SCIENTIFIC ACTUALITY OF THE RESEARCH

2. SCIENTIFIC ACTUALITY OF THE RESEARCH

A special attention is to be given to the investigation of biological adaptation to ²H₂O allowing cells to synthesize a deuterated forms of macromolecules (particulary interest have DNA and short-chain individual proteins both with well known amino acid sequence and conformation) with a certain structure allowing their functioning in ²H₂O environment.

Firstly, in this connection it would be very interesting to know, how the structure of fully deuterated macromolecules could be changed neganively or positively in a course of biological adaptation to ²H₂O requiring the presence of high concentrations of ²H₂O in growth media.

Secondly, if a cell will be growing on media containing the stepwise increasing concentrations of ²H₂O, for example starting up from zero up to 100% (v/v) ²H₂O, will the changes in the structure of [U -²H]labeled macromolecules to be corresponding to the ²H₂O content in media and what is a limit concentration of ²H₂O when the macromolecular structure keeps a stable constancy and how this fact corresponds with a limit of biological resistance to ²H₂O? For answers to these questions a number of modern consideration at the levels of the structure (primary, secondary, tertiary) and conformation of [U -²H]labeled DNA and individual proteins with using the methods of a special sequencing and modifications of deuterated macromolecules combined together with gel electrophoresis method as well as such powerful methods as NMR-spectroscopy to which will be taken a most part of proposed research, X-ray diffraction, IR-, laser- and CD-spectroscopy will be further involved.

An investigation will necessary mainly into the structure of [U -²H]labeled macromolecules in order to find at what level of macromolecular hierarchy a substitution of hydrogen atoms with deuterium ensued the consequence on the differences in the structure and the conformation of macromolecules and, therefore, the functional properties of the macromolecules in ²H₂O. In the frames of proposed research the developing of methods of biological adaptation to obtain [U -²H]labeled biological material with high levels of enrichment are also of a big interest. For this purpose the special biotechnological approaches based on using the strains with improved properties when growing on ²H₂O for obtaining fully deuterated DNA and individual proteins should be applied for allowing to prepare [U -²H]labeled macromolecules in gram scale quantities.

3. DISCUSSION

3.1. The methods for analyzing the structure and the conformation of [U -²H]labeled macromolecules.

The biological labelling with deuterium is an useful tool for investigating the structure and the conformational properties of macromolecules. The fundamental objectives have meant that living models have retained their importance for functional studies of such biological important macromolecules and can be used to obtain structural and dynamic information about the [U -²H]labeled macromolecules.

The method of X-ray diffraction should be noted as a indespencible tool for determing the details of the three-dimentional structure of globular proteins and other macromolecules (Mathews C. K., van Holde K. E., 1996). Yet this technique has the fundamental limitation that it can be employed only when the molecules are crystallized, and crystallization is not always easy or even possible. Furthermore, this method cannot easily be used to study the conformational changes in response to changes in the molecules environment.

Other methods, for example IR-spectroscopy, can provide direct information concerning the macromolecular structure. For example, the exact positions of infrared bands corresponding to vibrations in the polypeptide backbone are sensitive to the conformational state (a helix, b sheet et.) of the chain (Campbell I. D., and Dwek R. A., 1984). Thus, the studies in this region of the spectrum are often used to investigate the conformations of protein molecules.

Although, IR-, and absorption spectroscopy can be helpful in following molecular changes, such measurements are difficult to interpret directly in terms of changes of secondary structure. For this purpose, techniques of circular dichroism involving polarized light have become important (Johnson W. C., 1990). For example, if a protein is denatured so that its native structure, containing a helix and b sheet regions, is transformed into an unfolded, random-coil structure, this transformation will be reflected in a dramatic change in its CD spectrum. Circular dichroism can be used in another way, to estimate the content of a helix and b sheet in native proteins. The contributions of these different secondary structures to their circular dichroism at different wavelenghths are known, so we may attempt to match an observed spectrum of protein by a combination of such contributions.

