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Measuring localized redox enzyme electron transfer in a live cell with conducting atomic force microscopy.

Alfonta L, Meckes B, Amir L, Schlesinger O, Ramachandran S, Lal R - Anal. Chem. (2014)

Bottom Line: A quinone, an electron transfer mediator, was covalently attached site specifically to the displayed ADHII.An electrochemical comparison between two quinone containing mutants with different distances from the NAD(+) binding site in alcohol dehydrogenase II was performed.Electron transfer in redox active proteins showed increased efficiency when mediators are present closer to the NAD(+) binding site.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, ‡Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev , P.O. Box 653, Beer-Sheva, 84105, Israel.

ABSTRACT
Bacterial systems are being extensively studied and modified for energy, sensors, and industrial chemistry; yet, their molecular scale structure and activity are poorly understood. Designing efficient bioengineered bacteria requires cellular understanding of enzyme expression and activity. An atomic force microscope (AFM) was modified to detect and analyze the activity of redox active enzymes expressed on the surface of E. coli. An insulated gold-coated metal microwire with only the tip conducting was used as an AFM cantilever and a working electrode in a three-electrode electrochemical cell. Bacteria were engineered such that alcohol dehydrogenase II (ADHII) was surface displayed. A quinone, an electron transfer mediator, was covalently attached site specifically to the displayed ADHII. The AFM probe was used to lift a single bacterium off the surface for electrochemical analysis in a redox-free buffer. An electrochemical comparison between two quinone containing mutants with different distances from the NAD(+) binding site in alcohol dehydrogenase II was performed. Electron transfer in redox active proteins showed increased efficiency when mediators are present closer to the NAD(+) binding site. This study suggests that an integrated conducting AFM used for single cell electrochemical analysis would allow detailed understanding of enzyme electron transfer processes to electrodes, the processes integral to creating efficiently engineered biosensors and biofuel cells.

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AFM imagesof the different stages of surface and tip manipulations.(A) Large scan area with D314Az mutant bacteria. (B–D) Scansof single bacterium used in electrochemical measurements of V66Az(B), D314Az (C), and WT (D). (E) Light microscopy images of bacteriaattached to the gold substrate.
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fig1: AFM imagesof the different stages of surface and tip manipulations.(A) Large scan area with D314Az mutant bacteria. (B–D) Scansof single bacterium used in electrochemical measurements of V66Az(B), D314Az (C), and WT (D). (E) Light microscopy images of bacteriaattached to the gold substrate.

Mentions: Three different mutants of alcoholdehydrogenase II (ADHII) were displayed on the surface of E. coli. Each mutant was generated with varying distancesfrom the NAD+ binding pocket; mutants V66Az and P182Azwere generated with approximate distances of ∼5 Å eachfrom the binding pocket and mutant D314Az with a distance of ∼42Å from the binding pocket (Figure S1, SupportingInformation). In this study, we have excluded mutant P182Azas V66Az is a similar distance (∼5 Å) from the NAD+ binding pocket. The modified bacteria were covalently attachedto gold-sputtered glass slides via linker 1. Slides withattached bacteria were imaged with the wire AFM cantilever. Figure 1A shows an image of multiple bacteria on the surfaceof a slide. A bacterium was localized with the AFM for subsequentelectrochemical analysis. Figure 1B–Dshows individual bacterium of different mutants, V66Az, D314Az, andwild type bacteria (WT), selected for electrochemical measurements.The presence of bacteria on the gold substrates was confirmed withlight microscopy imaging (Figure 1E).


Measuring localized redox enzyme electron transfer in a live cell with conducting atomic force microscopy.

Alfonta L, Meckes B, Amir L, Schlesinger O, Ramachandran S, Lal R - Anal. Chem. (2014)

AFM imagesof the different stages of surface and tip manipulations.(A) Large scan area with D314Az mutant bacteria. (B–D) Scansof single bacterium used in electrochemical measurements of V66Az(B), D314Az (C), and WT (D). (E) Light microscopy images of bacteriaattached to the gold substrate.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215851&req=5

fig1: AFM imagesof the different stages of surface and tip manipulations.(A) Large scan area with D314Az mutant bacteria. (B–D) Scansof single bacterium used in electrochemical measurements of V66Az(B), D314Az (C), and WT (D). (E) Light microscopy images of bacteriaattached to the gold substrate.
Mentions: Three different mutants of alcoholdehydrogenase II (ADHII) were displayed on the surface of E. coli. Each mutant was generated with varying distancesfrom the NAD+ binding pocket; mutants V66Az and P182Azwere generated with approximate distances of ∼5 Å eachfrom the binding pocket and mutant D314Az with a distance of ∼42Å from the binding pocket (Figure S1, SupportingInformation). In this study, we have excluded mutant P182Azas V66Az is a similar distance (∼5 Å) from the NAD+ binding pocket. The modified bacteria were covalently attachedto gold-sputtered glass slides via linker 1. Slides withattached bacteria were imaged with the wire AFM cantilever. Figure 1A shows an image of multiple bacteria on the surfaceof a slide. A bacterium was localized with the AFM for subsequentelectrochemical analysis. Figure 1B–Dshows individual bacterium of different mutants, V66Az, D314Az, andwild type bacteria (WT), selected for electrochemical measurements.The presence of bacteria on the gold substrates was confirmed withlight microscopy imaging (Figure 1E).

Bottom Line: A quinone, an electron transfer mediator, was covalently attached site specifically to the displayed ADHII.An electrochemical comparison between two quinone containing mutants with different distances from the NAD(+) binding site in alcohol dehydrogenase II was performed.Electron transfer in redox active proteins showed increased efficiency when mediators are present closer to the NAD(+) binding site.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, ‡Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev , P.O. Box 653, Beer-Sheva, 84105, Israel.

ABSTRACT
Bacterial systems are being extensively studied and modified for energy, sensors, and industrial chemistry; yet, their molecular scale structure and activity are poorly understood. Designing efficient bioengineered bacteria requires cellular understanding of enzyme expression and activity. An atomic force microscope (AFM) was modified to detect and analyze the activity of redox active enzymes expressed on the surface of E. coli. An insulated gold-coated metal microwire with only the tip conducting was used as an AFM cantilever and a working electrode in a three-electrode electrochemical cell. Bacteria were engineered such that alcohol dehydrogenase II (ADHII) was surface displayed. A quinone, an electron transfer mediator, was covalently attached site specifically to the displayed ADHII. The AFM probe was used to lift a single bacterium off the surface for electrochemical analysis in a redox-free buffer. An electrochemical comparison between two quinone containing mutants with different distances from the NAD(+) binding site in alcohol dehydrogenase II was performed. Electron transfer in redox active proteins showed increased efficiency when mediators are present closer to the NAD(+) binding site. This study suggests that an integrated conducting AFM used for single cell electrochemical analysis would allow detailed understanding of enzyme electron transfer processes to electrodes, the processes integral to creating efficiently engineered biosensors and biofuel cells.

Show MeSH