scholarly journals Single molecule turnover of fluorescent ATP by myosin and actomyosin unveil elusive enzymatic mechanisms

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Marko Ušaj ◽  
Luisa Moretto ◽  
Venukumar Vemula ◽  
Aseem Salhotra ◽  
Alf Månsson

AbstractBenefits of single molecule studies of biomolecules include the need for minimal amounts of material and the potential to reveal phenomena hidden in ensembles. However, results from recent single molecule studies of fluorescent ATP turnover by myosin are difficult to reconcile with ensemble studies. We found that key reasons are complexities due to dye photophysics and fluorescent contaminants. After eliminating these, through surface cleaning and use of triple state quenchers and redox agents, the distributions of ATP binding dwell times on myosin are best described by 2 to 3 exponential processes, with and without actin, and with and without the inhibitor para-aminoblebbistatin. Two processes are attributable to ATP turnover by myosin and actomyosin respectively, whereas the remaining process (rate constant 0.2–0.5 s−1) is consistent with non-specific ATP binding to myosin, possibly accelerating ATP transport to the active site. Finally, our study of actin-activated myosin ATP turnover without sliding between actin and myosin reveals heterogeneity in the ATP turnover kinetics consistent with models of isometric contraction.


Author(s):  
Marko Usaj ◽  
Luisa Moretto ◽  
Venukumar Vemula ◽  
Aseem Salhotra ◽  
Alf Månsson

AbstractSingle molecule enzymology using fluorescent substrate requires truly minimal amounts of proteins. This is highly beneficial when the protein source is either advanced expression systems or samples from humans/animals with ethical and economic implications. Further benefits of single molecule analysis is the potential to reveal phenomena hidden in ensemble studies. However, dye photophysics and fluorescent contaminants complicate interpretation of the single molecule data. We here corroborate the importance of such complexities using fluorescent Alexa647 ATP to study ATP turnover by myosin and actomyosin. We further show that the complexities are largely eliminated by aggressive surface cleaning and use of a range of triple state quenchers and redox agents with minor effects on actin-myosin function. Using optimized assay conditions, we then show that the distributions of ATP binding dwell times on myosin are best described by the sum of 2 to 3 exponential processes. This applies in the presence and absence of actin and in the presence and absence of the drug para-aminoblebbistatin. Two of the processes are attributable to ATP turnover by myosin and actomyosin, respectively. A remaining process with rate constant in the range 0.2-0.5 s-1 is consistent with non-specific ATP binding to myosin and bioinformatics modelling suggests that such binding may be important for accelerated ATP transport to the active site. Finally, we report studies of the actin-activated myosin ATP turnover under conditions with no sliding between actin and myosin, as in isometrically contracting muscle, revealing heterogeneity in the ATP turnover kinetics between different molecules.



2004 ◽  
Vol 32 (1) ◽  
pp. 41-45 ◽  
Author(s):  
S.A. McKinney ◽  
E. Tan ◽  
T.J. Wilson ◽  
M.K. Nahas ◽  
A.-C. Déclais ◽  
...  

Branched helical junctions are common in nucleic acids. In DNA, the four-way junction (Holliday junction) is an essential intermediate in homologous recombination and is a highly dynamic structure, capable of stacking conformer transitions and branch migration. Our single-molecule fluorescence studies provide unique insight into the energy landscape of Holliday junctions by visualizing these processes directly. In the hairpin ribozyme, an RNA four-way junction is an important structural element that enhances active-site formation by several orders of magnitude. Our single-molecule studies suggest a plausible mechanism for how the junction achieves this remarkable feat; the structural dynamics of the four-way junction bring about frequent contacts between the loops that are needed to form the active site. The most definitive evidence for this is the observation of three-state folding in single-hairpin ribozymes, the intermediate state of which is populated due to the intrinsic properties of the junction.



2017 ◽  
Author(s):  
Wenfa Ng

Scale transcends multiple levels of biological and physical organization, and is the critical factor that determines success of any scientific investigation. Specifically, the scale at which a question is posed plays a crucial role in influencing the type of experiments and apparatuses needed. Single cell and single molecule experiments came to the fore of experiment science due to its capability at addressing a fundamental problem in biology and physical science: does the same behavior in cells and molecules transcend different length and population scales? Thus far, single cell experiments could be achieved with trapping of single cell using optical tweezer traps and microfluidic channels. The same, however, is not true for single molecule studies, which remains in the realm of theoretical and simulation studies. Specifically, single molecule experiment remains at the hundreds to thousands of molecules level, where possible skew in the population of molecules sampled could provide a false depiction of molecular reality of a larger population. But, what do scientists learn from single cell and single molecule studies? Is it the uncovering of mysteries of the probabilistic behavior at the single entity level, guided by perhaps quantum mechanics? The answer is no for single cell studies, given that cellular decision making require the input of tens to hundreds of molecular sensors and effectors within a cell. Hence, single cell behavior is not random, but directed at a nutrient or concentration gradient or signaling source. On the other hand, enzymatic catalysis of a single molecule substrate with the active site involves a quantum mechanical crosstalk. Thus, reaction between the substrate molecule and the active site proceeds if suitable energy levels (i.e., quantum mechanical states) are found for both parties. Given that distribution of quantum mechanical states is probabilistic, stochasticity rules single molecule interaction such as a covalent bond formation reaction between reactant A and B. Thus, single cell and single molecule studies do hold relevance in biological and physical sciences research if the correct experiment tool is used for a pertinent question at an appropriate length and population scale. For example, while tremendous amount of basic understanding could be derived from single cell experiments, single cell perspective is not relevant to questions examining the interactions between two large subpopulations of cells. Single molecule experiments, on the other hand, remains in the theoretical and simulation realm for highlighting the effect of quantum mechanics in guiding the behavior of molecules at the nanoscale.



