Limits...
Single-molecule enzymatic conformational dynamics: spilling out the product molecules.

Zheng D, Lu HP - J Phys Chem B (2014)

Bottom Line: Our results have shown a wide distribution of the multiple conformational states involved in active-site interacting with the product molecules during the product releasing.We have identified that there is a significant pathway in which the product molecules are spilled out from the enzymatic active site, driven by a squeezing effect from a tight active-site conformational state, although the conventional pathway of releasing a product molecule from an open active-site conformational state is still a primary pathway.Our study provides new insight into the enzymatic reaction dynamics and mechanism, and the information is uniquely obtainable from our combined time-resolved single-molecule spectroscopic measurements and analyses.

View Article: PubMed Central - PubMed

Affiliation: Center for Photochemical Sciences, Department of Chemistry, Bowling Green State University , Bowling Green, Ohio 43403, United States.

ABSTRACT
Product releasing is an essential step of an enzymatic reaction, and a mechanistic understanding primarily depends on the active-site conformational changes and molecular interactions that are involved in this step of the enzymatic reaction. Here we report our work on the enzymatic product releasing dynamics and mechanism of an enzyme, horseradish peroxidase (HRP), using combined single-molecule time-resolved fluorescence intensity, anisotropy, and lifetime measurements. Our results have shown a wide distribution of the multiple conformational states involved in active-site interacting with the product molecules during the product releasing. We have identified that there is a significant pathway in which the product molecules are spilled out from the enzymatic active site, driven by a squeezing effect from a tight active-site conformational state, although the conventional pathway of releasing a product molecule from an open active-site conformational state is still a primary pathway. Our study provides new insight into the enzymatic reaction dynamics and mechanism, and the information is uniquely obtainable from our combined time-resolved single-molecule spectroscopic measurements and analyses.

Show MeSH

Related in: MedlinePlus

Single-molecule fluorescence experimental scheme.(A) Schematicrepresentation of the enzymatic reaction on (3-aminopropyl) trimethoxysilanemodified cover glass. Maleimide-activated HRP is linked to the sulfhydryl(−SH) group of 3-mercaptopropyl-trimethoxysilane. Nonfluorescentsubstrate Amplex red in PBS buffer is converted to fluorescent resorufinproduct by a single HRP molecule in the presence of hydrogen peroxideinitiator, the single-molecule fluorogenic assay. (B) Schematic representationof the total internal reflection fluorescence microscopy imaging-guidedconfocal fluorescence spectroscopy (TIRFM-CFS). M1: reflection mirror.DM1–DM2: dichroic mirror beam splitters. SPP: side port prismfor left/vis obs. TL: tube lens. EF1–EF2: emission filters.L1–L2: lens. PBS: Polarization beam splitter. SPAD1–SPAD2:single photon avalanche photodiode. (C), (D) The typical raw dataof single-molecule photon time-stamping spectroscopy of each detectorchannel with the perpendicular and parallel polarization components,respectively. Each data point represents a detected photon plottedby its arrival time (t) and delay time (Δt).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4126733&req=5

fig1: Single-molecule fluorescence experimental scheme.(A) Schematicrepresentation of the enzymatic reaction on (3-aminopropyl) trimethoxysilanemodified cover glass. Maleimide-activated HRP is linked to the sulfhydryl(−SH) group of 3-mercaptopropyl-trimethoxysilane. Nonfluorescentsubstrate Amplex red in PBS buffer is converted to fluorescent resorufinproduct by a single HRP molecule in the presence of hydrogen peroxideinitiator, the single-molecule fluorogenic assay. (B) Schematic representationof the total internal reflection fluorescence microscopy imaging-guidedconfocal fluorescence spectroscopy (TIRFM-CFS). M1: reflection mirror.DM1–DM2: dichroic mirror beam splitters. SPP: side port prismfor left/vis obs. TL: tube lens. EF1–EF2: emission filters.L1–L2: lens. PBS: Polarization beam splitter. SPAD1–SPAD2:single photon avalanche photodiode. (C), (D) The typical raw dataof single-molecule photon time-stamping spectroscopy of each detectorchannel with the perpendicular and parallel polarization components,respectively. Each data point represents a detected photon plottedby its arrival time (t) and delay time (Δt).

