Analyzing reactions of single mechanoenzyme molecules by light microscopy

1992 ◽  
Vol 50 (1) ◽  
pp. 426-427
Author(s):  
Jeff Gelles
Keyword(s):  
Image Processing ◽  
Reaction Mechanisms ◽  
Transient State ◽  
Nanometer Scale ◽  
Reaction Intermediates ◽  
Enzyme Molecule ◽  
Directed Movement ◽  
Enzyme Molecules ◽  
Individual Enzyme ◽  
Single Reaction

Mechanoenzymes are enzymes which use a chemical reaction to power directed movement along biological polymer. Such enzymes include the cytoskeletal motors (e.g., myosins, dyneins, and kinesins) as well as nucleic acid polymerases and helicases. A single catalytic turnover of a mechanoenzyme moves the enzyme molecule along the polymer a distance on the order of 10−9 m We have developed light microscope and digital image processing methods to detect and measure nanometer-scale motions driven by single mechanoenzyme molecules. These techniques enable one to monitor the occurrence of single reaction steps and to measure the lifetimes of reaction intermediates in individual enzyme molecules. This information can be used to elucidate reaction mechanisms and determine microscopic rate constants. Such an approach circumvents difficulties encountered in the use of traditional transient-state kinetics techniques to examine mechanoenzyme reaction mechanisms.

scholarly journals Bottom-up single-molecule strategy for understanding subunit function of tetrameric β-galactosidase

2018 ◽  
Vol 115 (33) ◽  
pp. 8346-8351 ◽  
Author(s):  
Xiang Li ◽  
Yu Jiang ◽  
Shaorong Chong ◽  
David R. Walt
Keyword(s):  
Single Molecule ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Single Molecule Level ◽  
Subunit Function ◽  
Enzyme Molecules ◽  
Individual Enzyme ◽  
Kinetics Of

In this paper, we report an example of the engineered expression of tetrameric β-galactosidase (β-gal) containing varying numbers of active monomers. Specifically, by combining wild-type and single-nucleotide polymorphism plasmids at varying ratios, tetrameric β-gal was expressed in vitro with one to four active monomers. The kinetics of individual enzyme molecules revealed four distinct populations, corresponding to the number of active monomers in the enzyme. Using single-molecule-level enzyme kinetics, we were able to measure an accurate in vitro mistranslation frequency (5.8 × 10−4 per base). In addition, we studied the kinetics of the mistranslated β-gal at the single-molecule level.

scholarly journals Conformational change in individual enzyme molecules

2015 ◽  
Vol 93 (6) ◽  
pp. 611-618 ◽  
Author(s):  
Jeremie J. Crawford ◽  
Frannie Itzkow ◽  
Joanna MacLean ◽  
Douglas B. Craig
Keyword(s):  
Heat Shock ◽  
Electrophoretic Mobility ◽  
Conformational Changes ◽  
Average Rate ◽  
Electrophoretic Mobilities ◽  
Significant Difference ◽  
Average Change ◽  
Before And After ◽  
Enzyme Molecules ◽  
Individual Enzyme

Single β-galactosidase molecule assays were performed using a capillary electrophoresis based protocol, employing post-column laser-induced fluorescence detection. In a first set of experiments, the distribution of single β-galactosidase molecule catalytic rates and electrophoretic mobilities were determined from lysates of Escherichia coli strains containing deletions for different heat shock proteins and grown under normal and heat shock conditions. There was no clear observed pattern of effect of heat shock protein expression on these distributions. In a second set of experiments, individual enzyme molecule catalytic rates were determined at 21 °C before and after 2 sequential brief periods of incubation at 50, 28, and 10 °C. The brief incubations at 50 °C caused a change in the enzyme molecules resulting in a different catalytic rate. Any given molecule was just as likely to show an increase in rate as a decrease, resulting in no significant difference in the average rate of the population. The average change in individual molecule rate was dependent upon the temperature of the brief incubation period, with a lesser average change occurring at 28 °C and negligible change at 10 °C. A third set of experiments was similar to that of the second with the exception that it was electrophoretic mobility that was considered. This provided a similar result. Incubation at higher temperature resulted in a change in electrophoretic mobility. The probability of an individual molecules switching to a higher mobility was approximately equal to that of switching to a lower mobility, resulting in no net average change in the population. The magnitude of the changes in electrophoretic mobilities suggest that the associated conformational changes are subtle.

