Mu Gao

Steered molecular dynamics and mechanical functions of proteins

Atomic force microscopy of single molecules, steered molecular dynamics and the theory of stochastic processes have established a new field that investigates mechanical functions of proteins, such as ligand-receptor binding/unbinding and elasticity of muscle proteins during stretching. The combination of these methods yields information on the energy landscape that controls mechanical function and on the force-bearing components of proteins, as well as on the underlying physical mechanisms.

Structure and functional significance of mechanically unfolded fibronectin type III1 intermediates.

Fibronectin (FN) forms fibrillar networks coupling cells to the extracellular matrix. The formation of FN fibrils, fibrillogenesis, is a tightly regulated process involving the exposure of cryptic binding sites in individual FN type III (FN-III) repeats presumably exposed by mechanical tension. The FN-III1 module has been previously proposed to contain such cryptic sites that promote the assembly of extracellular matrix FN fibrils. We have combined NMR and steered molecular dynamics simulations to study the structure and mechanical unfolding pathway of FN-III1. This study finds that FN-III1 consists of a β-sandwich structure that unfolds to a mechanically stable intermediate about four times the length of the native folded state. Considering previous experimental findings, our studies provide a structural model by which mechanical stretching of FN-III1 may induce fibrillogenesis through this partially unfolded intermediate.

Identifying unfolding intermediates of FN-III10 by steered molecular dynamics

Experimental studies have indicated that FN-III modules undergo reversible unfolding as a mechanism of elasticity. The unfolding of FN-III modules, including the cell-binding FN-III(10) module, has further been suggested to be functionally relevant by exposing buried cryptic sites or modulating cell binding. While steered molecular dynamics (SMD) simulations have provided one tool to investigate this process, computational requirements so far have limited detailed analysis to the early stages of unfolding. Here, we use an extended periodic box to probe the unfolding of FN-III(10) for extensions longer than 60A. Up to three plateaus, corresponding to three metastable intermediates, were observed in the extension-time profile from SMD stretching of FN-III(10). The first and second plateaus correspond to a twisted and an aligned state prior to unraveling FN-III(10) beta-strands. The third plateau, at an extension of approximately 100A, follows unraveling of FN-III(10) A and B-strands and precedes breaking of inter-strand hydrogen bonds between F and G-strands. The simulations revealed three forced unfolding pathways of FN-III(10), one of which is preferentially selected under physiological conditions. Implications for fibronectin fibrillogenesis are discussed.

How the headpiece hinge angle is opened: new insights into the dynamics of integrin activation

How the integrin head transitions to the high-affinity conformation is debated. Although experiments link activation with the opening of the hinge angle between the βA and hybrid domains in the ligand-binding headpiece, this hinge is closed in the liganded αvβ3 integrin crystal structure. We replaced the RGD peptide ligand of this structure with the 10th type III fibronectin module (FnIII10) and discovered through molecular dynamics (MD) equilibrations that when the conformational constraints of the leg domains are lifted, the βA/hybrid hinge opens spontaneously. Together with additional equilibrations on the same nanosecond timescale in which small structural variations impeded hinge-angle opening, these simulations allowed us to identify the allosteric pathway along which ligand-induced strain propagates via elastic distortions of the α1 helix to the βA/hybrid domain hinge. Finally, we show with steered MD how force accelerates hinge-angle opening along the same allosteric pathway. Together with available experimental data, these predictions provide a novel framework for understanding integrin activation.

Steered molecular dynamics studies of titin I1 domain unfolding.

The cardiac muscle protein titin, responsible for developing passive elasticity and extensibility of muscle, possesses about 40 immunoglobulin-like (Ig) domains in its I-band region. Atomic force microscopy (AFM) and steered molecular dynamics (SMD) have been successfully combined to investigate the reversible unfolding of individual Ig domains. However, previous SMD studies of titin I-band modules have been restricted to I27, the only structurally known Ig domain from the distal region of the titin I-band. In this paper we report SMD simulations unfolding I1, the first structurally available Ig domain from the proximal region of the titin I-band. The simulations are carried out with a view toward upcoming atomic force microscopy experiments. Both constant velocity and constant force stretching have been employed to model mechanical unfolding of oxidized I1, which has a disulfide bond bridging beta-strands C and E, as well as reduced I1, in which the disulfide bridge is absent. The simulations reveal that I1 is protected against external stress mainly through six interstrand hydrogen bonds between its A and B beta-strands. The disulfide bond enhances the mechanical stability of oxidized I1 domains by restricting the rupture of backbone hydrogen bonds between the A- and G-strands. The disulfide bond also limits the maximum extension of I1 to approximately 220 A. Comparison of the unfolding pathways of I1 and I27 are provided and implications to AFM experiments are discussed.

