Hai Pan , Meng Qin , Wei Meng , Yi Cao, and Wei Wang
Owing to their many outstanding features, such as small size, large surface area, and cell penetration ability, nanoparticles have been increasingly used in medicine and biomaterials as drug carriers and diagnostic or therapeutic agents. However, our understanding of the interactions of biological entities, especially proteins, with nanoparticles is far behind the explosive development of nanotechnology. In typical protein–nanoparticle interactions, two processes (i.e., surface binding and conformational changes in proteins) are intermingled with each other and have not yet been quantitatively described. Here, by using a stopped-flow fast mixing technique, we were able to shed light on the kinetics of the adsorption-induced protein unfolding on nanoparticle surfaces in detail. We observed a biphasic denaturation behavior of protein GB1 on latex nanoparticle surfaces. Such kinetics can be adequately described by a fast equilibrium adsorption followed by a slow reversible unfolding of GB1. On the basis of this model, we quantitatively measured all rate constants that are involved in this process, from which the free-energy profile is constructed. This allows us to evaluate the effects of environmental factors, such as pH and ionic strength, on both the adsorption and the conformational change in GB1 on the latex nanoparticle surface. These studies provide a general physical picture of the adsorption-induced unfolding of proteins on nanoparticle surfaces and a quantitative description of the energetics of each transition. We anticipate that it will greatly advance our current understanding of protein–nanoparticle interactions and will be helpful for the rational control of such interactions in biomedical applications.
DOI
Journal: Langmuir
Jordi Ribas-Arino and Dominik Marx
DOI
Journal: Chemical Reviews
Hao Yu, Amar Nath Gupta, Xia Liu,, Krishna Neupane, Angela M. Brigley, Iveta Sosova, and Michael T. Woodside
Protein folding is described conceptually in terms of diffusion over a configurational free-energy landscape, typically reduced to a one-dimensional profile along a reaction coordinate. In principle, kinetic properties can be predicted directly from the landscape profile using Kramers theory for diffusive barrier crossing, including the folding rates and the transition time for crossing the barrier. Landscape theory has been widely applied to interpret the time scales for protein conformational dynamics, but protein folding rates and transition times have not been calculated directly from experimentally measured free-energy profiles. We characterized the energy landscape for native folding of the prion protein using force spectroscopy, measuring the change in extension of a single protein molecule at high resolution as it unfolded/refolded under tension. Key parameters describing the landscape profile were first recovered from the distributions of unfolding and refolding forces, allowing the diffusion constant for barrier crossing and the transition path time across the barrier to be calculated. The full landscape profile was then reconstructed from force-extension curves, revealing a double-well potential with an extended, partially unfolded transition state. The barrier height and position were consistent with the previous results. Finally, Kramers theory was used to predict the folding rates from the landscape profile, recovering the values observed experimentally both under tension and at zero force in ensemble experiments. These results demonstrate how advances in single-molecule theory and experiment are harnessing the power of landscape formalisms to describe quantitatively the mechanics of folding.
DOI
Journal: Proceedings of the National Academy of Sciences
Ronen Berkovich, Rodolfo I. Hermans, Ionel Popa, Guillaume Stirnemann, Sergi Garcia-Manyes, Bruce J. Berne, and Julio M. Fernandez
The elastic restoring force of tissues must be able to operate over the very wide range of loading rates experienced by living organisms. It is surprising that even the fastest events involving animal muscle tissues do not surpass a few hundred hertz. We propose that this limit is set in part by the elastic dynamics of tethered proteins extending and relaxing under a changing load. Here we study the elastic dynamics of tethered proteins using a fast force spectrometer with sub-millisecond time resolution, combined with Brownian and Molecular Dynamics simulations. We show that the act of tethering a polypeptide to an object, an inseparable part of protein elasticity in vivo and in experimental setups, greatly reduces the attempt frequency with which the protein samples its free energy. Indeed, our data shows that a tethered polypeptide can traverse its free-energy landscape with a surprisingly low effective diffusion coefficient Deff ∼ 1,200 nm2/s. By contrast, our Molecular Dynamics simulations show that diffusion of an isolated protein under force occurs at Deff ∼ 108 nm2/s. This discrepancy is attributed to the drag force caused by the tethering object. From the physiological time scales of tissue elasticity, we calculate that tethered elastic proteins equilibrate in vivo withDeff ∼ 104–106 nm2/s which is two to four orders magnitude smaller than the values measured for untethered proteins in bulk.