scholarly journals Hook-length of the bacterial flagellum is optimized for maximal stability of the flagellar bundle

2018 ◽  
Author(s):  
Imke Spöring ◽  
Vincent A. Martinez ◽  
Christian Hotz ◽  
Jana Schwarz-Linek ◽  
Keara L. Grady ◽  
...  
Keyword(s):  
Basal Body ◽  
Swimming Speed ◽  
Self Assembly ◽  
Nanometer Scale ◽  
Hook Length ◽  
Optimal Length ◽  
Directed Movement ◽  
Bacterial Flagellum ◽  
Evolutionary Forces ◽  
Molecular Ruler

AbstractMost bacteria swim in liquid environments by rotating one or several flagella. The long external filament of the flagellum is connected to a membrane-embedded basal-body by a flexible universal joint, the hook, which allows the transmission of motor torque to the filament. The length of the hook is controlled on a nanometer-scale by a sophisticated molecular ruler mechanism. However, why its length is stringently controlled has remained elusive. We engineered and studied a diverse set of hook-length variants ofSalmonella enterica. Measurements of plate-assay motility, single-cell swimming speed and directional persistence in quasi 2D and population-averaged swimming speed and body angular velocity in 3D revealed that the motility performance is optimal around the wild type hook-length. We conclude that too short hooks may be too stiff to function as a junction and too long hooks may buckle and create instability in the flagellar bundle. Accordingly, peritrichously flagellated bacteria move most efficiently as the distance travelled per body rotation is maximal and body wobbling is minimized. Thus, our results suggest that the molecular ruler mechanism evolved to control flagellar hook growth to the optimal length consistent with efficient bundle formation. The hook-length control mechanism is therefore a prime example of how bacteria evolved elegant, but robust mechanisms to maximize their fitness under specific environmental constraints.Author summaryMany bacteria use flagella for directed movement in liquid environments. The flexible hook connects the membrane-embedded basal-body of the flagellum to the long, external filament. Flagellar function relies on self-assembly processes that define or self-limit the lengths of major parts. The length of the hook is precisely controlled on a nanometer-scale by a molecular ruler mechanism. However, the physiological benefit of tight hook-length control remains unclear. Here, we show that the molecular ruler mechanism evolved to control the optimal length of the flagellar hook, which is consistent with efficient motility performance. These results highlight the evolutionary forces that enable flagellated bacteria to optimize their fitness in diverse environments and might have important implications for the design of swimming micro-robots.

scholarly journals Inactivation precedes changes in allosteric properties and conformation of d-glyceraldehyde-3-phosphate dehydrogenase and fructose-1,6-bisphosphatase during denaturation by guanidinium chloride

1994 ◽  
Vol 303 (1) ◽  
pp. 241-245 ◽  
Author(s):  
R F Jiang ◽  
C L Tsou
Keyword(s):  
Conformational Changes ◽  
Enzyme Molecule ◽  
Guanidinium Chloride ◽  
Fructose 1,6 Bisphosphatase ◽  
Enzyme Molecules ◽  
Amp Inhibition ◽  
Allosteric Properties

It has been shown that inactivation of several enzymes precedes overall conformational changes of the enzyme molecules as a whole during denaturation [Tsou (1993) Science, 262, 380-381]. However, the relation between inactivation, loss of allosteric properties of oligomeric enzymes and unfolding of the enzyme molecule during denaturation remain little explored. These have now been compared for D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose-1,6-bisphosphatase (FruP2ase) during denaturation by guanidinium chloride (GdmCl). GAPDH is completely inactivated at 0.3 M GdmCl but at this GdmCl concentration it still binds NAD+ with negative co-operativity. At 0.4 M GdmCl, inactivation of FruP2ase reaches completion whereas its allosteric properties, including the heterotropic effect of AMP inhibition and K+ activation with positive co-operativity, are only partially affected. Much higher GdmCl concentrations are required to bring about unfolding of the overall structures of both enzymes.

