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Abstract：
Neutral mutational drift is an important source of biological diversity that remains underexploited in fundamental studies of protein biophysics. This study uses a synthetic transcriptional circuit to study neutral drift in protein tyrosine phosphatase 1B (PTP1B), a mammalian signaling enzyme for which conformational changes are rate limiting. Kinetic assays of purified mutants indicate that catalytic activity, rather than thermodynamic stability, guides enrichment under neutral drift, where neutral or mildly activating mutations can mitigate the effects of deleterious ones. In general, mutants show a moderate activity-stability tradeoff, an indication that minor improvements in the activity of PTP1B do not require concomitant losses in its stability. Multiplexed sequencing of large mutant pools suggests that substitutions at allosterically influential sites are purged under biological selection, which enriches for mutations located outside of the active site. Findings indicate that the positional dependence of neutral mutations within drifting populations can reveal the presence of allosteric networks and illustrate an approach for using synthetic transcriptional systems to explore these mutations in regulatory enzymes.
摘要：
中性突变漂移是生物多样性的重要来源，在蛋白质生物物理学的基础研究中仍未得到充分利用。本研究使用合成转录电路研究蛋白酪氨酸磷酸酶1B（PTP1B）的中性漂移，构象变化是限速的哺乳动物信号酶。纯化突变体的动力学分析表明催化活性，而不是热力学稳定性，引导在中性漂移下富集，中性或轻度激活突变可以减轻有害突变的影响。总的来说,突变体表现出中等的活性-稳定性权衡，这表明PTP1B活性的微小改善不需要伴随其稳定性的损失。大型突变池的多重测序表明，在生物选择下清除了变构影响位点的替换，富集位于活性位点之外的突变。研究结果表明，漂移种群中中性突变的位置依赖性可以揭示变构网络，并说明了使用合成转录系统探索调节酶中这些突变的方法。本文受版权保护。保留所有权利。
