Computational Studies of Mechanical Signal Transduction in Proteins

Cellular signaling is a system of complex interplay of communications that guides cellular processes and coordinates cell behavior. In this dissertation, united by three main themes, investigations of mechanisms of mechanical signal transduction in proteins are presented. The first theme focuses on chemical to mechanical signal transduction. Using molecular dynamics simulations, aspects of thin filament calcium-induced regulation are investigated. The calcium-dependent behavior of skeletal troponin, and key conformational events subsequent to calcium expulsion from troponin C regulatory sites are described. Dynamics of cardiac troponin C, and details of residue coordination leading to calcium binding in the regulatory site are elucidated. These findings are incorporated into a model integrating the calcium dependent behavior of troponin to its ability to interact with actin and regulate muscle contraction. The second theme focuses on steered molecular dynamics (SMD) investigations of force induced mechanical unfolding of slipknot proteins. Unfolding of slipknot AFV3-109 occurs via either two-state, or three-state process involving the formation of a stable intermediate state. The results demonstrate a mechanical unfolding pathway bifurcation and potential gearbox mechanism with non similar responses to pulling force that may enable differential mechanical signal transduction. The third theme focuses on two aspects of mechanical proteins stabilization to mechanical unfolding - the solvent environment effect and neighboring beta strands effect. The solvent environment plays an integral role in cellular processes. SMD unfolding of I27 and solvent substitution were combined to reveal that solvent environment modulates the force resistance of I27. During unfolding, solvent molecules interact with I27's force bearing patch, in solvent molecule geometry-dependent mode multiplicity. Protein topology and pulling geometry play important roles in determining protein mechanical stability. The critical importance of neighboring beta strands stabilization effect is explored. The proteins Top7 and barstar have similar force-bearing topology but different mechanical stability. SMD simulations of barstar reveal that barstar unfolds by beta strand peeling whereas Top7, which has two additional beta strands in its force bearing patch unfolds via substructure-sliding. This neighboring beta strands stabilization effect may be a general mechanism in protein mechanics and de-novo design guideline for mechanically stable proteins with novel topology

Hydrophobic core and hydrophobic residue re-packing following release of Ca²⁺.

<p>In all panels the saturated, open state N-lobe TnC is shown in red and the depleted, closed state N-lobe TnC is shown in blue. <b>A.</b> A hydrophobic PHE scaffold remains undisturbed in the open to closed transition. Residues PHE74, PHE77 and PHE25 are shown as vdW spheres. Top panel shows the TnC open state, bottom panel shows the TnC closed state. Green arrows point to PHE74 in each panel. <b>B.</b> Expulsion of residue PHE28 into the solvent and sliding of VAL44 into the hydrophobic core in the open to closed transition. Top panel shows the TnC open state, bottom panel shows the TnC closed state. Orange arrows point to PHE28 in each panel. <b>C.</b> Tight repacking of MET81, MET80 and MET45, which finish in a tight formation upon closure of the hydrophobic pocket, takes place in the open to closed transition. Top panel shows the TnC open state, bottom panel shows the TnC closed state. Violet arrows point to MET80 and MET81 in each panel.</p

Calcium Induced Regulation of Skeletal Troponin — Computational Insights from Molecular Dynamics Simulations

<div><p>The interaction between calcium and the regulatory site(s) of striated muscle regulatory protein troponin switches on and off muscle contraction. In skeletal troponin binding of calcium to sites I and II of the TnC subunit results in a set of structural changes in the troponin complex, displaces tropomyosin along the actin filament and allows myosin-actin interaction to produce mechanical force. In this study, we used molecular dynamics simulations to characterize the calcium dependent dynamics of the fast skeletal troponin molecule and its TnC subunit in the calcium saturated and depleted states. We focused on the N-lobe and on describing the atomic level events that take place subsequent to removal of the calcium ion from the regulatory sites I and II. A main structural event - a closure of the A/B helix hydrophobic pocket results from the integrated effect of the following conformational changes: the breakage of H-bond interactions between the backbone nitrogen atoms of the residues at positions 2, 9 and sidechain oxygen atoms of the residue at position 12 (N²-OE¹²/N⁹-OE¹²) in sites I and II; expansion of sites I and II and increased site II N-terminal end-segment flexibility; strengthening of the β-sheet scaffold; and the subsequent re-packing of the N-lobe hydrophobic residues. Additionally, the calcium release allows the N-lobe to rotate relative to the rest of the Tn molecule. Based on the findings presented herein we propose a novel model of skeletal thin filament regulation.</p> </div

The core domain of the troponin molecule.

<p>The Ca²⁺ sensor troponin consist of 3 subunits. The TnI subunit is shown in blue, the TnC subunit is shown in red and the TnT subunit is show in yellow. Ca²⁺ ions located in sites I through IV are shown as green vdW spheres. Sites III and IV located in the IT arm have a higher affinity to Ca²⁺ and do not serve a regulatory role. The N-terminal of TnT, the C-terminal of TnT and the C-terminal of TnI are not present in the crystal structure and are not displayed herein. Molecular coordinates were obtained from 1YTZ.pdb.</p

Mechanically Untying a Protein Slipknot: Multiple Pathways Revealed by Force Spectroscopy and Steered Molecular Dynamics Simulations

Protein structure is highly diverse when considering a wide range of protein types, helping to give rise to the multitude of functions that proteins perform. In particular, certain proteins are known to adopt a knotted or slipknotted fold. How such proteins undergo mechanical unfolding was investigated utilizing a combination of single molecule atomic force microscopy (AFM), protein engineering, and steered molecular dynamics (SMD) simulations to show the mechanical unfolding mechanism of the slipknotted protein AFV3-109. Our results reveal that the mechanical unfolding of AFV3-109 can proceed via multiple parallel unfolding pathways that all cause the protein slipknot to untie and the polypeptide chain to completely extend. These distinct unfolding pathways proceed via either a two- or three-state unfolding process involving the formation of a well-defined, stable intermediate state. SMD simulations predict the same contour length increments for different unfolding pathways as single molecule AFM results, thus providing a plausible molecular mechanism for the mechanical unfolding of AFV3-109. These SMD simulations also reveal that two-state unfolding is initiated from both the N- and C-termini, while three-state unfolding is initiated only from the C-terminus. In both pathways, the protein slipknot was untied during unfolding, and no tightened slipknot conformation was observed. Detailed analysis revealed that interactions between key structural elements lock the knotting loop in place, preventing it from shrinking and the formation of a tightened slipknot conformation. Our results demonstrate the bifurcation of the mechanical unfolding pathway of AFV3-109 and point to the generality of a kinetic partitioning mechanism for protein folding/unfolding

Additional file 1 of Clinical and genetic characterization of 47 Chinese pediatric patients with Pitt–Hopkins syndrome: a retrospective study

Additional file 1: Table S1. A table detailing the clinical signs and genomic defects