Macromolecular interactions

SRP 3. Macromolecular interactions

3.1 Molecular System Bioenergetics

Molecular System Bioenergetics approach is aimed to study intracellular structural interactions in the regulation of energy metabolism in healthy cells as well as in pathology. System Biology paradigm assumes the description of complicated biological system through the study of relatively independent subsystems; describing their structure, function and interactions between them.
In the case of cardiac muscle cell bioenergetics the approach requires the kinetic co-functioning description of the system comprising of respiratory chain, ATP sythasome in the mitochondrial inner membrane (including ATP synthase, adenosine-nucleotide translocase and phosphate transporters), mitochondrial creatine kinase, porine channel in the mitochondrial outer membrane (through which the metabolites are exchanged to cytosol) and protein factors modulating the channel, one of which is assumed to be tubulin heterodimer.
Alterations of intracellular structural interactions and formation of mature energy metabolism during postnatal development is an ideal model to study highly organised intracellular systems, where the bioenergetic regulation of cells varies according to their structure. Changes in the cell bioenergetics are one of the first signs of the cell pathology; therefore the studies of the bioenergetics of the malignant cells are of great importance.
The program gives us theoretical background to understand the bioenergetics of healthy muscle cells, as well as cellular pathologies like ischemia, heart failure, myocardial infarction, neurodegenerative diseases, bioenergetic mechanisms of cancer, and mechanisms of reperfusion injury.

3.2 Interactions of Biomolecules

Cellular processes and their regulation are based on recognition, binding and co-operation of bioactive molecules, and are determined by fundamental interactions in a very special cellular medium that is highly crowded with biomolecules. These basic interactions tend to yield non-traditional and unexpected manifestations.
The aim of the research is to understand the interactions and to give their predictable description combining experimental studies with corresponding theoretical modelling of processes based on structural data.
Basic principles of molecular recognition and complex formation are studied in systems: protein-protein, protein-DNA, protein-cell membrane, protein-bioactive peptide, and protein low molecular ligands.
Our focus in these studies is finding and isolating new catalytic, gene regulating, diagnostic and pharmacological active substances from different natural sources and the overall biochemical characterization of these substances.
The results of the studies have fundamental character as they clarify the mechanism of molecular recognition between proteins and other signalling molecules in normal and pathological processes. The practical importance of the investigations is based on the potential role of the new unique proteins in understanding and resolving of contemporary health problems such as cancer, neurodegenerative, heart and blood diseases. The knowledge of the structure – function relationships of biomolecules is necessary in medicine and in pharmacology for designing new drugs and diagnostics and these data are helpful for synthesis of new bioactive substances.

3.3 Two-photon Absorption (2PA) in Biomolecules

Two-photon fluorescence microscopy is becoming one of the standard and most informative methods in biological research because it facilitates increased spatial resolution and increased depth of tissue penetration. These useful attributes occur due to special physical properties of two-photon absorption (2PA) phenomena, which include quadratic dependence of the excitation probability on instantaneous photon flux density, and which also allow using near-infrared wavelengths to excite visible fluorescence.
There is however at least one more unique physical property of 2PA that can and should be exploited to obtain important novel information, especially in order to address numerous critical questions regarding structure and function of biopolymers. Namely, because 2PA constitutes a higher-order interaction between light and molecular chromophore, the probability of this process depends not only on transition dipole moments between different molecular energy levels, but also depends on the value of permanent electric dipole moments of the same chromophore, which itself varies as a function of local electric field. Recently it was shown that quantitative measurement of 2PA cross section in biological chromophores such as fluorescent proteins can be used to determine accurate value of the corresponding dipole moment difference parameter, and thus determine the strength and direction of the local electric field acting inside 3-nm diameter barrel protein. This type of novel physical measurement is uniquely valuable because it allows to begin shedding light on the very fundamental, but still largely unknown properties of local electrostatic interactions in- and between biopolymers on nanometre scale.
The purpose of the research work in this area is two-fold. The first goal is to continue developing physical principles of local electric field sensing by two-photon spectroscopy and microscopy. This is addressed by investigating 2PA properties in broad range of different fluorescent as well as non-fluorescent biomolecular constructs and probes, in order to create and characterize novel type of molecular multi-photon optical sensors that are specially designed to detect and quantify local electric fields. Improving accuracy and reliability of 2PA data, as well as improving acquisition speed, optimizing wavelength range, integration with existing microscope systems etc. are examples of numerous critical technical issues that also need to be addressed. The second and at this time a more distant goal is to initiate R&D level work on specialized hardware and software that, in combination with the specialized 2PA-optimized molecular probes can be used by other researches’ for a broad range of biomolecular investigations.
The utmost purpose of this strategic research direction is to develop new experimental methods and physical instruments or tools that will allow us and other researchers worldwide to understand how biopolymers perform their most amazing complex functions, and perhaps how man-made technology could augment or mimic these functions. All this will ultimately allow us better understand physical principles of life itself.

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