Single-Molecule Tracking of Collagenase on Native Type I Collagen Fibrils Reveals Degradation Mechanism

SummaryBackgroundCollagen, the most abundant human protein, is the principal component of the extracellular matrix and plays important roles in maintaining tissue and organ integrity. Highly resistant to proteolysis, fibrillar collagen is degraded by specific matrix metalloproteases (MMPs). Degradation of fibrillar collagen underlies processes including tissue remodeling, wound healing, and cancer metastasis. However, the mechanism of native collagen fibril degradation remains poorly understood.ResultsHere we present the results of high-resolution tracking of individual MMPs degrading type I collagen fibrils. MMP1 exhibits cleavage-dependent biased and hindered diffusion but spends 90% ± 3% of the time in one of at least two distinct pause states. One class of exponentially distributed pauses (class I pauses) occurs randomly along the fibril, whereas a second class of pauses (class II pauses) exhibits multistep escape kinetics and occurs periodically at intervals of 1.3 ± 0.2 μm and 1.5 ± 0.2 μm along the fibril. After these class II pauses, MMP1 moved faster and farther in one direction along the fibril, indicative of biased motion associated with cleavage. Simulations indicate that 5% ± 2% of the class II pauses result in the initiation of processive collagen degradation, which continues for bursts of 15 ± 4 consecutive cleavage events.ConclusionsThese findings provide a mechanistic paradigm for type I collagen degradation by MMP1 and establish a general approach to investigate MMP-fibrillar collagen interactions. More generally, this work demonstrates the fundamental role of enzyme-substrate interactions including binding and motion in determining the activity of an enzyme on an extended substrate

Vibrational Study of 13C-enriched C60 Crystals

The infrared (IR) spectrum of solid C60 exhibits many weak vibrational modes. Symmetry breaking due to 13C isotopes provides a possible route for optically activating IR-silent vibrational modes. Experimental spectra and a semi-empirical theory on natural abundance and 13C-enriched single crystals of C60 are presented. By comparing the experimental results with the theoretical results, we exclude this isotopic activation mechanism from the explanation for weakly active fundamentals in the spectra.Comment: Accepted for Phys. Rev. B, typeset in REVTEX v3.0 in LaTeX. Postscript file including figures is available at http://insti.physics.sunysb.edu/~mmartin/papers/c13twocol2.ps File with figures will be e-mailed by reques

Mixed-state twin observables

Twin observables, i.e. opposite subsystem observables A+ and A- that are indistinguishable in measurement in a given mixed or pure state W, are investigated in detail algebraicly and geometrically. It is shown that there is a far-reaching correspondence between the detectable (in W) spectral entities of the two operators. Twin observables are state-dependently quantum-logically equivalent, and direct subsystem measurement of one of them ipso facto gives rise to the indirect (i.e. distant) measurement of the other. Existence of nontrivial twins requires singularity of W. Systems in thermodynamic equilibrium do not admit subsystem twins. These observables may enable one to simplify the matrix representing W.Comment: 13 page

Nucleic Acids Res

Type II topoisomerases are essential enzymes that regulate DNA topology through a strand-passage mechanism. Some type II topoisomerases relax supercoils, unknot and decatenate DNA to below thermodynamic equilibrium. Several models of this non-equilibrium topology simplification phenomenon have been proposed. The kinetic proofreading (KPR) model postulates that strand passage requires a DNA-bound topoisomerase to collide twice in rapid succession with a second DNA segment, implying a quadratic relationship between DNA collision frequency and relaxation rate. To test this model, we used a single-molecule assay to measure the unlinking rate as a function of DNA collision frequency for Escherichia coli topoisomerase IV (topo IV) that displays efficient non-equilibrium topology simplification activity, and for E. coli topoisomerase III (topo III), a type IA topoisomerase that unlinks and unknots DNA to equilibrium levels. Contrary to the predictions of the KPR model, topo IV and topo III unlinking rates were linearly related to the DNA collision frequency. Furthermore, topo III exhibited decatenation activity comparable with that of topo IV, supporting proposed roles for topo III in DNA segregation. This study enables us to rule out the KPR model for non-equilibrium topology simplification. More generally, we establish an experimental approach to systematically control DNA collision frequency

Femtosecond Laser-Produced Plasma X-Rays from Periodically Modulated Surface Targets

We have studied theoretically and experimentally the x-ray production above 1 keV from femtosecond laser plasmas generated on periodically modulated surface targets. Laser energy coupling to plasma surface waves has been modeled using a numerical differential method. Almost total absorption of incident laser radiation is predicted for optimized interaction conditions. Silicon gratings have been irradiated by a 120fs Ti:sapphire laser at irradiances in excess of 1016 W/cm2. X-ray intensities above 1.5 keV (K-shell lines) have been measured as a function of the incidence angle. Results show a distinct x-ray emission maximum for the first order diffraction angle and are in good qualitative agreement with our theoretical predictions