Supramolecular Hemoprotein Assembly with a Periodic Structure Showing Heme–Heme Exciton Coupling

A supramolecular assembly of units of cytochrome <i>b</i>₅₆₂ with externally attached heme having intermolecular linkages formed via the heme–heme pocket interaction was investigated in an effort to construct a well-defined structure. The engineered site for surface attachment of heme at Cys80 in an N80C mutant of cytochrome <i>b</i>₅₆₂ provides the primary basis for the formation of the periodic assembly structure, which is characterized herein by circular dichroism (CD) spectroscopy and high-speed atomic force microscopy (AFM). This assembly represents the first example of the observation of a split-type Cotton effect by heme–heme exciton coupling in an artificial hemoprotein assembly system. Molecular dynamics simulations validated by simulated CD spectra, AFM images, and mutation experiments reveal that the assembly has a periodic helical structure with 3 nm pitches, suggesting the formation of the assembled structure is driven not only by the heme–heme pocket interaction but also by additional secondary hydrogen bonding and/or electrostatic interactions at the protein interfaces of the assembly

Supramolecular Hemoprotein Assembly with a Periodic Structure Showing Heme–Heme Exciton Coupling

A supramolecular assembly of units of cytochrome <i>b</i>₅₆₂ with externally attached heme having intermolecular linkages formed via the heme–heme pocket interaction was investigated in an effort to construct a well-defined structure. The engineered site for surface attachment of heme at Cys80 in an N80C mutant of cytochrome <i>b</i>₅₆₂ provides the primary basis for the formation of the periodic assembly structure, which is characterized herein by circular dichroism (CD) spectroscopy and high-speed atomic force microscopy (AFM). This assembly represents the first example of the observation of a split-type Cotton effect by heme–heme exciton coupling in an artificial hemoprotein assembly system. Molecular dynamics simulations validated by simulated CD spectra, AFM images, and mutation experiments reveal that the assembly has a periodic helical structure with 3 nm pitches, suggesting the formation of the assembled structure is driven not only by the heme–heme pocket interaction but also by additional secondary hydrogen bonding and/or electrostatic interactions at the protein interfaces of the assembly

Supramolecular Hemoprotein Assembly with a Periodic Structure Showing Heme–Heme Exciton Coupling

A supramolecular assembly of units of cytochrome <i>b</i>₅₆₂ with externally attached heme having intermolecular linkages formed via the heme–heme pocket interaction was investigated in an effort to construct a well-defined structure. The engineered site for surface attachment of heme at Cys80 in an N80C mutant of cytochrome <i>b</i>₅₆₂ provides the primary basis for the formation of the periodic assembly structure, which is characterized herein by circular dichroism (CD) spectroscopy and high-speed atomic force microscopy (AFM). This assembly represents the first example of the observation of a split-type Cotton effect by heme–heme exciton coupling in an artificial hemoprotein assembly system. Molecular dynamics simulations validated by simulated CD spectra, AFM images, and mutation experiments reveal that the assembly has a periodic helical structure with 3 nm pitches, suggesting the formation of the assembled structure is driven not only by the heme–heme pocket interaction but also by additional secondary hydrogen bonding and/or electrostatic interactions at the protein interfaces of the assembly

Real-Time Dynamic Adsorption Processes of Cytochrome <i>c</i> on an Electrode Observed through Electrochemical High-Speed Atomic Force Microscopy

