186 research outputs found
Antilipolytic effects of N6-phenylisopropyladenosine and prostaglandin E2 in fat-cells of obese volunteers before and during energy restriction
Influence of Hydrodynamic Interactions on Mechanical Unfolding of Proteins
We incorporate hydrodynamic interactions in a structure-based model of
ubiquitin and demonstrate that the hydrodynamic coupling may reduce the peak
force when stretching the protein at constant speed, especially at larger
speeds. Hydrodynamic interactions are also shown to facilitate unfolding at
constant force and inhibit stretching by fluid flows.Comment: to be published in Journal of Physics: Condensed Matte
Knotting and unknotting of a protein in single molecule experiments.
Spontaneous folding of a polypeptide chain into a knotted structure remains one of the most puzzling and fascinating features of protein folding. The folding of knotted proteins is on the timescale of minutes and thus hard to reproduce with atomistic simulations that have been able to reproduce features of ultrafast folding in great detail. Furthermore, it is generally not possible to control the topology of the unfolded state. Single-molecule force spectroscopy is an ideal tool for overcoming this problem: by variation of pulling directions, we controlled the knotting topology of the unfolded state of the 52-knotted protein ubiquitin C-terminal hydrolase isoenzyme L1 (UCH-L1) and have therefore been able to quantify the influence of knotting on its folding rate. Here, we provide direct evidence that a threading event associated with formation of either a 31 or 52 knot, or a step closely associated with it, significantly slows down the folding of UCH-L1. The results of the optical tweezers experiments highlight the complex nature of the folding pathway, many additional intermediate structures being detected that cannot be resolved by intrinsic fluorescence. Mechanical stretching of knotted proteins is also of importance for understanding the possible implications of knots in proteins for cellular degradation. Compared with a simple 31 knot, we measure a significantly larger size for the 52 knot in the unfolded state that can be further tightened with higher forces. Our results highlight the potential difficulties in degrading a 52 knot compared with a 31 knot.N.C.H.L. is supported by a UBD Chancellor’s Scholarship from the Brunei Government. W.N. was supported by the Ministry of Education (MoE) Singapore. S.S.M acknowledges funding from the Alexander von Humboldt Foundation. This work was supported by an SFB 863 A2 grant of Deutsche Forschungsgemeinschaft to M.R
Theory of mechanical unfolding of homopolymer globule: all-or-none transition in force-clamp mode vs phase coexistence in position-clamp mode
Equilibrium mechanical unfolding of a globule formed by long flexible
homopolymer chain collapsed in a poor solvent and subjected to an extensional
force f (force-clamp mode) or extensional deformation D (position-clamp mode)
is studied theoretically. Our analysis, like all previous analysis of this
problem, shows that the globule behaves essentially differently in two modes of
extension. In the force-clamp mode, mechanical unfolding of the globule with
increasing applied force occurs without intramolecular microphase segregation,
and at certain threshold value of the pulling force the globule unfolds as a
whole ("all-or-none" transition). The value of the threshold force and the
corresponding jump in the distance between the chain ends increase with a
deterioration of the solvent quality and/or with an increase in the degree of
polymerization. In the position-clamp mode, the globule unfolding occurs via
intramolecular microphase coexistence of globular and extended microphases
followed by an abrupt unraveling transition. Reaction force in the microphase
segregation regime demonstrates an "anomalous" decrease with increasing
extension. Comparison of deformation curves in force and position-clamp modes
demonstrates that at weak and strong extensions the curves for two modes
coincide, differences are observed in the intermediate extension range. Another
unfolding scenario is typical for short globules: in both modes of extension
they unfold continuously, without jumps or intramolecular microphase
coexistence, by passing a sequence of uniformly elongated configurations.Comment: 19 pages, 13 figures, 1 tabl
Human Factors Operability Timeline Analysis to Improve the Processing Flow of the Orion Spacecraft
The Constellation Program (CxP) Orion vehicle goes through several areas and stages of processing before its launched at the Kennedy Space Center. In order to have efficient and effective processing, all of the activities need to be analyzed. This was accomplished by first developing a timeline of events that included each activity, and then each activity was analyzed by operability experts and human factors experts with spacecraft processing experience. This papers focus is to explain the results and the process for developing this human factors operability timeline analysis to improve the processing flow of Orion
