受挫，并且具有对突变更敏感的折叠机制。因此，古老的四聚体酶可能已经准备好突变成动力学稳定的人类转运蛋白。在TTR和EcTRP二聚体-二聚体界面内的关键位点交换残基的广泛突变研究表明，TTR中的酪氨酸111被苏氨酸取代，是EcTRP中挫折的看门人，因为它对功能至关重要。沮丧,在TTR中几乎不存在，发生在EcTRP的多个位点，甚至协同某些突变对。我们提供的证据表明，TTRC末端的进化是维持先前存在的动力学稳定性，同时减少挫败感和对突变的敏感性的代偿性事件。我们提出了从EcTRPs到功能性杂种再到现代TTRs的“过度补偿”途径，这与此处讨论的生物物理学一致。还提出了另一种可能的途径。
