Background— An open-irrigated radiofrequency (RF) ablation catheter was developed to measure contact force (CF). Three optical fibers measure microdeformation of the catheter tip. The purpose of this study was to (1) validate the accuracy of CF sensor (CFS) (bench test); and (2) determine the relationship between CF and tissue temperatures, lesion size, steam pop, and thrombus during RF ablation using a canine thigh muscle preparation. Methods and Results— CFS measurements (total 1409) from 2 catheters in 3 angles (perpendicular, parallel, and 45°) were compared with a certified balance (range, 0 to 50 g). CFS measurements correlated highly (R2≥0.988; mean error, ≤1.0 g). In 10 anesthetized dogs, a skin cradle over the thigh muscle was superfused with heparinized blood at 37°C. A 7F catheter with 3.5-mm saline-irrigated electrode and CFS (Endosense) was held perpendicular to the muscle at CF of 2, 10, 20, 30, and 40 g. RF was delivered (n=100) for 60 seconds at 30 or 50 W (irrigation 17 or 30 mL/min). Tissue temperature (3 and 7 mm depths), lesion size, thrombus, and steam pop increased significantly with increasing CF at each RF power. Lesion size was greater with applications of lower power (30 W) and greater CF (30 to 40 g) than at high power (50 W) with lower CF (2 to 10 g). Conclusions— This novel ablation catheter, which accurately measures CF, confirmed CF is a major determinant of RF lesion size. Steam pop and thrombus incidence also increases with CF. CFS in an open-irrigated ablation catheter that may optimize the selection of RF power and application time to maximize lesion formation and reduce the risk of steam pop and thrombus

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Essential for DNA biosynthesis and repair, ribonucleotide reductases (RNRs) convert ribonucleotides to deoxyribonucleotides via radical-based chemistry. Although long known that allosteric regulation of RNR activity is vital for cell health, the molecular basis of this regulation has been enigmatic, largely due to a lack of structural information about how the catalytic subunit (α 2 ) and the radical-generation subunit (β 2 ) interact. Here we present the first structure of a complex between α 2 and β 2 subunits for the prototypic RNR from Escherichia coli. Using four techniques (small-angle X-ray scattering, X-ray crystallography, electron microscopy, and analytical ultracentrifugation), we describe an unprecedented α 4 β 4 ring-like structure in the presence of the negative activity effector dATP and provide structural support for an active α 2 β 2 configuration. We demonstrate that, under physiological conditions, E. coli RNR exists as a mixture of transient α 2 β 2 and α 4 β 4 species whose distributions are modulated by allosteric effectors. We further show that this interconversion between α 2 β 2 and α 4 β 4 entails dramatic subunit rearrangements, providing a stunning molecular explanation for the allosteric regulation of RNR activity in E. coli.allostery | protein-protein interactions | conformational equilibria | nucleotide metabolism I mportant targets of anticancer and antiviral drugs, ribonucleotide reductases (RNRs) are classified by the metallocofactor used to generate a thiyl radical (1) that initiates reduction of ribonucleotides to deoxyribonucleotides (2, 3). Class Ia RNRs are found in all eukaryotes and many aerobic bacteria, with the Escherichia coli enzyme serving as the prototype. These RNRs reduce ribonucleoside 5′-diphosphates and are composed of two homodimeric subunits: α 2 and β 2 (Fig. 1A). The α 2 subunit contains the active site, where ribonucleotide reduction occurs, and two types of allosteric effector binding sites (4, 5). One effector site tunes the specificity for all four ribonucleotide substrates in response to intracellular levels of deoxyribonucleoside 5′-triphosphates (dATP, dTTP, dGTP) and ATP (6, 7) such that balanced pools of deoxyribonucleotides are maintained (8). The second effector site controls the rate of reduction, binding ATP to turn the enzyme on or dATP to turn it off (4, 6). This activity site is housed in an N-terminal cone domain (5, 9) and provides a means for negative feedback regulation to safeguard against cytotoxic elevation of deoxyribonucleotide levels (2, 3, 10). The β 2 subunit harbors the essential diferric-tyrosyl radical (Y122• in E. coli) cofactor (11) that initiates radical chemistry.Active RNR has long been proposed to be a transient α 2 β 2 complex (Fig. 1B) with enhanced subunit affinity upon binding of substrates and effectors (12-15). For each turnover, α 2 , β 2 , substrate and effector must interact, triggering long-range proton-coupled electron transfer (PCET) reducing Y122• in β 2 and oxidizing C439 to a thiyl radical in the active s...

Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides and requires a diferric-tyrosyl radical (Y•) cofactor to initiate catalysis. The initiation process requires long range proton-coupled electron transfer (PCET) over 35 Å between the two subunits by a specific pathway (Y 122

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