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Register a new userOn-chip Acousto Thermal Shift Assay for Rapid and Sensitive Assessment of Protein Thermodynamic Stability
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Thermal shift assays (TSAs) have been extensively used to study thermodynamics of proteins and provide an efficient means to assess protein-ligand binding or protein-protein interaction. However, existing TSAs have limitations such as time consuming, labor intensive, or low sensitivity. Here we introduce a novel acousto thermal shift assay (ATSA), the first ultrasound enabled TSA, for real-time analysis of protein thermodynamic stability. It capitalizes the novel coupling of unique acoustic mechanisms to achieve protein unfolding, concentration, and measurement on a single microfluidic chip within minutes. Compared to conventional TSA methods, our ATSA technique enabled ultra-fast (at least 30 times faster), highly sensitive (7-34 folds higher), and label-free monitoring of protein-ligand interactions and protein stability. ATSA paves new avenues for protein analysis in biology, medicine and fast diagnosis.
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We propose a novel method for refining force-field parameters of protein systems. In this method, the agreement of the secondary-structure stability and instability between the protein conformations obtained by experiments and those obtained by molecular dynamics simulations is used as a criterion for the optimization of force-field parameters. As an example of the applications of the present method, we refined the force-field parameter set of the AMBER ff99SB force field by searching the torsion-energy parameter spaces of $psi$ (N-C$^{alpha}$-C-N) and $zeta$ (C$^{beta}$-C$^{alpha}$-C-N) of the backbone dihedral angles. We then performed folding simulations of $alpha$-helical and $beta$-hairpin peptides, using the optimized force field. The results showed that the new force-field parameters gave structures more consistent with the experimental implications than the original AMBER ff99SB force field.
We review the background, theory and general equations for the analysis of equilibrium protein unfolding experiments, focusing on denaturant and heat-induced unfolding. The primary focus is on the thermodynamics of reversible folding/unfolding transitions and the experimental methods that are available for extracting thermodynamic parameters. We highlight the importance of modelling both how the folding equilibrium depends on a perturbing variable such as temperature or denaturant concentration, and the importance of modelling the baselines in the experimental observables.
We adopt the point of view that analysis of the stability of the protein folding process is central to understanding the underlying physics of folding. Stability of the folding process means that many perturbations do not disrupt the progress from the random coil to the native state. In this paper we explore the stability of folding using established methods from physics and mathematics. Our result is a preliminary theory of the physics of folding. We suggest some tests of these ideas using folding simulations. We begin by supposing that folding events are related in some way to mechanical waves on the molecule. We adopt an analytical approach to the physics which was pioneered by M.V. Berry, (in another context), based upon mathematics developed mainly by R. Thom and V.I. Arnold. We find that the stability of the folding process can be understood in terms of structures known as caustics, which occur in many kinds of wave phenomena. The picture that emerges is that natural selection has given us a set of protein molecules which have mechanical waves that propagate according to several mathematically specific restrictions. Successful simulations of folding can be used to test and constrain these wave motions. With some additional assumptions the theory explains or is consistent with a number of experimental facts about folding. We emphasize that this wave-based approach is fundamentally different from energy-based approaches.
Approximately 1% of the known protein structures display knotted configurations in their native fold but their function is not understood. It has been speculated that the entanglement may inhibit mechanical protein unfolding or transport, e.g., as in cellular threading or translocation processes through narrow biological pores. Here we investigate tigh peptide knot (TPK) characteristics in detail by pulling selected 3_1 and 4_1-knotted peptides using all-atom molecular dynamics computer simulations. We find that the 3_1 and 4_1-TPK lengths are typically Delta l~4.7 nm and 6.9 nm, respectively, for a wide range of tensions (F < 1.5 nN), pointing to a pore diameter of ~2 nm below which a translocated knotted protein might get stuck. The 4_1-knot length is in agreement with recent AFM pulling experiments. Detailed TPK characteristics however, may be sequence-specific: we find a different size and structural behavior in polyglycines, and, strikingly, a strong hydrogen bonding and water molecule trapping capability of hydrophobic TPKs due to side chain shielding of the polar TPK core. Water capturing and release is found to be controlla ble by the tightening force in a few cases. These mechanisms result into a sequence-specific locking and metastability of TPKs what might lead to a blocking of knotted peptide transport at designated sequence-positions. Intriguingly, macroscopic tight 4_1-knot structures are reproduced microscopically (figure-of-eight vs. the pretzel) and can be tuned by sequence in contrast to mathematical predictions. Our findings may explain a function of knots in native proteins, challenge previous studies on macromolecular knots, and may find use in bio- and nanotechnology.
The protein folding problem must ultimately be solved on all length scales from the atomic up through a hierarchy of complicated structures. By analyzing the stability of the folding process using physics and mathematics, this paper shows that features without length scales, i.e. topological features, are potentially of central importance. Topology is a natural mathematical tool for the study of shape and we avail ourselves of that tool to examine the relationship between the amino acid sequence and the shapes of protein molecules. We apply what we learn to conjectures about their biological evolution.
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