Escherichia coli β-galactosidase is heterogeneous with respect to the activity of individual molecules

1998 ◽  
Vol 76 (5) ◽  
pp. 623-626 ◽  
Author(s):  
Douglas B Craig ◽  
Norman J Dovichi
Keyword(s):  
Escherichia Coli ◽  
Capillary Electrophoresis ◽  
Single Molecule ◽  
Enzyme Assay ◽  
Laser Induced Fluorescence ◽  
Enzyme Molecule ◽  
Fluorogenic Substrate ◽  
Individual Enzyme

Escherichia coli β-galactosidase molecules were incubated with fluorogenic substrate in a capillary. Upon flushing the reactor contents past the detector, individual randomly distributed peaks of product were observed, each representing the activity of an individual enzyme molecule. Individual molecules were found to differ with respect to their activities. Molecules showed a 23-fold distribution of activities with the majority of molecules within a 4-fold distribution.Key words: single-molecule chemistry, β-galactosidase, capillary electrophoresis, laser-induced fluorescence, enzyme assay.

A Study on the Monitoring of Nano Scale EHL Film Formation with Ultra Low Aspect Ratio

2005 ◽  
Vol 297-300 ◽  
pp. 257-262
Author(s):  
Si Youl Jang ◽  
Hyun Sang Kong
Keyword(s):  
Image Processing ◽  
Aspect Ratio ◽  
Film Thickness ◽  
Digital Image Processing ◽  
Digital Image ◽  
Processing Technology ◽  
Fluid Film ◽  
Nanometer Scale ◽  
Low Aspect Ratio ◽  
Fluid Film Thickness

The measurement of ultra low aspect ratio fluid film thickness is very crucial technique both for the verification of lubrication media characteristics and for the clearance design in many precision components such as MEMS, precision bearings and other slideways. Many technologies are applied to the measurement of ultra low aspect ratio fluid film thickness (i.e. elastohydrodynamic lubrication film thickness). In particular, in-situ optical interferometric method has many advantages in making the actual contact behaviors realized with the experimental apparatus. This measurement method also does the monitoring of the surface defects and fractures happening during the contact behavior, which are delicately influenced by the surface conditions such as load, velocity, lubricant media as well as surface roughness. Careful selection of incident lights greatly enhances the fringe resolutions up to ~1.0 nanometer scale with digital image processing technology. In this work, it is found that coaxial aligning trichromatic incident light filtering system developed by the author can provide much finer resolution of ultra low aspect ratio fluid film thickness than monochromatic or dichromatic incident lights, because it has much more spectrums of color components to be discriminated according the variations of film thickness. For the measured interferometric images of ultra low aspect ratio fluid film thickness it is shown how the film thickness is finely digitalized and measured in nanometer scale with digital image processing technology and space layer method. The developed measurement system can make it possible to visualize the contact deformations and possible fractures of contacting surface under the repeated loading condition.

scholarly journals Reactions of 2-carbonyl and 2-hydroxy(or methoxy)alkyl substituted benzimidazoles with arenes in the superacid CF3SO3H. NMR and DFT study of dicationic electophilic species

2019 ◽  
Author(s):  
Dmitry Ryabukhin ◽  
Alexey Turdakov ◽  
Natalia Soldatova ◽  
Mikhail Kompanets ◽  
Alexander Ivanov ◽  
...  
Keyword(s):  
Dft Calculations ◽  
Reaction Mechanisms ◽  
Dft Study ◽  
Reaction Intermediates ◽  
Reaction Products

Interaction of 2-carbonyl and 2-hydroxy(or methoxy)alkyl benzimidazoles with arenes in the Brønsted superacid TfOH resulted in the formation of the corresponding Friedel-Crafts reaction products, 2-diarylmethyl and 2-arylmethyl substituted benzimidazoles in yields up to 90%. The reaction intermediates, N,O-diproptonated species derived from starting benzimidazoles in TfOH, were thorougly studied by means of NMR and DFT calculations. The plausible reaction mechanisms have been discussed.

Sign in / Sign up

Export Citation Format

Share Document