Unfolding of titin domains studied by molecular dynamics simulations

Titin, a ∼1 μm long protein found in striated muscle myofibrils, possesses unique elastic properties. The extensible behavior of titin has been demonstrated in atomic force microscopy and optical tweezer experiments to involve the reversible unfolding of individual immunoglobulin-like (Ig) domains. We have used steered molecular dynamics (SMD), a novel computer simulation method, to investigate the mechanical response of single titin Ig domains upon stress. Simulations of stretching Ig domains I1 and I27 have been performed in a solvent of explicit water molecules. The SMD approach provides a detailed structural and dynamic description of how Ig domains react to external forces. Validation of SMD results includes both qualitative and quantitative agreement with AFM recordings. Furthermore, combining SMD with single molecule experimental data leads to a comprehensive understanding of Ig domains mechanical properties. A set of backbone hydrogen bonds that link the domains terminal β-strands play a key role in the mechanical resistance to external forces. Slight differences in architecture permit a mechanical unfolding intermediate for I27, but not for I1. Refolding simulations of I27 demonstrate a locking mechanism.

Simulated Refolding of Stretched Titin Immunoglobulin Domains

Steered molecular dynamics (SMD) is used to investigate forced unfolding and spontaneous refolding of immunoglobulin I27, a domain of the muscle protein titin. Previous SMD simulations revealed the events leading to stretch-induced unfolding of I27, the rupture of hydrogen bonds bridging beta-strands A and B, and those bridging beta-strands A and G, the latter rupture occurring at an extension of approximately 15 A and preceding the complete unfolding. Simulations are now used to study the refolding of partially unfolded I27 domains. The results reveal that stretched domains with ruptured interstrand hydrogen bonds shrink along the extension direction. Two types of refolding patterns are recognized: for separated beta-strands A and G, in most simulations five of the six hydrogen bonds between A and G stably reformed in 2 ns, whereas for separated beta-strands A and B hydrogen bonds seldom reformed in eight 2-ns simulations. The mechanical stability of the partially refolded intermediates has been tested by re-stretching.

Large Scale Simulation of Protein Mechanics and Function

Publisher Summary The chapter illustrates the state of art in large-scale biomolecular modeling. Such modeling has become feasible only through parallel computing utilizing hundreds and soon thousands of processors. Fortunately, the necessary computers are available to many researchers and the needed new generation of molecular dynamics and molecular visualization programs has been developed and these resources are widely shared. Harnessing them effectively requires great effort, to which purpose the authors group has developed the molecular dynamics program Not (just) Another Molecular Dynamics program (NAMD). For the graphical analysis of the ensuing gigabytes and terabytes of data the group has developed the program Visual Molecular Dynamics (VMD). These programs are widely used today since the increasing availability of protein structures has led to most biomedical researchers using structural information for the design and analysis of their experiments. The chapter describes NAMD and VMD and illustrates three exemplary simulations of large-scale systems that are presently the subject of intense research. The functions of all three proteins investigated are mainly mechanical. NAMD is primarily designed to work with Chemistry at HARvard Molecular Mechanics (CHARMM) force field parameters. Input files for NAMD can be generated using CHARMM, X-PLOR, or VMD. NAMD has also been extended to read Assisted Model Building with Energy Refinement (AMBER) and GROningen MAchine for Chemical Simulations (GROMACS) input file formats. NAMD running on 768 processors has enabled a record-breaking two million atom 5 ns simulation of the ribosome using an AMBER force field. To relate the observed mechanics to the architecture of proteins is the domain of so-called steered molecular dynamics simulations, contributing to the founding of the new field of mechanobiology, which studies the role of forces in cellular processes.