edoc

Combining in Vitro and in Silico Single-Molecule Force Spectroscopy to Characterize and Tune Cellulosomal Scaffoldin Mechanics

Verdorfer, Tobias and Bernardi, Rafael C. and Meinhold, Aylin and Ott, Wolfgang and Luthey-Schulten, Zaida and Nash, Michael A. and Gaub, Hermann E.. (2017) Combining in Vitro and in Silico Single-Molecule Force Spectroscopy to Characterize and Tune Cellulosomal Scaffoldin Mechanics. Journal of the American Chemical Society, 139 (49). pp. 17841-17852.

Full text not available from this repository.

Official URL: http://edoc.unibas.ch/58786/

Downloads: Statistics Overview

Abstract

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.
Faculties and Departments:05 Faculty of Science > Departement Chemie > Chemie > Synthetic Systems (Nash)
UniBasel Contributors:Nash, Michael
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:American Chemical Society
ISSN:0002-7863
e-ISSN:1520-5126
Note:Publication type according to Uni Basel Research Database: Journal article
Identification Number:
Last Modified:15 Jan 2018 09:59
Deposited On:15 Jan 2018 09:59

Repository Staff Only: item control page