DOI
Journal: Proceedings of the National Academy of Sciences
Raymond W. Friddle, Aleksandr Noy, and James J. De Yoreo
Single molecule force spectroscopy probes the strength, lifetime, and energetic details of intermolecular interactions in a simple experiment. A growing number of these studies have reported distinctly nonlinear trends in rupture force with loading rate that are typically explained in conventional models by invoking complex escape pathways. Recent analyses suggested that these trends should be expected even for simple barriers based on the basic assumptions of bond rupture dynamics and thus may represent the norm rather than the exception. Here we explore how these nonlinear trends reflect the two fundamental regimes of bond rupture: (i) a near-equilibrium regime, produced either by bond reforming in the case of a single bond or by asynchronized rupture of multiple individual bonds, and (ii) a kinetic regime produced by fast, non-equilibrium bond rupture. We analyze both single- and multi-bonded cases, describe the full evolution of the system as it transitions between near- and far-from-equilibrium loading regimes, and show that both interpretations produce essentially identical force spectra. Data from 10 different molecular systems show that this model provides a comprehensive description of force spectra for a diverse suite of bonds over experimentally relevant loading rates, removes the inconsistencies of previous interpretations of transition state distances, and gives ready access to both kinetic and thermodynamic information about the interaction. These results imply that single-molecule binding free energies for a vast number of bonds have already been measured.
DOI
Journal: Proceedings of the National Academy of Sciences
Krishna Neupane, Dustin B. Ritchie, Hao Yu1, Daniel A. N. Foster, Feng Wang, and Michael T. Woodside
The duration of structural transitions in biopolymers is only a fraction of the time spent searching diffusively over the configurational energy landscape. We found the transition time, τTP, and the diffusion constant, D, for DNA and RNA folding using energy landscapes obtained from single-molecule trajectories under tension in optical traps. DNA hairpins, RNA pseudoknots, and a riboswitch all had τTP∼10 μs and D∼10-13–14 m2/s, despite widely differing unfolding rates. These results show how energy-landscape analysis can be harnessed to characterize brief but critical events during folding reactions.
DOI
Journal: Physical Review Letters
Ibrahim Mohammad, Pamma D. Arora, Yeganeh Naghibzadeh, Yongqiang Wang, Jeff Li, Wendall Mascarenhas, Paul A. Janmey, John F. Dawson and Christopher A. McCulloch
The role of adhesion-associated actin-binding proteins in cell migration is not well defined. In mouse fibroblasts we screened for focal adhesion-associated proteins that were isolated with collagen-coated beads and detected by tandem mass spectrometry. We identified flightless I (FliI) as an actin-binding protein in focal adhesion fractions, which was verified by immunoblotting. By confocal microscopy most FliI was distributed throughout the cytosol and in focal adhesions. By sedimentation assays and in vitro binding assays, we found that FliI associates with actin filaments and actin monomers. Assays using purified proteins showed that FliI inhibits actin polymerization and caps but does not sever actin filaments. Cells with FliI knockdown or cells overexpressing FliI migrated more or less rapidly, respectively, than wild-type controls. Compared with controls, cells with FliI knockdown were less adherent than wild-type cells, exhibited reduced numbers of focal adhesions containing activated β1 integrins and vinculin, and exhibited increased incorporation of actin monomers into nascent filaments at focal adhesions. These data indicate that FliI regulates cell migration through its localization to focal adhesions and its ability to cap actin filaments, which collectively affect focal adhesion maturation.—Mohammad, I., Arora, P. D., Naghibzadeh, Y., Wang, Y., Li, J., Mascarenhas, W., Janmey, P. A., Dawson, J. F., McCulloch, C. A. Flightless I is a focal adhesion-associated actin-capping protein that regulates cell migration.
DOI
Journal: The FASEB Journal
