44 research outputs found
The role of binding site on the mechanical unfolding mechanism of ubiquitin.
We apply novel atomistic simulations based on potential energy surface exploration to investigate the constant force-induced unfolding of ubiquitin. At the experimentally-studied force clamping level of 100 pN, we find a new unfolding mechanism starting with the detachment between Ξ²5 and Ξ²3 involving the binding site of ubiquitin, the Ile44 residue. This new unfolding pathway leads to the discovery of new intermediate configurations, which correspond to the end-to-end extensions previously seen experimentally. More importantly, it demonstrates the novel finding that the binding site of ubiquitin can be responsible not only for its biological functions, but also its unfolding dynamics. We also report in contrast to previous single molecule constant force experiments that when the clamping force becomes smaller than about 300 pN, the number of intermediate configurations increases dramatically, where almost all unfolding events at 100 pN involve an intermediate configuration. By directly calculating the life times of the intermediate configurations from the height of the barriers that were crossed on the potential energy surface, we demonstrate that these intermediate states were likely not observed experimentally due to their lifetimes typically being about two orders of magnitude smaller than the experimental temporal resolution
Theoretical studies of the kinetics of mechanical unfolding of cross-linked polymer chains and their implications for single molecule pulling experiments
We have used kinetic Monte Carlo simulations to study the kinetics of
unfolding of cross-linked polymer chains under mechanical loading. As the ends
of a chain are pulled apart, the force transmitted by each crosslink increases
until it ruptures. The stochastic crosslink rupture process is assumed to be
governed by first order kinetics with a rate that depends exponentially on the
transmitted force. We have performed random searches to identify optimal
crosslink configurations whose unfolding requires a large applied force
(measure of strength) and/or large dissipated energy (measure of toughness). We
found that such optimal chains always involve cross-links arranged to form
parallel strands. The location of those optimal strands generally depends on
the loading rate. Optimal chains with a small number of cross-links were found
to be almost as strong and tough as optimal chains with a large number of
cross-links. Furthermore, optimality of chains with a small number of
cross-links can be easily destroyed by adding cross-links at random. The
present findings are relevant for the interpretation of single molecule force
probe spectroscopy studies of the mechanical unfolding of load-bearing
proteins, whose native topology often involves parallel strand arrangements
similar to the optimal configurations identified in the study
Dynamical Response of Nanomechanical Resonators to Biomolecular Interactions
We studied the dynamical response of a nanomechanical resonator to
biomolecular (e.g. DNA) adsorptions on a resonator's surface by using a
theoretical model, which considers the Hamiltonian H such that the potential
energy consists of elastic bending energy of a resonator and the potential
energy for biomolecular interactions. It was shown that the resonant frequency
shift of a resonator due to biomolecular adsorption depends on not only the
mass of adsorbed biomolecules but also the biomolecular interactions.
Specifically, for dsDNA adsorption on a resonator's surface, the resonant
frequency shift is also dependent on the ionic strength of a solvent, implying
the role of molecular interactions on the dynamic behavior of a resonator. This
indicates that nanomechanical resonators may enable one to quantify the
biomolecular mass, implying the enumeration of biomolecules, as well as gain
insight into intermolecular interactions between adsorbed biomolecules on the
surface.Comment: 17 page, 4 figures, accepted for publication at PRB. Physical Review
B, accepte
Nanomechanical In Situ Monitoring of Proteolysis of Peptide by Cathepsin B
Characterization and control of proteolysis of peptides by specific cellular protease is a priori requisite for effective drug discovery. Here, we report the nanomechanical, in situ monitoring of proteolysis of peptide chain attributed to protease (Cathepsin B) by using a resonant nanomechanical microcantilever immersed in a liquid. Specifically, the detection is based on measurement of resonant frequency shift arising from proteolysis of peptides (leading to decrease of cantilever's overall mass, and consequently, increases in the resonance). It is shown that resonant microcantilever enables the quantification of proteolysis efficacy with respect to protease concentration. Remarkably, the nanomechanical, in situ monitoring of proteolysis allows us to gain insight into the kinetics of proteolysis of peptides, which is well depicted by Langmuir kinetic model. This implies that nanomechanical biosensor enables the characterization of specific cellular protease such as its kinetics
Aptamer-functionalized nano-pattern based on carbon nanotube for sensitive, selective protein detection
We have developed a horizontally aligned carbon nanotube sensor that enables not only the specific detection of biomolecules with ultra-sensitivity, but also the quantitative characterization of binding affinity between biomolecules and/or interaction between a carbon nanotube and a biomolecule, for future applications in early diagnostics. In particular, we have fabricated horizontally aligned carbon nanotubes, which were functionalized with specific aptamers that are able to specifically bind to biomolecules (i.e. thrombin). Our detection system is based on scanning probe microscopy (SPM) imaging for horizontally aligned aptamer-conjugated carbon nanotubes (ACNTs) that specifically react with target biomolecules at an ultra-low concentration. It is shown that the binding affinity between thrombin molecule and ACNT can be quantitatively characterized using SPM imaging. It is also found that the smart carbon nanotube sensor coupled with SPM imaging permits us to achieve the high detection sensitivity even up to similar to 1 pM, which is much higher than that of other bioassay methods. Moreover, we have shown that our method enables a quantitative study on small molecule-mediated inhibition of specific biomolecular interactions. In addition, we have shown that our ACNT-based system allows for the quantitative study of the effect of chemical environment (e.g. pH and ion concentration) on the binding affinity. Our study sheds light on carbon nanotube sensor coupled with SPM imaging, which opens a new avenue to early diagnostics and drug screening with high sensitivity.close2
Experimental and Computational Characterization of Biological Liquid Crystals: A Review of Single-Molecule Bioassays
Quantitative understanding of the mechanical behavior of biological liquid crystals such as proteins is essential for gaining insight into their biological functions, since some proteins perform notable mechanical functions. Recently, single-molecule experiments have allowed not only the quantitative characterization of the mechanical behavior of proteins such as protein unfolding mechanics, but also the exploration of the free energy landscape for protein folding. In this work, we have reviewed the current state-of-art in single-molecule bioassays that enable quantitative studies on protein unfolding mechanics and/or various molecular interactions. Specifically, single-molecule pulling experiments based on atomic force microscopy (AFM) have been overviewed. In addition, the computational simulations on single-molecule pulling experiments have been reviewed. We have also reviewed the AFM cantilever-based bioassay that provides insight into various molecular interactions. Our review highlights the AFM-based single-molecule bioassay for quantitative characterization of biological liquid crystals such as proteins