2017 ◽  
Author(s):  
Wenfa Ng

Scale transcends multiple levels of biological and physical organization, and is the critical factor that determines success of any scientific investigation. Specifically, the scale at which a question is posed plays a crucial role in influencing the type of experiments and apparatuses needed. Single cell and single molecule experiments came to the fore of experiment science due to its capability at addressing a fundamental problem in biology and physical science: does the same behavior in cells and molecules transcend different length and population scales? Thus far, single cell experiments could be achieved with trapping of single cell using optical tweezer traps and microfluidic channels. The same, however, is not true for single molecule studies, which remains in the realm of theoretical and simulation studies. Specifically, single molecule experiment remains at the hundreds to thousands of molecules level, where possible skew in the population of molecules sampled could provide a false depiction of molecular reality of a larger population. But, what do scientists learn from single cell and single molecule studies? Is it the uncovering of mysteries of the probabilistic behavior at the single entity level, guided by perhaps quantum mechanics? The answer is no for single cell studies, given that cellular decision making require the input of tens to hundreds of molecular sensors and effectors within a cell. Hence, single cell behavior is not random, but directed at a nutrient or concentration gradient or signaling source. On the other hand, enzymatic catalysis of a single molecule substrate with the active site involves a quantum mechanical crosstalk. Thus, reaction between the substrate molecule and the active site proceeds if suitable energy levels (i.e., quantum mechanical states) are found for both parties. Given that distribution of quantum mechanical states is probabilistic, stochasticity rules single molecule interaction such as a covalent bond formation reaction between reactant A and B. Thus, single cell and single molecule studies do hold relevance in biological and physical sciences research if the correct experiment tool is used for a pertinent question at an appropriate length and population scale. For example, while tremendous amount of basic understanding could be derived from single cell experiments, single cell perspective is not relevant to questions examining the interactions between two large subpopulations of cells. Single molecule experiments, on the other hand, remains in the theoretical and simulation realm for highlighting the effect of quantum mechanics in guiding the behavior of molecules at the nanoscale.



2019 ◽  
Vol 116 (3) ◽  
pp. 128a-129a ◽  
Author(s):  
Sushi Madhira ◽  
Don C. Lamb ◽  
Promod R. Pratap


2014 ◽  
Vol 42 (8) ◽  
pp. 5177-5190 ◽  
Author(s):  
Amit Sharma ◽  
Robert N. Leach ◽  
Christopher Gell ◽  
Nan Zhang ◽  
Patricia C. Burrows ◽  
...  


2021 ◽  
Vol 22 (5) ◽  
pp. 2398
Author(s):  
Wooyoung Kang ◽  
Seungha Hwang ◽  
Jin Young Kang ◽  
Changwon Kang ◽  
Sungchul Hohng

Two different molecular mechanisms, sliding and hopping, are employed by DNA-binding proteins for their one-dimensional facilitated diffusion on nonspecific DNA regions until reaching their specific target sequences. While it has been controversial whether RNA polymerases (RNAPs) use one-dimensional diffusion in targeting their promoters for transcription initiation, two recent single-molecule studies discovered that post-terminational RNAPs use one-dimensional diffusion for their reinitiation on the same DNA molecules. Escherichia coli RNAP, after synthesizing and releasing product RNA at intrinsic termination, mostly remains bound on DNA and diffuses in both forward and backward directions for recycling, which facilitates reinitiation on nearby promoters. However, it has remained unsolved which mechanism of one-dimensional diffusion is employed by recycling RNAP between termination and reinitiation. Single-molecule fluorescence measurements in this study reveal that post-terminational RNAPs undergo hopping diffusion during recycling on DNA, as their one-dimensional diffusion coefficients increase with rising salt concentrations. We additionally find that reinitiation can occur on promoters positioned in sense and antisense orientations with comparable efficiencies, so reinitiation efficiency depends primarily on distance rather than direction of recycling diffusion. This additional finding confirms that orientation change or flipping of RNAP with respect to DNA efficiently occurs as expected from hopping diffusion.



2010 ◽  
Vol 63 (4) ◽  
pp. 624
Author(s):  
Michael J. Serpe ◽  
Jason R. Whitehead ◽  
Stephen L. Craig

Single molecule atomic force microscopy (AFM) studies of oligonucleotide-based supramolecular polymers on surfaces are used to examine the molecular weight distribution of the polymers formed between a functionalized surface and an AFM tip as a function of monomer concentration. For the concentrations examined here, excellent agreement with a multi-stage open association model of polymerization is obtained, without the need to invoke additional contributions from secondary steric interactions at the surface.



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