High-speed AFM reveals accelerated binding of agitoxin-2 to a K+ channel by induced fit.

Agitoxin-2 (AgTx2) from scorpion venom is a potent blocker of K+ channels. The docking model has been elucidated, but it remains unclear whether binding dynamics are described by a two-state model (AgTx2-bound and AgTx2-unbound) or a more complicated mechanism, such as induced fit or conformational selection. Here, we observed the binding dynamics of AgTx2 to the KcsA channel using high-speed atomic force microscopy. From images of repeated binding and dissociation of AgTx2 to the channel, single-molecule kinetic analyses revealed that the affinity of the channel for AgTx2 increased during persistent binding and decreased during persistent dissociation. We propose a four-state model, including high- and low-affinity states of the channel, with relevant rate constants. An induced-fit pathway was dominant and accelerated binding by 400 times. This is the first analytical imaging of scorpion toxin binding in real time, which is applicable to various biological dynamics including channel ligands, DNA-modifier proteins, and antigen-antibody complexes.

Inorganic polyphosphate induces osteoblastic differentiation.

Inorganic polyphosphate [Poly(P)] is especially prevalent in osteoblasts. We tested the hypothesis that Poly(P) stimulates osteoblastic differentiation and polyphosphate metabolism for bone formation. The osteoblast-like cell line, MC 3T3-E1, was cultured with Poly(P), and gene expression was evaluated by real-time reverse-transcription polymerase chain-reaction. Phosphatase activity and extracellular matrix mineralization were also determined. The role of Poly(P) was assessed in a beagle dog alveolar bone regeneration model. Poly(P) increased osteocalcin, osterix, bone sialoprotein, and tissue non-specific alkaline phosphatase gene expression, with a high level of end-polyphosphatase activity, resulting in low-chain-length Poly(P), inorganic pyrophosphate, and inorganic phosphate production. MC3T3-E1 cells differentiated into mature osteoblasts and showed expression of ectonucleotide pyrophosphatase phosphodiesterase 1, while mouse progressive ankylosis gene expression remained unchanged. Promotion of alveolar bone regeneration was observed in Poly(P)-treated beagle dogs. These findings suggest that Poly(P) induces osteoblastic differentiation and bone mineralization, and acts as a resource for mineralization.

Inorganic polyphosphate: a possible stimulant of bone formation.

Inorganic polyphosphates [Poly(P)] are often distributed in osteoblasts. We undertook the present study to verify the hypothesis that Poly(P) stimulates osteoblasts and facilitates bone formation. The osteoblast-like cell line MC 3T3-E1 was cultured with Poly(P), and gene expression and potential mineralization were evaluated by reverse-transcription polymerase chain-reaction. Alkaline phosphatase activity, von Kossa staining, and resorption pit formation analyses were also determined. The potential role of Poly(P) in bone formation was assessed in a rat alveolar bone regeneration model. Poly(P) induced osteopontin, osteocalcin, collagen 1alpha, and osteoprotegerin expression and increased alkaline phosphatase activity in MC 3T3-E1 cells. Dentin slice pit formation decreased with mouse osteoblast and bone marrow macrophage co-cultivation in the presence of Poly(P). Promotion of alveolar bone regeneration was observed locally in Poly(P)-treated rats. These findings suggest that Poly(P) plays a role in osteoblastic differentiation, activation, and bone mineralization. Thus, local poly(P) delivery may have a therapeutic benefit in periodontal disease.

Atomic force microscopy of RecA--DNA complexes using a carbon nanotube tip.

We report high resolution images of RecA-double stranded (ds) DNA complexes obtained by atomic force microscopy (AFM). When a carbon nanotube (CNT) tip was used, AFM images visualized the 10-nm pitch of RecA-dsDNA complexes and RecA filaments as three-dimensional surface topography without reconstruction analysis. The depth of the notch between two pitches was less than 1 nm. When adsorbed on a soft surface covered with proteins, naked DNA, RecA monomers, RecA hexamers, and short RecA filaments were all clearly resolved in one image. The high resolution images with a CNT tip provided valuable information on the initiation process of RecA-dsDNA complex formation.

RecA-double stranded DNA complexes studied by atomic force microscopy.

RecA-double stranded (ds) DNA complexes have been studied by atomic force microscopy (AFM). When the complexes were prepared in the presence of ATP gamma S, fully covered RecA-dsDNA filaments were observed by AFM. When the concentration of RecA proteins was lower, various lengths of filaments were found. The variation of the observed structures may directly reflect the real distribution of the intermediate complexes in the reaction mixture, as the mixture was simply deposited on a mica surface for AFM observation without special fixation or staining. The use of a carbon nanotube (CNT) AFM tip enabled high resolution to reveal the periodicity of RecA-dsDNA filaments. Our observations demonstrated the potential of the AFM method for the structural studies of the RecA-dsDNA complexes, especially their intermediate states.