气候变化领域集成服务门户(CCPortal): Protein design-scapes generated by microfluidic DNA assembly elucidate domain coupling in the bacterial histidine kinase CpxA

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DOI	10.1073/pnas.2017719118
	Protein design-scapes generated by microfluidic DNA assembly elucidate domain coupling in the bacterial histidine kinase CpxA
	Clark I.C.; Mensa B.; Ochs C.J.; Schmidt N.W.; Mravic M.; Quintana F.J.; DeGrado W.F.; Abate A.R.
发表日期	2021
ISSN	00278424
卷号	118 期号:12
英文摘要	The randomization and screening of combinatorial DNA libraries is a powerful technique for understanding sequence–function relationships and optimizing biosynthetic pathways. Although it can be difficult to predict a priori which sequence combinations encode functional units, it is often possible to omit undesired combinations that inflate library size and screening effort. However, defined library generation is difficult when a complex scan through sequence space is needed. To overcome this challenge, we designed a hybrid valve- and droplet-based microfluidic system that deterministically assembles DNA parts in picoliter droplets, reducing reagent consumption and bias. Using this system, we built a combinatorial library encoding an engineered histidine kinase (HK) based on bacterial CpxA. Our library encodes designed transmembrane (TM) domains that modulate the activity of the cytoplasmic domain of CpxA and variants of the structurally distant “S helix” located near the catalytic domain. We find that the S helix sets a basal activity further modulated by the TM domain. Surprisingly, we also find that a given TM motif can elicit opposing effects on the catalytic activity of different S-helix variants. We conclude that the intervening HAMP domain passively transmits signals and shapes the signaling response depending on subtle changes in neighboring domains. This flexibility engenders a richness in functional outputs as HKs vary in response to changing evolutionary pressures. © 2021 National Academy of Sciences. All rights reserved.
英文关键词	Droplet microfluidics \| rational library design \| protein engineering \| histidine kinase \| signal transduction
语种	英语
scopus关键词	bacterial protein; protein CpxA; protein histidine kinase; unclassified drug; Article; cell membrane; controlled study; DNA synthesis; enzyme active site; enzyme activity; microfluidics; nonhuman; priority journal; protein domain; protein engineering; protein structure; protein synthesis; protein transport; signal transduction
来源期刊	Proceedings of the National Academy of Sciences of the United States of America
文献类型	期刊论文
条目标识符	http://gcip.llas.ac.cn/handle/2XKMVOVA/180181
作者单位	Department of Bioengineering, California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720, United States; Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158, United States; Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco, CA 94158, United States; Broad Institute of MIT and Harvard, Cambridge, MA 02142, United States; Ann Romney Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, United States; Chan Zuckerberg Biohub, San Francisco, CA 94158, United States
推荐引用方式 GB/T 7714	Clark I.C.,Mensa B.,Ochs C.J.,et al. Protein design-scapes generated by microfluidic DNA assembly elucidate domain coupling in the bacterial histidine kinase CpxA[J],2021,118(12).
APA	Clark I.C..,Mensa B..,Ochs C.J..,Schmidt N.W..,Mravic M..,...&Abate A.R..(2021).Protein design-scapes generated by microfluidic DNA assembly elucidate domain coupling in the bacterial histidine kinase CpxA.Proceedings of the National Academy of Sciences of the United States of America,118(12).
MLA	Clark I.C.,et al."Protein design-scapes generated by microfluidic DNA assembly elucidate domain coupling in the bacterial histidine kinase CpxA".Proceedings of the National Academy of Sciences of the United States of America 118.12(2021).