Resolving molecular determinants of mechanical stability of proteins is crucial in the rational design of advanced biomaterials for use in biomedical and nanotechnological applications. Here we present an interdisciplinary study combining bioinformatics screening, steered molecular dynamics simulations, protein engineering, and single-molecule force spectroscopy that explores the mechanical properties of a macro domain protein with mixed α þ β topology. The unique architecture is defined by a single seven-stranded β-sheet in the core of the protein flanked by five α-helices. Unlike mechanically stable proteins studied thus far, the macro domain provides the distinct advantage of having the key load-bearing hydrogen bonds (H bonds) buried in the hydrophobic core protected from water attacks. This feature allows direct measurement of the force required to break apart the load-bearing H bonds under locally hydrophobic conditions. Steered molecular dynamics simulations predicted extremely high mechanical stability of the macro domain by using constant velocity and constant force methods. Single-molecule force spectroscopy experiments confirm the exceptional mechanical strength of the macro domain, measuring a rupture force as high as 570 pN. Furthermore, through selective deletion of shielding peptide segments, we examined the same key H bonds under hydrophilic environments in which the β-strands are exposed to solvent and verify that the high mechanical stability of the macro domain results from excellent shielding of the load-bearing H bonds from competing water. Our study reveals that shielding water accessibility to the load-bearing strands is a critical molecular determinant for enhancing the mechanical stability of proteins.atomic force microscopy | biomimetic materials | molecular dynamics simulations | protein engineering | single-molecule force spectroscopy S ince the advent of steered molecular dynamics (SMD) simulations and single-molecule force spectroscopy (SMFS) using atomic force microscopy (AFM), elucidating nature's engineering principles for advanced biomaterials design has come to the forefront of biophysical and materials research (1-5). Combined SMFS and SMD protein unfolding studies characterizing the mechanical strength of elastomeric proteins have suggested that mechanical stability is primarily governed by two key parameters: the protein's secondary structure (6-10) and pulling geometry (11,12). In effect, domains having β-sandwich and β-grasp topologies stretched from their terminal, parallel H-bonded β-strands are most mechanically robust because of shearing of the strands upon forced unfolding, whereas α-helical domains are least mechanically stable (6-12). The dependence of mechanical stability on native topology has been verified by recent studies on proteins that have no natural load-bearing function (13-17) and even de novo designed proteins (18).Although the significance of protein sequence, topology, pulling geometry, and unfolding kinetics has emerged as molecular determinants of p...

This article describes results on using steered molecular dynamics (SMD) simulations and experimental single molecule force spectroscopy (SMFS) to investigate the relationship between hydrogen bonding and mechanical stability of a series of homodimeric β-sheet mimics. The dimers consisting of 4, 6, and 8 H-bonding sites were modeled in explicit chloroform solvent and the rupture force was studied using constant velocity SMD. The role of solvent structuring on the conformation of the dimers was analyzed and showed no significant contribution of chloroform molecules in the rupture event. The simulated stability of the dimers was validated by force data obtained with atomic force microscopy (AFM)-based SMFS in toluene. The computational model for the 8H dimer also offered insight into a possible mismatched dimer intermediate that may contribute to the lower than expected mechanical stability observed by single molecule AFM force studies. In addition, atomic level analysis of the rupture mechanism verified the dependence of mechanical strength on pulling trajectory due to the directional nature of chemical bonding under an applied force. The knowledge gained from this basic study will be used to guide further design of modular polymers having folded nanostructures through strategic programming of weak, non-covalent interactions into polymer backbones.

Native load-bearing proteins, such as the muscle protein titin, exhibit a remarkable degree of combined toughness, strength, and elasticity which have yet to be matched by synthetic materials. Single molecule nanomechanical studies on titin and other modular proteins suggest that these exceptional properties arise from a modular elongation mechanism. The sequential unfolding allows modular biopolymers to sustain a large force over the whole extension of the chain, which makes the polymer strong, along with a large area under the forceextension curve, making it tough as well. In addition, when the external force is removed, the unfolded domains of modular proteins will refold automatically, making them elastic. Inspired by nature, one research effort in my group is aimed at designing synthetic macromolecules that form high order structures by programming non-covalent interactions into polymer chain. The goal is to achieve synthetic biomaterials with combined strength, toughness and elasticity. Three classes of well-defined modular polymers have been synthesized in our laboratory: (1) using quadruple hydrogen-bonding motif 2-ureidon-4-pyrimidone (Upy) to direct the formation loops along a polymer chain (J. Am. Chem. Soc. 2004, 126, 2058; (2) using a peptidomimetic betasheet based double-closed loop (DCL) as module (J. Am. Chem. Soc. 2004, 126, 14328); and (3) an engineered protein G domain III as module. Single molecule force-extension experiments revealed the sequential unfolding of the loops or domains as these modular polymers are stretched, resulting in sawtooth-patterned curves similar to those seen in titin and other biopolymers. In this paper, we will discuss our designs, syntheses and single-molecule studies of polymers having modular domain structures. n K4.9.1 Mater. Res. Soc. Symp. Proc. Vol. 873E

No abstract