Mentions: Horseradishperoxidase immobilized on the cover glass was used in our experiments.The cover glass (Gold seal, 3419) was first washed in fresh preparedsulfuric acid dichromate cleaning solution for 1 h to eliminate greaseand possible fluorescent spots. After washing with water and dryingwith nitrogen gas, the cover glass was treated overnight with a mixturesolution of 3-mercaptopropyl-trimethoxysilane (Fluka, 09324), isobutyltrimethoxysilane(Sigma, 444065), and dimethyl sulfoxide (Sigma, D4540) with a volumeratio of 1:300:6000. After baking at 110 °C for 10 min, the silanatedcover glass was washed with methanol and water. Then the coverslipswere incubated in 50 mM PBS buffer (pH 8.0) with about 1 nM maleimide-activatedHRP (Thermo scientific, 31485) for 2 h and followed by rinsing withwater and PBS buffer. Phosphate buffer (PBS) was prepared with potassiumphosphate monobasic solution (Sigma-Aldrich, P8709) and potassiumphosphate dibasic solution (Sigma-Aldrich, P8584). Maleimide-activatedHRP was linked to the sulfhydryl (−SH) group of 3-mercaptopropyl-trimethoxysilaneon the cover glass as shown in Figure 1A. SubstrateAmplex Red (Invitrogen, A12222) was dissolved in dimethyl sulfoxide(DMSO) at 5 mg/mL and stored at −20 °C in the dark beforeuse. The reaction solution was prepared just prior to experimentation,with 200 nM Amplex Red and 2 mM H2O2 in PBSbuffer (pH 7.4). All chemicals were used without further purification.In our experiment, about 0.5 mL of reaction solution was filled ina home-built magnetic chamber that is composed with the coverglasstethered with HRP at the bottom and a lid on the top of the chamberto eliminate the evaporation.


Single-molecule enzymatic conformational dynamics: spilling out the product molecules.

Zheng D, Lu HP - J Phys Chem B (2014)

Single-molecule fluorescence experimental scheme.(A) Schematicrepresentation of the enzymatic reaction on (3-aminopropyl) trimethoxysilanemodified cover glass. Maleimide-activated HRP is linked to the sulfhydryl(−SH) group of 3-mercaptopropyl-trimethoxysilane. Nonfluorescentsubstrate Amplex red in PBS buffer is converted to fluorescent resorufinproduct by a single HRP molecule in the presence of hydrogen peroxideinitiator, the single-molecule fluorogenic assay. (B) Schematic representationof the total internal reflection fluorescence microscopy imaging-guidedconfocal fluorescence spectroscopy (TIRFM-CFS). M1: reflection mirror.DM1–DM2: dichroic mirror beam splitters. SPP: side port prismfor left/vis obs. TL: tube lens. EF1–EF2: emission filters.L1–L2: lens. PBS: Polarization beam splitter. SPAD1–SPAD2:single photon avalanche photodiode. (C), (D) The typical raw dataof single-molecule photon time-stamping spectroscopy of each detectorchannel with the perpendicular and parallel polarization components,respectively. Each data point represents a detected photon plottedby its arrival time (t) and delay time (Δt).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126733&req=5

