Modelling the unfolding pathway of biomolecules: theoretical approach and experimental prospect

We analyse the unfolding pathway of biomolecules comprising several independent modules in pulling experiments. In a recently proposed model, a critical velocity $v_{c}$ has been predicted, such that for pulling speeds $v>v_{c}$ it is the module at the pulled end that opens first, whereas for $v<v_{c}$ it is the weakest. Here, we introduce a variant of the model that is closer to the experimental setup, and discuss the robustness of the emergence of the critical velocity and of its dependence on the model parameters. We also propose a possible experiment to test the theoretical predictions of the model, which seems feasible with state-of-art molecular engineering techniques.Comment: Accepted contribution for the Springer Book "Coupled Mathematical Models for Physical and Biological Nanoscale Systems and Their Applications" (proceedings of the BIRS CMM16 Workshop held in Banff, Canada, August 2016), 16 pages, 6 figure

A New, Modular Mass Calibrant for High-Mass MALDI-MS

The application of matrix-assisted laser desorption/ionization mass spectrometry (MALDIMS) for the analysis of high-mass proteins requires suitable calibration standards at high m/z ratios. Several possible candidates were investigated, and concatenated polyproteins based on recombinantly expressed maltodextrin-binding protein (MBP) are shown here to be well suited for this purpose. Introduction of two specific recognition sites into the primary sequence of the polyprotein allows for the selective cleavage of MBP3 into MBP and MBP2. Moreover, these MBP2 and MBP3 oligomers can be dimerized specifically, such that generation of MPB4 and MBP6 is possible as well. With the set of calibrants presented here, the m/z range of 40–400 kDa is covered. Since all calibrants consist of the same species and differ only in mass, the ionization efficiency is expected to be similar. However, equimolar mixtures of these proteins did not yield equal signal intensities on a detector specifically designed for detecting high-mass molecules

Mechanical Unfolding of Acylphosphatase Studied by Single-Molecule Force Spectroscopy and MD Simulations

Single-molecule manipulation methods provide a powerful means to study protein transitions. Here we combined single-molecule force spectroscopy and steered molecular-dynamics simulations to study the mechanical properties and unfolding behavior of the small enzyme acylphosphatase (AcP). We find that mechanical unfolding of AcP occurs at relatively low forces in an all-or-none fashion and is decelerated in the presence of a ligand, as observed in solution measurements. The prominent energy barrier for the transition is separated from the native state by a distance that is unusually long for α/β proteins. Unfolding is initiated at the C-terminal strand (βT) that lies at one edge of the β-sheet of AcP, followed by unraveling of the strand located at the other. The central strand of the sheet and the two helices in the protein unfold last. Ligand binding counteracts unfolding by stabilizing contacts between an arginine residue (Arg-23) and the catalytic loop, as well as with βT of AcP, which renders the force-bearing units of the protein resistant to force. This stabilizing effect may also account for the decelerated unfolding of ligand-bound AcP in the absence of force