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
Single-molecule fluorescenceintensity trajectories and the intensitydistributions of HRP-catalyzed oxidation of Amplex Red, binning with50 ms. (A1) Intensity trajectory of the perpendicular polarizationcomponent relative to the excitation polarization after the compensationby the G factor of photon detection. (B1) Intensitytrajectory of the parallel polarization component relative to theexcitation polarization. (A2) and (B2) The distributions of florescenceintensity of the trajectories in A1 and B1, respectively. The blackcurve denotes the background intensity distribution from control experiment,and the inset is the zoom-in near the threshold of the background.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Single-molecule fluorescenceintensity trajectories and the intensitydistributions of HRP-catalyzed oxidation of Amplex Red, binning with50 ms. (A1) Intensity trajectory of the perpendicular polarizationcomponent relative to the excitation polarization after the compensationby the G factor of photon detection. (B1) Intensitytrajectory of the parallel polarization component relative to theexcitation polarization. (A2) and (B2) The distributions of florescenceintensity of the trajectories in A1 and B1, respectively. The blackcurve denotes the background intensity distribution from control experiment,and the inset is the zoom-in near the threshold of the background.

Mentions: Fluorescence intensity is themost straightforward parameter tocharacterize fluorescence signals of fluorogenic enzymatic reactions.Fluorescence from a single-molecule product can only be detected andidentified while it is still confined in the enzyme. After being releasedfrom the enzymatic active site, the fluorescent product molecule diffusesout of the laser excitation confocal volume within submilliseconds.50 By the single-molecule photon time stampingspectroscopy technique, we record the intensity trajectories of differentpolarization channels. Figures 2 A1 and 2 B1 show perpendicular channel and parallel channelfluorescence intensity trajectories, respectively, with a binningtime of 50 ms, of the fluorescent products from a single HRP enzymetethered to the coverglass in PBS buffer solution at pH 7.4. The fluctuationsof fluorescence bursts recorded from both parallel and perpendicularpolarization channels show similar behaviors and photon count distributions.On average, the intensity of the parallel component is higher thanthat of the perpendicular component even after the G-factor compensation. The corresponding intensity distributions ofdifferent polarizations are shown in Figures 2 A2 and 2 B2, respectively. The black curveis from the intensity distribution of the background, which was takenat the same conditions but without enzyme in the focus volume. Thiscurve clearly shows the profile of the photon time stamping measurementwithout fluorescence turnovers. The insets show the intensity distributionsabove the edge of the background. The intensity distribution of thebackground shows a narrow band below 120 photons, whereas the distributionsof intensity beyond the background are asymmetric and elongated towardthe higher intensity side. For the distribution portion of intensitybeyond the background, it is evident that the distribution likelycontains more than one component since the distribution is significantlywider than the highest intensity shot noise.


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

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

Single-molecule fluorescenceintensity trajectories and the intensitydistributions of HRP-catalyzed oxidation of Amplex Red, binning with50 ms. (A1) Intensity trajectory of the perpendicular polarizationcomponent relative to the excitation polarization after the compensationby the G factor of photon detection. (B1) Intensitytrajectory of the parallel polarization component relative to theexcitation polarization. (A2) and (B2) The distributions of florescenceintensity of the trajectories in A1 and B1, respectively. The blackcurve denotes the background intensity distribution from control experiment,and the inset is the zoom-in near the threshold of the background.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Single-molecule fluorescenceintensity trajectories and the intensitydistributions of HRP-catalyzed oxidation of Amplex Red, binning with50 ms. (A1) Intensity trajectory of the perpendicular polarizationcomponent relative to the excitation polarization after the compensationby the G factor of photon detection. (B1) Intensitytrajectory of the parallel polarization component relative to theexcitation polarization. (A2) and (B2) The distributions of florescenceintensity of the trajectories in A1 and B1, respectively. The blackcurve denotes the background intensity distribution from control experiment,and the inset is the zoom-in near the threshold of the background.
Mentions: Fluorescence intensity is themost straightforward parameter tocharacterize fluorescence signals of fluorogenic enzymatic reactions.Fluorescence from a single-molecule product can only be detected andidentified while it is still confined in the enzyme. After being releasedfrom the enzymatic active site, the fluorescent product molecule diffusesout of the laser excitation confocal volume within submilliseconds.50 By the single-molecule photon time stampingspectroscopy technique, we record the intensity trajectories of differentpolarization channels. Figures 2 A1 and 2 B1 show perpendicular channel and parallel channelfluorescence intensity trajectories, respectively, with a binningtime of 50 ms, of the fluorescent products from a single HRP enzymetethered to the coverglass in PBS buffer solution at pH 7.4. The fluctuationsof fluorescence bursts recorded from both parallel and perpendicularpolarization channels show similar behaviors and photon count distributions.On average, the intensity of the parallel component is higher thanthat of the perpendicular component even after the G-factor compensation. The corresponding intensity distributions ofdifferent polarizations are shown in Figures 2 A2 and 2 B2, respectively. The black curveis from the intensity distribution of the background, which was takenat the same conditions but without enzyme in the focus volume. Thiscurve clearly shows the profile of the photon time stamping measurementwithout fluorescence turnovers. The insets show the intensity distributionsabove the edge of the background. The intensity distribution of thebackground shows a narrow band below 120 photons, whereas the distributionsof intensity beyond the background are asymmetric and elongated towardthe higher intensity side. For the distribution portion of intensitybeyond the background, it is evident that the distribution likelycontains more than one component since the distribution is significantlywider than the highest intensity shot noise.

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