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

The toposiomerase IIIalpha-RMI1-RMI2 complex orients human Bloom’s syndrome helicase for efficient disruption of D-loops

Homologous recombination (HR) is a ubiquitous and efficient process that serves the repair of severe forms of DNA damage and the generation of genetic diversity during meiosis. HR can proceed via multiple pathways with different outcomes that may aid or impair genome stability and faithful inheritance, underscoring the importance of HR quality control. Human Bloom’s syndrome (BLM, RecQ family) helicase plays central roles in HR pathway selection and quality control via unexplored molecular mechanisms. Here we show that BLM’s multi-domain structural architecture supports a balance between stabilization and disruption of displacement loops (D-loops), early HR intermediates that are key targets for HR regulation. We find that this balance is markedly shifted toward efficient D-loop disruption by the presence of BLM’s interaction partners Topoisomerase IIIα-RMI1-RMI2, which have been shown to be involved in multiple steps of HR-based DNA repair. Our results point to a mechanism whereby BLM can differentially process D-loops and support HR control depending on cellular regulatory mechanisms

Direct observation of topoisomerase IA gate dynamics

Type IA topoisomerases cleave single-stranded DNA and relieve negative supercoils in discrete steps corresponding to the passage of the intact DNA strand through the cleaved strand. Although type IA topoisomerases are assumed to accomplish this strand passage via a protein-mediated DNA gate, opening of this gate has never been observed. We developed a single-molecule assay to directly measure gate opening of the Escherichia coli type IA topoisomerases I and III. We found that after cleavage of single-stranded DNA, the protein gate opens by as much as 6.6 nm and can close against forces in excess of 16 pN. Key differences in the cleavage, ligation, and gate dynamics of these two enzymes provide insights into their different cellular functions. The single-molecule results are broadly consistent with conformational changes obtained from molecular dynamics simulations. These results allowed us to develop a mechanistic model of interactions between type IA topoisomerases and single-stranded DNA

Surface Modification of Fluorescent Nanodiamonds for Biological Applications

Fluorescent nanodiamonds (FNDs) are a new class of carbon nanomaterials that offer great promise for biological applications such as cell labeling, imaging, and sensing due to their exceptional optical properties and biocompatibility. Implementation of these applications requires reliable and precise surface functionalization. Although diamonds are generally considered inert, they typically possess diverse surface groups that permit a range of different functionalization strategies. This review provides an overview of nanodiamond surface functionalization methods including homogeneous surface termination approaches (hydrogenation, halogenation, amination, oxidation, and reduction), in addition to covalent and non-covalent surface modification with different functional moieties. Furthermore, the subsequent coupling of biomolecules onto functionalized nanodiamonds is reviewed. Finally, biomedical applications of nanodiamonds are discussed in the context of functionalization