Biological adaptation to heavy water (on the three-dimentional structure and the function of macromolecules in the presence of ²H₂O)

1. Actuality of the work proposed.

1.1. Deuterium (²H), the hydrogen isotope with mass 2, was discovered by Urey. In the years immediately following this discovery, there developed a keen interest in a biological enrichment of a cell with ²H, which resulted in a miscellany of rather confusing data (Katz J., Crespy H. L. 1972). The main conclusion that can be derived from the most competent and comprehensive of the early studies is that high concentrations of ²H₂O are incompatible with life and reproduction. Nevertheless, a many cells could be quite well adapted to ²H₂O, so that a discussion about the role of macromolecules in biolodical adaptation to ²H₂O is still actual through more than four decades of years after the first description of the biological consequences of hydrogen replacement by deuterium. For example, the presence of deuterium in biological systems probably be manifested by changes in the structure and the conformation of macromolecules. On the other side, the screw parameters of the proton helix are changed by the presence of deuterium so that ordinary proteins dissolved in ²H₂O exhibit a more stable helical structure. While ²H₂O exerts a stabilizing effect upon the hydrogen-bonded helix, the presence of nonexchangeable deuterium in amino acid side chains causes a decrease in protein stability. These opposing effects do not cancel, and complete deuteriation of a protein results in destabilization. Deuterium substitution can thus be expected to modify, to a greater or lesser degree of order of a cell. There is every prospect that continued investigation of deuterium isotope effects in living organisms will yield results of both scientific and practical importance, for it is precisely. That is why the studies of the three-dimentional structure and the functioning of such biolodical important macromolecules as DNA and proteins, and/or enzymes obtained via biological adaptaition to high (maximum) concentrations of ²H₂O are most attract an attention of scientists as the way for creating a deuterated forms of DNA and enzymes could well be working in many biotechnological processes required the presence of ²H₂O. Thus, the main purpose of the present project is the studies of the three-dimentional structure and the function of macromolecules (particularly DNA and individual proteins and/or enzymes) obtained via biological adaptation to high concentrations of ²H₂O.

1.2. To carry out the studies with deuterated macromolecules one must firstly to obtain the appropriate deuterated material with high level of enrichment for extraction the DNA and proteins to whom the various methods of stable isotope detection further can be applyed. For example, the three dimentional nuclear magnetic resonanse (NMR) combined together with the method of X-ray diffraction, infrared (IR)-, laser spectrometry and circular dichroism (CD) is a well proved method for the studies of the structure and the functioning of deuterated macromolecules, and for investigations of various aspects of the biophysical behavior. Taking into account the ecological aspect of deuterated compounds it should be noted, that the preferable properties of deuterium for biochemical studies are caused mainly by the absence of radioactivity of deuterium that is the most important fact for carring out the biological incorporation of deuterium into organism.

2. The scientific aims

A special aim is the investigation of biological adaptation to deuterium allowing to cells to synthesize a deuterated forms of macromolecules (DNA, proteins) with a three-dimentional structure allowing their functioning in ²H₂O environment. With scientific point of view it would be very interesting to know, how the deuterated macromolecules could change positively the conformation and the structure in the presence of high concentrations of ²H₂O. For answers to these questions a number of modern consideration at the levels of three-dimentional structure of DNA and proteins (and/or enzymes) with using the methods of NMR-, X-ray diffraction, IR-, laserand CD-spectroscopy will be involved. An investigation will be necessary into the structure of isotopic altered macromolecules in order to find whether the substitution of hydrogen atoms with deuterium ensued consequence to the diffrences in the structure and the conformation of macromolecules and the functioning. In the frames of research the developing of methods of biological adaptation to obtain the deuterated biologival material with highly (maximum§) levels of enrichment are also of interest. For this purpose the special biotechnological approaches on obtaining deuterated DNA and proteins should be applied for allowing to prepare them in gram scale quantities.

The research plan is based on a combination of microbiological and genetical research combaining with investigation of three-dimentional structure and the conformation of macromolecules with using NMR-, X-ray diffraction, IR-, laserand CD-spectroscopy technique (which of them is available there): 2.1. Study of certain aspects of synthesis of deuterated macromolecules by cells adapted to ²H₂O; 2.2. Investigations of functional properties of deuterated macromolecules; 2.4. The preparation of deuterated macromolecules (particularly individual proteins) with using adapted cells.

