期刊
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
卷 139, 期 49, 页码 17841-17852出版社
AMER CHEMICAL SOC
DOI: 10.1021/jacs.7b07574
关键词
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资金
- EU seventh Framework Programme [NMP4-SL-2013-604530]
- Nanosystems Initiative Munich
- ERC Advanced Grant CelluFuel
- Society in Science - The Branco Weiss Fellowship from ETH Zurich
- ERC [715207]
- Human Frontier Science Program [RGY0080/2015]
- National Institutes of Health (NIH) [9P41GM104601]
- National Science Foundation (NSF) [MCB-1616590]
- Energy Biosciences Institute (Regents of the University of California Berkeley) [EBI 231 UCB BP 2014OO4J01]
- National Science Foundation [ACI-1440026, OCI-0725070, ACI-1238993]
- state of Illinois
- DOE Office of Science User Facility [DE-AC02-06CH11357]
- European Research Council (ERC) [715207] Funding Source: European Research Council (ERC)
Cellulosomes are polyprotein machineries that efficiently degrade cellulosic material. Crucial to their function are scaffolds consisting of highly homologous cohesin domains, which serve a dual role by coordinating a multiplicity of enzymes as well as anchoring the microbe to its substrate. Here we combined two approaches to elucidate the mechanical properties of the main scaffold ScaA of Acetivibrio cellulolyticus. A newly developed parallelized one-pot in vitro transcription-translation and protein pull-down protocol enabled high-throughput atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) measurements of all cohesins from ScaA with a single cantilever, thus promising improved relative force comparability. Albeit very similar in sequence, the hanging cohesins showed considerably lower unfolding forces than the bridging cohesins, which are subjected to force when the microbe is anchored to its substrate. Additionally, all-atom steered molecular dynamics (SMD) simulations on homology models offered insight into the process of cohesin unfolding under force. Based on the differences among the individual force propagation pathways and their associated correlation communities, we designed mutants to tune the mechanical stability of the weakest hanging cohesin. The proposed mutants were tested in a second high-throughput AFM SMFS experiment revealing that in one case a single alanine to glycine point mutation suffices to more than double the mechanical stability. In summary, we have successfully characterized the force induced unfolding behavior of all cohesins from the scaffoldin ScaA, as well as revealed how small changes in sequence can have large effects on force resilience in cohesin domains. Our strategy provides an efficient way to test and improve the mechanical integrity of protein domains in general.
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