Technologies based on pulsed electric field (PEF) are increasingly pervasive in medical and industrial applications. However, the detailed understanding of how PEF acts on biosamples including proteins at the molecular level is missing. There are indications that PEF might act on biomolecules via electrogenerated reactive oxygen species (ROS). However, it is unclear how this action is modulated by the pro- and antioxidants, which are naturally present components of biosamples. This knowledge gap is often due to insufficient sensitivity of the conventionally utilized detection assays. To overcome this limitation, here we employed an endogenous (bio)chemiluminescence sensing platform, which enables sensitive detection of PEF-generated ROS and oxidative processes in proteins, to inspect effects of pro-and antioxidants. Taking bovine serum albumin (BSA) as a model protein, we found that the chemiluminescence signal arising from its solution is greatly enhanced in the presence of H 2 O 2 as a prooxidant, especially during PEF treatment. In contrast, the chemiluminescence signal decreases in the presence of antioxidant enzymes (catalase, superoxide dismutase), indicating the involvement of both H 2 O 2 and electrogenerated superoxide anion in oxidation-reporting chemiluminescence signal before, during, and after PEF treatment. We also performed additional biochemical and biophysical assays, which confirmed that BSA underwent structural changes after H 2 O 2 treatment, with PEF having only a minor effect. We proposed a scheme describing the reactions leading from interfacial charge transfer at the anode by which ROS are generated to the actual photon emission. Results of our work help to elucidate the mechanisms of action of PEF on proteins via electrogenerated reactive oxygen species and open up new avenues for the application of PEF technology. The developed chemiluminescence technique enables label-free, in-situ and non-destructive sensing of interactions between ROS and proteins. The technique may be applied to study oxidative damage of other classes of biomolecules such as lipids, nucleic acids or carbohydrates.

• MeSH
• antioxidancia * metabolismus   MeSH
• elektřina MeSH
• katalasa metabolismus   MeSH
• luminiscence MeSH
• luminiscenční měření * metody   MeSH
• oxidace-redukce * MeSH
• peroxid vodíku metabolismus   MeSH
• reaktivní formy kyslíku * metabolismus   MeSH
• sérový albumin hovězí * metabolismus   MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antioxidancia * MeSH
• katalasa MeSH
• peroxid vodíku MeSH
• reaktivní formy kyslíku * MeSH
• sérový albumin hovězí * MeSH

Electrochemical methods can be used not only for the sensitive analysis of proteins but also for deeper research into their structure, transport functions (transfer of electrons and protons), and sensing their interactions with soft and solid surfaces. Last but not least, electrochemical tools are useful for investigating the effect of an electric field on protein structure, the direct application of electrochemical methods for controlling protein function, or the micromanipulation of supramolecular protein structures. There are many experimental arrangements (modalities), from the classic configuration that works with an electrochemical cell to miniaturized electrochemical sensors and microchip platforms. The support of computational chemistry methods which appropriately complement the interpretation framework of experimental results is also important. This text describes recent directions in electrochemical methods for the determination of proteins and briefly summarizes available methodologies for the selective labeling of proteins using redox-active probes. Attention is also paid to the theoretical aspects of electron transport and the effect of an external electric field on the structure of selected proteins. Instead of providing a comprehensive overview, we aim to highlight areas of interest that have not been summarized recently, but, at the same time, represent current trends in the field.

• Klíčová slova
• Electrode, Microdevice, Peptide, Protein, Sensor,
• MeSH
• elektrochemické techniky * metody   MeSH
• elektrochemie MeSH
• oxidace-redukce MeSH
• proteiny * MeSH
• transport elektronů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• proteiny * MeSH

Kinesin is a motor protein essential in cellular functions, such as intracellular transport and cell-division, as well as for enabling nanoscopic transport in bio-nanotechnology. Therefore, for effective control of function for nanotechnological applications, it is important to be able to modify the function of kinesin. To circumvent the limitations of chemical modifications, here we identify another potential approach for kinesin control: the use of electric forces. Using full-atom molecular dynamics simulations (247,358 atoms, total time ∼ 4.4 μs), we demonstrate, for the first time, that the kinesin-1 motor domain can be detached from a microtubule by an intense electric field within the nanosecond timescale. We show that this effect is field-direction dependent and field-strength dependent. A detailed analysis of the electric forces and the work carried out by electric field acting on the microtubule-kinesin system shows that it is the combined action of the electric field pulling on the β-tubulin C-terminus and the electric-field-induced torque on the kinesin dipole moment that causes kinesin detachment from the microtubule. It is shown, for the first time in a mechanistic manner, that an electric field can dramatically affect molecular interactions in a heterologous functional protein assembly. Our results contribute to understanding of electromagnetic field-biomatter interactions on a molecular level, with potential biomedical and bio-nanotechnological applications for harnessing control of protein nanomotors.

• Klíčová slova
• Electric field, Microtubules, Molecular dynamics simulation, Proteins, Tubulin,
• Publikační typ
• časopisecké články MeSH