History, rare and multiple events of mechanical unfolding of repeat proteins

Fidan Sumbul , Arin Marchesi , Felix Rico (1)

1 Bio-AFM-Lab - BIO-AFM-LAB Bio Atomic Force Microscopy Laboratory

Mechanical unfolding of proteins consisting of repeat domains is an excellent tool to obtain large statistics. Force spectroscopy experiments using AFM on proteins presenting multiple domains have revealed that unfolding forces depend on the number of folded domains (history) and have reported intermediate states and rare events. However, the common use of unspecific attachment approaches to pull the protein of interest holds important limitations to study unfolding history and may lead to discarding rare and multiple probing events due to the presence of unspecific adhesion and uncertainty on the pulling site. Site-specific methods recently emerged minimize this uncertainty and would be excellent tools to probe unfolding history and rare events. However, detail characterization of these approaches is required to identify their advantages and limitations. Here, we characterize a site-specific binding approach based on the ultrastable complex dockerin/cohesin III revealing its advantages and limitations to assess the unfolding history and to investigate rare and multiple events during the unfolding of repeated domains. We show that this approach is more robust, reproducible and provide larger statistics than conventional unspecific methods. We show that the method is optimal to reveal the history of unfolding from the very first domain, and to detect rare events, while being more limited to assess intermediate states. Finally, we quantify the unfolding forces required to unfold two molecules pulled in parallel, difficult using unspecific approaches. The proposed method represents a step forward towards more reproducible measurements to probe protein unfolding history and opens the door to systematic probing of rare and multiple molecule unfolding mechanisms.

Domaines: Biochimie, Biologie Moléculaire

Fidan Sumbul, Arin Marchesi, Felix Rico. History, rare and multiple events of mechanical unfolding of repeat proteins. Journal of Chemical Physics, 2018, 148 (12), ⟨10.1063/1.5013259⟩. ⟨hal-01792492⟩