Single Molecule Michaelis-Menten Equation beyond Quasi-Static Disorder

The classic Michaelis-Menten equation describes the catalytic activities for ensembles of enzyme molecules very well. But recent single-molecule experiment showed that the waiting time distribution and other properties of single enzyme molecule are not consistent with the prediction based on the viewpoint of ensemble. It has been contributed to the slow inner conformational changes of single enzyme in the catalytic processes. In this work we study the general dynamics of single enzyme in the presence of dynamic disorder. We find that at two limiting cases, the slow reaction and nondiffusion limits, Michaelis-Menten equation exactly holds although the waiting time distribution has a multiexponential decay behaviors in the nondiffusion limit.Particularly, the classic Michaelis-Menten equation still is an excellent approximation other than the two limits.Comment: 10 pages, 1 figur

Repurposing designed mutants: a valuable strategy for computer-aided laccase engineering – the case of POXA1b

The broad specificity of laccases, a direct consequence of their shallow binding site, makes this class of enzymes a suitable template to build specificity toward putative substrates. In this work, a computational methodology that accumulates beneficial interactions between the enzyme and the substrate in productive conformations is applied to oxidize 2,4-diamino-benzenesulfonic acid with POXA1b laccase. Although the experimental validation of two designed variants yielded negative results, most likely due to the hard oxidizability of the target substrate, molecular simulations suggest that a novel polar binding scaffold was designed to anchor negatively charged groups. Consequently, the oxidation of three such molecules, selected as representative of different classes of substances with different industrial applications, significantly improved. According to molecular simulations, the reason behind such an improvement lies in the more productive enzyme–substrate binding achieved thanks to the designed polar scaffold. In the future, mutant repurposing toward other substrates could be first carried out computationally, as done here, testing molecules that share some similarity with the initial target. In this way, repurposing would not be a mere safety net (as it is in the laboratory and as it was here) but rather a powerful approach to transform laccases into more efficient multitasking enzymes.This work was funded by INDOX (KBBE-2013-7-613549) European project and CTQ2013-48287-R Spanish National Project. V. G. and E. M. acknowledge Università degli Studi di Napoli and Generalitat de Catalunya for their respective predoctoral fellowships.Peer ReviewedPostprint (author's final draft

Drug transport mechanism of P-glycoprotein monitored by single molecule fluorescence resonance energy transfer

In this work we monitor the catalytic mechanism of P-glycoprotein (Pgp) using single-molecule fluorescence resonance energy transfer (FRET). Pgp, a member of the ATP binding cassette family of transport proteins, is found in the plasma membrane of animal cells where it is involved in the ATP hydrolysis driven export of hydrophobic molecules. When expressed in the plasma membrane of cancer cells, the transport activity of Pgp can lead to the failure of chemotherapy by excluding the mostly hydrophobic drugs from the interior of the cell. Despite ongoing effort, the catalytic mechanism by which Pgp couples MgATP binding and hydrolysis to translocation of drug molecules across the lipid bilayer is poorly understood. Using site directed mutagenesis, we have introduced cysteine residues for fluorescence labeling into different regions of the nucleotide binding domains (NBDs) of Pgp. Double-labeled single Pgp molecules showed fluctuating FRET efficiencies during drug stimulated ATP hydrolysis suggesting that the NBDs undergo significant movements during catalysis. Duty cycle-optimized alternating laser excitation (DCO-ALEX) is applied to minimize FRET artifacts and to select the appropriate molecules. The data show that Pgp is a highly dynamic enzyme that appears to fluctuate between at least two major conformations during steady state turnover.Comment: 10 pages, 7 figure

Label-free optical detection of single enzyme-reactant reactions and associated conformational changes

