Efficient and simplified nanomechanical analysis of intrinsically disordered proteins

Abstract

Intrinsically disordered proteins (IDPs) lack a tertiary structure. Amyloidogenic IDPs (aIDPs) in particular have attracted great interest due to their implication in several devastating diseases as well as in critical biological functions. However, the conformational changes that trigger amyloid formation in aIDPs are largely unknown. aIDPs' conformational polymorphism at the monomer level encumbers their study using bulk techniques. Single-molecule techniques like atomic force microscopy-based single-molecule force spectroscopy represent a promising approach and a "carrier-guest" strategy, in which the protein of interest is mechanically protected, was developed to overcome the spurious signals from the noisy proximal region. However, since the carrier and single-molecule markers have similar mechanostabilities, their signals can intermingle in the force-extension recordings, making peak selection and analysis very laborious, cumbersome and prone to error for the non-expert. Here we have developed a new carrier, the c8C module from the CipC scaffoldin, with a higher mechanostability so that the signals from the protected protein will appear at the end of the recordings. This assures an accurate, more efficient and expert-independent analysis, simplifying both the selection and analysis of the single-molecule data. Furthermore, this modular design can be integrated into any SMFS polyprotein-based vector, thus constituting a useful utensil in the growing toolbox of protein nanomechanics.

Replica exchange molecular dynamics simulation of the coordination of Pt(ii)-Phenanthroline to amyloid-β

“…Another issue may be the use of an implicit solvent model, chosen here for the enhanced sampling that it enables, but which might also lead to imbalance in intramolecular over intermolecular interactions. This approach is widely used in study of intrinsically disordered proteins (see for instance 65,66 for recent examples). Moreover, the radius of gyration we report is in the range of values reported in literature, namely 9 to 13Å with a mean of 11.4Å, 67 which lends condence to the suitability of the approach we have taken.…”

Tau amyloidogenesis begins with a loss of its conformational polymorphism

“…The coding region of the K18 fragment from tau was amplified by PCR using a plasmid clone containing the full length human tau as a template for PCR amplification, which was kindly provided by Prof. Jesús Ávila (CBMSO-UAM). The selected restriction sites used to insert the guest fragment into the carrier ubiquitin of the pFS-2 vector and the minimal fusion proteins were AgeI-MluI (Oroz, Hervas, and Carrion-Vazquez 2012;Fernandez-Ramirez et al 2018). NheI-XhoI were selected for the insertion of K18 into pET28a (Novagen) expression vector.…”

“…When the OD 595 reached 0.4-1, the protein expression was induced by 1mM IPTG for 4 h. The lysis of bacterial pellets was done by 1% Triton X-100, 0.5% Tween-20, sonication pulses and the addition of 1mg/ml lysozyme. The purification by Ni 2+ affinity chromatography and size-exclusion was performed as previously described Fernandez-Ramirez et al 2018), using an FPLC apparatus (ÄKTA Purifier, GE Healthcare). The concentration of proteins was determined by their absorbance at 280 nm using their molar extinction coefficients.…”

“…This value represents the length of the carrier (≈26 nm) (Oroz, Hervas, and Carrion-Vazquez 2012) plus 50.4 nm from the 126-redidue K18 (Fernandez-Ramirez et al 2018), considering 0.4 nm/residue (Ainavarapu et al 2007).…”

Divergent CPEB prion-like domains reveal different assembly mechanisms for a generic amyloid-like fold

“…Next, we used single-molecule force spectroscopy based on atomic force microscopy (AFM-SMFS) using the length-clamp mode together with a protein engineering strategy, which was previously used to analyze conformational diversity of amyloid-forming proteins at the monomer level Fernandez-Ramirez et al 2018). Here, ApCPEB PLD is mechanically protected inside a carrier protein (I27 module from human cardiac titin) by cloning its code into the pFS-2 plasmid ( Figure 2B and S3A) (Oroz, Hervas, and Carrion-Vazquez 2012).…”