Although circular dichroism is an extremely useful technique, it is not a very discriminating one. That is, it cannot, at present, tell us what is happening at a particular point in a protein molecule. A method that has the great potential to do so is nuclear magnetic resonance. This advance now make it possible to use NMR to study a big varieties of DNA and proteins with more complex biological functions functioning in natural liquid environment. Often these proteins have more than one domain and more than one site of interaction. Allosteric systems, receptors and small molecule ligand-modulated DNA-binding proteins and DNA are some examples of the molecular systems which can now be analysed in molecular detail. For example, due to the development of two-dimentional Fourier transformation techniques, NMR spectroscopy has become a powerful tool for determining the protein structure and conformation (Fesic S. W. and Zuiderweg E. R., 1990).

3.2. The preparation of [U- ²H]labeled macromolecules.

Through technical advances of biotechnology, many macromolecules, for example a certain individual proteins are successfuly cloned and can be obtained in large quantities by expression in microbial and/or mammalian systems, so that an ever-increasing number of individual [U- ²H]labeled macromolecules from various biological objects are becoming commercially available. It should be noted, however, that the application of various methods for the preparation of [U -²H]labeled macromolecules (chemical or biosynthetical) often results in obtaining the forms of molecules with different number of protons substituted by deuterium, the phenomenon that is known as heterogenious labelling, so that the special methods for the preparation of [U -²H]labeled macromolecules should be applyed to minimaze this process. For example, the proteins containing only deuterium atoms in polypeptide chain of macromolecule can be produced biotechnologically with using the special genetically constructed strains of bacteria carrying the mutations of geens excluding the metabolic exchange between the parterns of unlabeled intermediators during the biosynthesis of [U -²H]labeled macromolecules.

I may briefly indicate three possibilities for deuterium enrichment:

(1) to grow the organism on a minium salt medium with content of ²H₂O 99% ²H;

(2) To grow the organism on a medium supplemented with 99% ²H₂O and [U -²H]labeled amino acid mixture.

(3) the isotopic exchange of susceptible protons in amino acid residues already incorporated into protein.

Method 1 is very useful for the preparation of [U- ²H]labeled macromolecules if only applyed strains of bacterial or different origin could well be grown on minimal media in the presence of high concentrations of ²H₂O. Very often in this case the biological adaptation to ²HO is required. Method 2, while generally applicable, is limited by the difficulty and expense of preparing fully deuterated amino acid mixtures from algae grown on ²H₂O. However, recently we proposed to use a fully deuterated biomass of methlotrophic bacterium B. methylicum with protein content about 55% (from dry weight) obtained via multistep adaptaition to 98% (v/v) ²H₂O and 2% (v/v) [U-²H]MetOH as growth substrates for growing the other bacterial strains to prepare a gram quantities of [U -²H]labeled amino acids, proteins and nucleosites with high levels of enrichment (90.0-97.5% ²H) (Mosin O. V., Karnaukhova E. N., Pshenichnikova A. B.; 1994; Skladnev D. A., Mosin O. V., et all; 1996; Shvets V. I., Yurkevich A. M., Mosin O. V.; 1995).

 Method 2 is also necessary when the organism will not grow on a minimal medium as it was in the case with the applying the bacteria requiring the complex composition media for their growth. This approach will also be necessary for the labeling of proteins expressed in systems other than E. coli (e.g. yeast, insect, and mammalian expression systems) which may be important for the proper folding of proteins from higher organisms. Since the protons of interest in proteins are most often carbon bound and thus do not exchange under mild conditions, method 3 is severely limited by stability of proteins under the harsh conditions necessary for (¹H-²H) exchange.

4. ADAPTATION TO ²H₂O AND BIOPHYSICAL PROPERTIES OF [U -²H]LABELED MACROMOLECULES

FIGURE

The imaginary principle of realization of biological adaptation

I II