Key Chemical Factors of Arginine Finger Catalysis of F₁‑ATPase Clarified by an Unnatural Amino Acid Mutation

A catalytically important arginine, called Arg finger, is employed in many enzymes to regulate their functions through enzymatic hydrolysis of nucleotide triphosphates. F₁-ATPase (F₁), a rotary motor protein, possesses Arg fingers which catalyze hydrolysis of adenosine triphosphate (ATP) for efficient chemomechanical energy conversion. In this study, we examined the Arg finger catalysis by single-molecule measurements for a mutant of F₁ in which the Arg finger is substituted with an unnatural amino acid of a lysine analogue, 2,7-diaminoheptanoic acid (Lyk). The use of Lyk, of which the side chain is elongated by one CH₂ unit so that its chain length to the terminal nitrogen of amine is set to be equal to that of arginine, allowed us to resolve key chemical factors in the Arg finger catalysis, i.e., chain length matching and chemical properties of the terminal groups. Rate measurements by single-molecule observations showed that the chain length matching of the side-chain length is not a sole requirement for the Arg finger to catalyze the ATP hydrolysis reaction step, indicating the crucial importance of chemical properties of the terminal guanidinium group in the Arg finger catalysis. On the other hand, the Lyk mutation prevented severe formation of an ADP inhibited state observed for a lysine mutant and even improved the avoidance of inhibition compared with the wild-type F₁. The present study demonstrated that incorporation of unnatural amino acids can widely extend with its high “chemical” resolution biochemical approaches for elucidation of the molecular mechanism of protein functions and furnishing novel characteristics

High-Speed Angle-Resolved Imaging of a Single Gold Nanorod with Microsecond Temporal Resolution and One-Degree Angle Precision

We developed two types of high-speed angle-resolved imaging methods for single gold nanorods (SAuNRs) using objective-type vertical illumination dark-field microscopy and a high-speed CMOS camera to achieve microsecond temporal and one-degree angle resolution. These methods are based on: (i) an intensity analysis of focused images of SAuNR split into two orthogonally polarized components and (ii) the analysis of defocused SAuNR images. We determined the angle precision (statistical error) and accuracy (systematic error) of the resultant SAuNR (80 nm × 40 nm) images projected onto a substrate surface (azimuthal angle) in both methods. Although both methods showed a similar precision of ∼1° for the azimuthal angle at a 10 μs temporal resolution, the defocused image analysis showed a superior angle accuracy of ∼5°. In addition, the polar angle was also determined from the defocused SAuNR images with a precision of ∼1°, by fitting with simulated images. By taking advantage of the defocused image method’s full revolution measurement range in the azimuthal angle, the rotation of the rotary molecular motor, F₁-ATPase, was measured with 3.3 μs temporal resolution. The time constants of the pauses waiting for the elementary steps of the ATP hydrolysis reaction and the torque generated in the mechanical steps have been successfully estimated. The high-speed angle-resolved SAuNR imaging methods will be applicable to the monitoring of the fast conformational changes of many biological molecular machines

Direct Measurement of Single-Molecule Adenosine Triphosphatase Hydrolysis Dynamics

F₁-ATPase (F₁) is a bidirectional molecular motor that hydrolyzes nearly all ATPs to fuel the cellular processes. Optical observation of labeled F₁ rotation against the α₃β₃ hexamer ring revealed the sequential mechanical rotation steps corresponding to ATP binding/ADP release and ATP hydrolysis/Pi release. These substeps originate from the F₁ rotation but with heavy load on the γ shaft due to fluorescent labeling and the photophysical limitation of an optical microscope, which hampers better understanding of the intrinsic kinetic behavior of ATP hydrolysis. In this work, we present a method capable of electrically monitoring ATP hydrolysis of a single label-free F₁ in real time by using a high-gain silicon nanowire-based field-effect transistor circuit. We reproducibly observe the regular current signal fluctuations with two distinct levels, which are induced by the binding dwell and the catalytic dwell, respectively, in both concentration- and temperature-dependent experiments. In comparison with labeled F₁, the hydrolysis rate of nonlabeled F₁ used in this study is 1 order of magnitude faster (1.69 × 10⁸ M^–1 s^–1 at 20 °C), and the differences between two sequential catalytic rates are clearer, demonstrating the ability of nanowire nanocircuits to directly probe the intrinsic dynamic processes of the biological activities with single-molecule/single-event sensitivity. This approach is complementary to traditional optical methods, offering endless opportunities to unravel molecular mechanisms of a variety of dynamic biosystems under realistic physiological conditions

Observation of viral particles with rapid-freeze, freeze-dry, platinum-replica TEM.

<p>(A) Images of viral particles. Scale bar, 100 nm. (B) Distribution of the diameter of viral particles visualized in TEM images.</p

Correlation between scattering and SEM images of viruses

<p>. (A) Scattering image. (B) SEM image. (C) False-color superimposed image of binarized scattering (red) and SEM (green) images shown in A and B. (D) Distribution of the distance between scattering spots (spot in SEM) and nearest-neighbor spots in SEM (scattering spot). We considered scattering and SEM spots within 400 nm (dashed line) as signals from the same particle. (E) FE-SEM image. The area enclosed by the black dashed square in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049208#pone-0049208-g004" target="_blank">Figure 4B</a> is shown. The particles are indicated by circles. (F) Distribution of the diameter of each particle measured in the FE-SEM images.</p

Effects of D13-9001 on FDG degradation determined by the microfluidic channel device.

<p>(A) Bright-field (top) and fluorescence images (bottom) of the <i>E. coli</i> wild-type, ΔB, and ΔC cells treated with different concentrations of PP. (B) Distributions of the fluorescence intensities of the cells and channels measured in the image shown in (A).</p

Experimental setup for label-free imaging of viral particles.

<p>(A) Schematic representation of the optical system of objective-type TIRDFM. ND, neutral density filter; BE, beam expander; FS, field stop; M1, mirror; L1, lens; PM, perforated mirror; L2, second objective lens inside microscope. (B) Expanded drawings depicting the sample chamber and objective lens.</p

Dependence of spot density on the virus concentration as observed by TIRDFM.

<p>(A) Scattering image of viral particles at different concentrations. The virus concentrations were 0, 1.0×10⁴, 3.0×10⁴, 1.0×10⁵, and 3.0×10⁵ pfu/mL. (B) Spot density plotted against virus concentration. The LOD was found to be 1.2×10⁴ pfu/mL. (C) Distribution of the intensity of scattering spots.</p

Effects of PAβN on FDG degradation determined by the microfluidic channel device.

<p>(A) Bright-field (top) and fluorescence images (bottom) of the <i>E. coli</i> wild-type, ΔB, and ΔC cells treated with different concentrations of PAβN. (B) Distributions of the fluorescence intensities of the cells and channels measured in the image shown in (A).</p

Effect of inhibitors on SYTOX Green uptake in <i>E. coli</i> MG1655 and its pump deletion mutants.

<p>Exponentially growing cells harvested in PBS containing 0.4% glucose were used. Membrane integrity was determined by measuring SYTOX Green uptake after exposure to D13-9001, PAβN, and PMB for 20 min. Fluorescence was determined (Ex/Em: 504/523 nm) after an incubation of 10 min with SYTOX Green in a black 384-well plate with a plate reader.</p