Monitoring the kinetics and conformational dynamics of single enzymes is crucial in order to better understand their biological functions as these motions and structural dynamics are usually unsynchronized among the molecules. Detecting the enzyme-reactant interactions and associated conformational changes of the enzyme on a single molecule basis, however, remain as a challenge with established optical techniques due to the commonly required labeling of the reactants or the enzyme itself. The labeling process is usually non-trivial and the labels themselves might skew the physical properties of the enzyme. Here we demonstrate an optical, label-free method capable of observing enzymatic interactions and the associated conformational changes on the single molecule level. We monitor polymerase/DNA interactions via the strong near-field enhancement provided by plasmonic nanorods resonantly coupled to whispering gallery modes in microcavities. Specifically, we employ two different recognition schemes: one in which the kinetics of polymerase/DNA interactions are probed in the vicinity of DNA-functionalized nanorods, and the other in which these interactions are probed via the magnitude of conformational changes in the polymerase molecules immobilized on nanorods. In both approaches we find that low and high polymerase activities can be clearly discerned via their characteristic signal amplitude and signal length distributions. Furthermore, the thermodynamic study of the monitored interactions suggests the occurrence of DNA polymerization. This work constitutes a proof-of-concept study of enzymatic activities via plasmonically enhanced microcavities and establishes an alternative and label-free method capable of investigating structural changes in single molecules

Influence of ellagitannins extracted by pomegranate fruit on disulfide isomerase PDIA3 activity

Pomegranate fruit is a functional food of high interest for human health due to its wide range of phytochemicals with antioxidant properties are implicated in the prevention of inflammation and cancer. Ellagitannins, such as punicalagin and ellagic acid, play a role as anti-atherogenic and neuroprotective molecules in the complex fighting against the degenerative diseases. The aim of this work was to evaluate the composition in punicalagins and ellagic acid of differently obtained extracts from whole fruit, peels and juices, prepared by squeezing or by centrifugation, of pomegranate belonging to different cultivars. Moreover, a wider phenolic fingerprint was also determined. The bioactivity of the extracts was tested on the redox activity of PDIA3 disulfide isomerase, an enzyme involved in the regulation of several cellular functions and associated with different diseases such as cancer, prion disorders, Alzheimer’s and Parkinson’s diseases. The results demonstrate that the different ratios between punicalagin and ellagic acid modulate the enzyme activity and other ellagitannins could interfere with this activity

Geometric Universality of Currents

We discuss a non-equilibrium statistical system on a graph or network. Identical particles are injected, interact with each other, traverse, and leave the graph in a stochastic manner described in terms of Poisson rates, possibly dependent on time and instantaneous occupation numbers at the nodes of the graph. We show that under the assumption of constancy of the relative rates, the system demonstrates a profound statistical symmetry, resulting in geometric universality of the statistics of the particle currents. This phenomenon applies broadly to many man-made and natural open stochastic systems, such as queuing of packages over the internet, transport of electrons and quasi-particles in mesoscopic systems, and chains of reactions in bio-chemical networks. We illustrate the utility of our general approach using two enabling examples from the two latter disciplines.Comment: 15 pages, 5 figure

Drug delivery in overcoming the blood-brain barrier: role of nasal mucosal grafting

The blood–brain barrier (BBB) plays a fundamental role in protecting and maintaining the homeostasis of the brain. For this reason, drug delivery to the brain is much more difficult than that to other compartments of the body. In order to bypass or cross the BBB, many strategies have been developed: invasive techniques, such as temporary disruption of the BBB or direct intraventricular and intracerebral administration of the drug, as well as noninvasive techniques. Preliminary results, reported in the large number of studies on the potential strategies for brain delivery, are encouraging, but it is far too early to draw any conclusion about the actual use of these therapeutic approaches. Among the most recent, but still pioneering, approaches related to the nasal mucosa properties, the permeabilization of the BBB via nasal mucosal engrafting can offer new potential opportunities. It should be emphasized that this surgical procedure is quite invasive, but the implication for patient outcome needs to be compared to the gold standard of direct intracranial injection, and evaluated whilst keeping in mind that central nervous system diseases and lysosomal storage diseases are chronic and severely debilitating and that up to now no therapy seems to be completely successful