<div><p>An understanding of dynamic processes of proteins on the electrode surface could enhance the efficiency of bioelectronics development and therefore it is crucial to gain information regarding both physical adsorption of proteins onto the electrode and its electrochemical property in real-time. We combined high-speed atomic force microscopy (HS-AFM) with electrochemical device for simultaneous observation of the surface topography and electron transfer of redox proteins on an electrode. Direct electron transfer of cytochrome <i>c</i> (cyt <i>c</i>) adsorbed on a self-assembled monolayers (SAMs) formed electrode is very attractive subject in bioelectrochemistry. This paper reports a real-time visualization of cyt <i>c</i> adsorption processes on an 11-mercaptoundecanoic acid-modified Au electrode together with simultaneous electrochemical measurements. Adsorbing cyt <i>c</i> molecules were observed on a subsecond time resolution simultaneously with increasing redox currents from cyt <i>c</i> using EC-HS-AFM. The root mean square roughness (<i>R</i>_RMS) from the AFM images and the number of the electrochemically active cyt <i>c</i> molecules adsorbed onto the electrode (<i>Γ</i>) simultaneously increased in positive cooperativity. Cyt <i>c</i> molecules were fully adsorbed on the electrode in the AFM images when the peak currents were steady. This use of electrochemical HS-AFM significantly facilitates understanding of dynamic behavior of biomolecules on the electrode interface and contributes to the further development of bioelectronics.</p></div

Real-time cyt <i>c</i> desorption processes from the MUA-modified gold electrode.

<p>(A) Continuous AFM images of desorbing cyt <i>c</i> molecules. Frame rate, 2 frames/s; image area, 150 × 150 nm². (B) Time evolution of <i>R</i>_RMS values at the higher ionic strengths. (C) Schematic of desorbing cyt <i>c</i> molecules from the MUA-modified electrode. </p

Time-course analysis of <i>R</i>_RMS and <i>Γ</i> values.

<p>(A) Time evolution of <i>R</i>_RMS values from the HS-AFM images (open circle) and the level of electrochemically active cyt <i>c</i> (<i>Γ</i>) from the cyclic voltammograms (open square). (B) Schematic of the adsorbing cyt <i>c</i> molecules on the MUA-modified electrode at each time point.</p

AFM images show (A) the MUA-modified gold surface and (B) cyt <i>c</i> adsorbed on the MUA SAM at 450 sec.

<p>Continuous AFM images of adsorbing cyt <i>c</i> molecules with real-time labels. Frame rate, 2 frames/s; image area, 150 × 150 nm². The cross sections of the images in (A) and (B), along the short white line, are shown in the lower right. </p

CVs of cyt <i>c</i> adsorbed on the electrode.

<p>(A) CVs of cyt <i>c</i> molecules adsorbed on the MUA electrode in a 10 mM phosphate buffer solution (pH 7.0) from 333 to 522 s (from −0.35 V to 0.1 V, each segment is 4.5 s). (B) Background-subtracted CVs from the voltammogram from 313 to 324 s and 333 to 522 s (from inside to outside, each segment is 4.5 s). The voltammograms were collected at a scan rate of 100 mVs⁻¹. Ag wire was used as a reference electrode. </p

Trade-off between Processivity and Hydrolytic Velocity of Cellobiohydrolases at the Surface of Crystalline Cellulose

Analysis of heterogeneous catalysis at an interface is difficult because of the variety of reaction sites and the difficulty of observing the reaction. Enzymatic hydrolysis of cellulose by cellulases is a typical heterogeneous reaction at a solid/liquid interface, and a key parameter of such reactions on polymeric substrates is the processivity, i.e., the number of catalytic cycles that can occur without detachment of the enzyme from the substrate. In this study, we evaluated the reactions of three closely related glycoside hydrolase family 7 cellobiohydrolases from filamentous fungi at the molecular level by means of high-speed atomic force microscopy to investigate the structure–function relationship of the cellobiohydrolases on crystalline cellulose. We found that high moving velocity of enzyme molecules on the surface is associated with a high dissociation rate constant from the substrate, which means weak interaction between enzyme and substrate. Moreover, higher values of processivity were associated with more loop regions covering the subsite cleft, which may imply higher binding affinity. Loop regions covering the subsites result in stronger interaction, which decreases the velocity but increases the processivity. These results indicate that there is a trade-off between processivity and hydrolytic velocity among processive cellulases

Kaplan-Meier event curves for clinical outcomes in patients with two-vessel or three-vessel disease including proximal LAD.

(TIF)</p