fig1: Single-molecule fluorescence experimental scheme.(A) Schematicrepresentation of the enzymatic reaction on (3-aminopropyl) trimethoxysilanemodified cover glass. Maleimide-activated HRP is linked to the sulfhydryl(−SH) group of 3-mercaptopropyl-trimethoxysilane. Nonfluorescentsubstrate Amplex red in PBS buffer is converted to fluorescent resorufinproduct by a single HRP molecule in the presence of hydrogen peroxideinitiator, the single-molecule fluorogenic assay. (B) Schematic representationof the total internal reflection fluorescence microscopy imaging-guidedconfocal fluorescence spectroscopy (TIRFM-CFS). M1: reflection mirror.DM1–DM2: dichroic mirror beam splitters. SPP: side port prismfor left/vis obs. TL: tube lens. EF1–EF2: emission filters.L1–L2: lens. PBS: Polarization beam splitter. SPAD1–SPAD2:single photon avalanche photodiode. (C), (D) The typical raw dataof single-molecule photon time-stamping spectroscopy of each detectorchannel with the perpendicular and parallel polarization components,respectively. Each data point represents a detected photon plottedby its arrival time (t) and delay time (Δt).
Mentions: Horseradishperoxidase immobilized on the cover glass was used in our experiments.The cover glass (Gold seal, 3419) was first washed in fresh preparedsulfuric acid dichromate cleaning solution for 1 h to eliminate greaseand possible fluorescent spots. After washing with water and dryingwith nitrogen gas, the cover glass was treated overnight with a mixturesolution of 3-mercaptopropyl-trimethoxysilane (Fluka, 09324), isobutyltrimethoxysilane(Sigma, 444065), and dimethyl sulfoxide (Sigma, D4540) with a volumeratio of 1:300:6000. After baking at 110 °C for 10 min, the silanatedcover glass was washed with methanol and water. Then the coverslipswere incubated in 50 mM PBS buffer (pH 8.0) with about 1 nM maleimide-activatedHRP (Thermo scientific, 31485) for 2 h and followed by rinsing withwater and PBS buffer. Phosphate buffer (PBS) was prepared with potassiumphosphate monobasic solution (Sigma-Aldrich, P8709) and potassiumphosphate dibasic solution (Sigma-Aldrich, P8584). Maleimide-activatedHRP was linked to the sulfhydryl (−SH) group of 3-mercaptopropyl-trimethoxysilaneon the cover glass as shown in Figure 1A. SubstrateAmplex Red (Invitrogen, A12222) was dissolved in dimethyl sulfoxide(DMSO) at 5 mg/mL and stored at −20 °C in the dark beforeuse. The reaction solution was prepared just prior to experimentation,with 200 nM Amplex Red and 2 mM H2O2 in PBSbuffer (pH 7.4). All chemicals were used without further purification.In our experiment, about 0.5 mL of reaction solution was filled ina home-built magnetic chamber that is composed with the coverglasstethered with HRP at the bottom and a lid on the top of the chamberto eliminate the evaporation.

Bottom Line: Our results have shown a wide distribution of the multiple conformational states involved in active-site interacting with the product molecules during the product releasing.We have identified that there is a significant pathway in which the product molecules are spilled out from the enzymatic active site, driven by a squeezing effect from a tight active-site conformational state, although the conventional pathway of releasing a product molecule from an open active-site conformational state is still a primary pathway.Our study provides new insight into the enzymatic reaction dynamics and mechanism, and the information is uniquely obtainable from our combined time-resolved single-molecule spectroscopic measurements and analyses.

View Article: PubMed Central - PubMed

Affiliation: Center for Photochemical Sciences, Department of Chemistry, Bowling Green State University , Bowling Green, Ohio 43403, United States.

ABSTRACT
Product releasing is an essential step of an enzymatic reaction, and a mechanistic understanding primarily depends on the active-site conformational changes and molecular interactions that are involved in this step of the enzymatic reaction. Here we report our work on the enzymatic product releasing dynamics and mechanism of an enzyme, horseradish peroxidase (HRP), using combined single-molecule time-resolved fluorescence intensity, anisotropy, and lifetime measurements. Our results have shown a wide distribution of the multiple conformational states involved in active-site interacting with the product molecules during the product releasing. We have identified that there is a significant pathway in which the product molecules are spilled out from the enzymatic active site, driven by a squeezing effect from a tight active-site conformational state, although the conventional pathway of releasing a product molecule from an open active-site conformational state is still a primary pathway. Our study provides new insight into the enzymatic reaction dynamics and mechanism, and the information is uniquely obtainable from our combined time-resolved single-molecule spectroscopic measurements and analyses.

Show MeSH
Related in: MedlinePlus