Effects of multiple-bond ruptures on kinetic parameters extracted from force spectroscopy measurements: Revisiting biotin-streptavidin interactions
Force spectroscopy measurements of the rupture of the molecular bond between biotin and streptavidin often results in a wide distribution of rupture forces. We attribute the long tail of high rupture forces to the nearly simultaneous rupture of more than one molecular bond. To decrease the number of possible bonds, we employed hydrophilic polymeric tethers to attach biotin molecules to the atomic force microscope probe. It is shown that the measured distributions of rupture forces still contain high forces that cannot be described by the forced dissociation from a deep potential well. We employed a recently developed analytical model of simultaneous rupture of two bonds connected by polymer tethers with uneven length to fit the measured distributions. The resulting kinetic parameters agree with the energy landscape predicted by molecular dynamics simulations. It is demonstrated that when more than one molecular bond might rupture during the pulling measurements there is a noise-limited range of probe velocities where the kinetic parameters measured by force spectroscopy correspond to the true energy landscape. Outside this range of velocities, the kinetic parameters extracted by using the standard most probable force approach might be interpreted as artificial energy barriers that are not present in the actual energy landscape. Factors that affect the range of useful velocities are discussed
Direct single-molecule observation of calcium-dependent misfolding in human neuronal calcium sensor-1
Single-molecule experiments in biological physics: methods and applications
I review single-molecule experiments (SME) in biological physics. Recent
technological developments have provided the tools to design and build
scientific instruments of high enough sensitivity and precision to manipulate
and visualize individual molecules and measure microscopic forces. Using SME it
is possible to: manipulate molecules one at a time and measure distributions
describing molecular properties; characterize the kinetics of biomolecular
reactions and; detect molecular intermediates. SME provide the additional
information about thermodynamics and kinetics of biomolecular processes. This
complements information obtained in traditional bulk assays. In SME it is also
possible to measure small energies and detect large Brownian deviations in
biomolecular reactions, thereby offering new methods and systems to scrutinize
the basic foundations of statistical mechanics. This review is written at a
very introductory level emphasizing the importance of SME to scientists
interested in knowing the common playground of ideas and the interdisciplinary
topics accessible by these techniques. The review discusses SME from an
experimental perspective, first exposing the most common experimental
methodologies and later presenting various molecular systems where such
techniques have been applied. I briefly discuss experimental techniques such as
atomic-force microscopy (AFM), laser optical tweezers (LOT), magnetic tweezers
(MT), biomembrane force probe (BFP) and single-molecule fluorescence (SMF). I
then present several applications of SME to the study of nucleic acids (DNA,
RNA and DNA condensation), proteins (protein-protein interactions, protein
folding and molecular motors). Finally, I discuss applications of SME to the
study of the nonequilibrium thermodynamics of small systems and the
experimental verification of fluctuation theorems. I conclude with a discussion
of open questions and future perspectives.Comment: Latex, 60 pages, 12 figures, Topical Review for J. Phys. C (Cond.
Matt
Calcium modulates force sensing by the von Willebrand factor A2 domain
von Willebrand factor (VWF) multimers mediate primary adhesion and aggregation of platelets. VWF potency critically depends on multimer size, which is regulated by a feedback mechanism involving shear-induced unfolding of the VWF-A2 domain and cleavage by the metalloprotease ADAMTS-13. Here we report crystallographic and single-molecule optical tweezers data on VWF-A2 providing mechanistic insight into calcium-mediated stabilization of the native conformation that protects A2 from cleavage by ADAMTS-13. Unfolding of A2 requires higher forces when calcium is present and primarily proceeds through a mechanically stable intermediate with non-native calcium coordination. Calcium further accelerates refolding markedly, in particular, under applied load. We propose that calcium improves force sensing by allowing reversible force switching under physiologically relevant hydrodynamic conditions. Our data show for the first time the relevance of metal coordination for mechanical properties of a protein involved in mechanosensing
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