One at a Time: Intramolecular Electron-Transfer Kinetics in Small Laccase Observed during Turnover

Single-molecule enzymology provides an unprecedented level of detail about aspects of enzyme mechanisms which have been very difficult to probe in bulk. One such aspect is intramolecular electron transfer (ET), which is a recurring theme in the research on oxidoreductases containing multiple redox-active sites. We measure the intramolecular ET rates between the copper centers of the small laccase from <i>Streptomyces coelicolor</i> at room temperature and pH 7.4, one molecule at a time, during turnover. The forward and backward rates across many molecules follow a log-normal distribution with means of 460 and 85 s^–1, respectively, corresponding to activation energies of 347 and 390 meV for the forward and backward rates. The driving force and the reorganization energy amount to 0.043 and 1.5 eV, respectively. The spread in rates corresponds to a spread of ∼30 meV in the activation energy. The second-order rate constant for reduction of the T1 site amounts to 2.9 × 10⁴ M^–1 s^–1. The mean of the distribution of forward ET rates is higher than the turnover rate from ensemble steady-state measurements and, thus, is not rate limiting

Super-resolution images of internalized α-syn aggregates in endosomal vesicles in time.

<p>(a) dSTORM image of a cell treated for half an hour with α-syn -Alexa532 aggregates. A detailed view of the aggregates in the cell membrane is shown below a). (b) After 2 hours of incubation, α-syn aggregates are internalized in vesicles. Detailed view of the aggregates in a vesicle shown in the image below b). (c) Internalized α-syn aggregates after 24 hours of incubation, with two different sized clusters highlighted bellow image c).</p

Characteristic properties of the optical setup.

<p>(a) Frame with the signal of several Alexa532 molecules. Scale bar = 2μm. (b) Histogram of the sigma of positional accuracy (Mean: 11 nm). (c) Zoom-in of the white square in Fig 1A showing the Gaussian intensity profile. (d) Histogram of the intensity of localizations (Mean: 447 photons).</p

Size distribution of α-syn aggregates in endosomal vesicles in time.

<p>(a)-(c) Histogram of FWHM of intracellular α-syn clusters in time. (ANOVA significance levels: (a)-(b): 10⁻⁴; (b)-(c):5×10⁻³; ((a)-(c):10⁻⁷). (d) A decrease in α-syn cluster size is observed in the mean average FWHM of α-syn clusters in time (median and 50% interval).</p

Internalization of α-syn sonicated fibrils in human neuroblastoma cells.

<p>Images show co-localization of Alexa 532 labeled α-syn aggregates (green) with LysoTracker Deep Red (red). SH-SY5Y cells were treated with 50 nM LysoTracker Deep Red, then washed, incubated further with Alexa532-labeled α-syn sonicated fibrils and imaged live on a confocal microscope.</p

Super-resolution imaging of the <i>in vitro</i> prepared α-syn fibrils.

<p>(a) AFM and (b) dSTORM images of intact wild-type α-syn fibrils covalently labeled with the NHS derivate of Alexa 532 fluorophore. (c) AFM and (d) dSTORM images of sonicated labeled α-syn fibrils.</p

Large Amplitude Conductance Gating in a Wired Redox Molecule

Further developments in the field of molecular electronics will require an understanding of the key relationships between chemical composition and subsequently observed electron transport characteristics. Although the relationship between redox activity and conductance gating has emerged in recent years, our ability to chemically engineer these characteristics (such as “on−off” switching magnitudes) is only now emerging. The report herein describes, to our knowledge, the first example of gated conductance in a single wired redox molecule in which a 3 orders of magnitude “on/off” switching ratio is observed. This switching magnitude significantly exceeds that observed with molecules weakly coupled to the supporting electrode and correlates directly with both electrochemical switching and an in situ determined heterogeneous electron transfer rate constant

Top-Down FTICR MS for the Identification of Fluorescent Labeling Efficiency and Specificity of the Cu-Protein Azurin

Fluorescent protein labeling has been an indispensable tool in many applications of biochemical, biophysical, and cell biological research. Although detailed information about the labeling stoichiometry and exact location of the label is often not necessary, for other purposes, this information is crucial. We have studied the potential of top-down electrospray ionization (ESI)-15T Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to study the degree and positioning of fluorescent labeling. For this purpose, we have labeled the Cu-protein azurin with the fluorescent label ATTO 655-N-hydroxysuccinimide­(NHS)-ester and fractionated the sample using anion exchange chromatography. Subsequently, individual fractions were analyzed by ESI-15T FTICR to determine the labeling stoichiometry, followed by top-down MS fragmentation, to locate the position of the label. Results showed that, upon labeling with ATTO 655-NHS, multiple different species of either singly or doubly labeled azurin were formed. Top-down fragmentation of different species, either with or without the copper, resulted in a sequence coverage of approximately 50%. Different primary amine groups were found to be (potential) labeling sites, and Lys-122 was identified as the major labeling attachment site. In conclusion, we have demonstrated that anion exchange chromatography in combination with ultrahigh resolution 15T ESI-FTICR top-down mass spectrometry is a valuable tool for measuring fluorescent labeling efficiency and specificity