First, in frames of the biological research with deuterium the studies of incorporation of deuterium into the macromolecules are necessary. A biosynthetic introduction of deuterium in conjunction with mass-spectrometry and NMR technique has been used to study the ²H-enrichment via convertion of low molecular weight substrates (²H₂O with isotopic purity 99,9 at.% ²H, C²H₃O²H with isotopic purity 97,5 at.% ²H) to the excreted amino acids, proteins and nucleotides by various strains of microbial and algae origin (green microalgae Chlorella sp. and Dunaliella salina Teod., blue green algae Spirulina sp., halophilic bacterium Halobacterium halobium, methylotrophic bacteria Methylobacillus flagellatum and Brevibacterium methylicum, bacills Bacillus subtilis and Bacillus amiloliqufanciens (Mosin O. V., Karnaukhova E. N., et all., 1993; Mosin O. V., Skladnev D. A., et all., 1996a). The influence of ²H₂O and other deuterated substrates (e.g. C²H₃O²H) on several growth characteristics of the microorganisms (time of generation, lag-phase, yield of biomass) and biosynthesis was investigated.

Second, these microorganisms were investigated to growth and biosynthesis on media containing highly concentrations of ²H₂O (90-98 v/v.%+ ). For example, an adaptation method for the preparation of high enriched cell protein (93% ²H) based on using methylotrophic bacterium B. methylicum grown on a medium containing 2 v/v.% C²H₃O²H and 98 v/v. % ²H₂O was developed and successfuly tested on other microoorganisms (Mosin O. V., et all., 1996d). The method called a “step by step” adaptation method, concists in incubation of cells by the plating on agaric media with 2 v/v. C²H₃O²H and increasily content of ²H₂O (starting from ordinary water, subsequently taking 24,5, 50, 73,5 up to 98 v/v.% ²H₂O) with following selection of cells colonies grown on those media#. The phenomenon of adaptation may implay a considerable alteration in the proton-deuteron composition of the macromolecules and those effects could cause a small hindering of growth in the presence of ²H₂O. The results indicated, however, that decreasing of cell production of B. methylicum (not more than 1,5 fold) in highly deuterated media was stipulated by increasing of the lag-phase as well as by increasing of the cell generation time. The method developed is very suitable for the preparation of [²H]-biomass from that a many macromolecules with high levels of enrichment could be extracted. A second advantage is that the deuterated biomass with protein content 55% (from dry weight) obtained via multistep adaptaition could be used as substrates for growing the other strains (Mosin O. V., et all., 1996c) .

Third, many organisms were investigated for the efficient biosynthetic preparation of deuterated cell compounds, including proteins. For example, amino acids, proteins and nucleosites with levels of enrichment (90-97,5% ²H ) were obtained from adapted species of Chlorella sp., H. halobium, B. methylicum, and B. subtilis and isolated in gramm quanteties with using reverse-phase high performance liquid chromatography (HPLC) and other standard methods, including precipitation, crystallization, extraction et. (Egorova T. A., Mosin O. V., et all., 1993)

Finally, to follow an early started research on the structure and the function of deuterated membrane protein bacteriorhodopsin, the efficiency of site-specific incorporation of [2,3,4,5,6²H₅]phenylalanine (90% ²H), [3,5²H₂]tyrosine (96% ²H), [2,4,5,6,7-²H₅]tryptophan (98% ²H) into the protein as well an attempt to obtain the bacteriorhodopsin with high level of enrichment (90% ²H) via using as growth substrates the hydrolysate of B. methylicum obtained after the adaptation on medium containing 2 v/v.% C²H₃O²H and 98 v/v.% ²H₂O (Mosin O. V., 1996a) were studied (Mosin O. V., et all., 1996b).

3. Principle statement of the scientific questions.

In the present investigation the following key questions (points) will be considered: 3.1. The studies of deuterium incorporation into biological macromolecules issuing the proposed chandes in three-dimentional structure and the conformation of macromolecules; 3.2. The efficiency of using the adapted cells for the technological preparation of deuterated macromolecules.

4. Explanations of what I plan to do.

The main goal of the project is the biological adaptation to ²H₂O for the growing of cells in the presence of high concentrations of ²H₂O (depending on experimental conditionsÄ) as a main stage to obtain a gram quantities of biological deuterated material for further structural and functional studies .

The concrete goal is the research of the three-dimentional structure and the conformation of deuterated macromolecules obtained from cells adapted to high concentrations of ²H₂O, combined with the evaluation of isotopic enrichment of macromolecules.

The second point is the study of functional properties of deuterated macromolecules.

5. Explanations of what I plan to investigate.

I plan to investigate the following questions: 5.1. How cells by the changes in the structure and the conformation of deuterated macromolecules could adapt themselves to high concentration of ²H₂O and 5.2. How functionate the deuterated macromoleculs ?

The main objects will be a different kinds of cells synthesized DNA and proteins (in the case with proteins the specially strains of bacteria producing the individual proteins should be applyed) both of microbial, plants and/or animal origin, that are available elsewhere. Cells will be adapted to high concentrations of ²H₂O by “step-by-step” adaptation method developed ealier so that to obtain the preparative quantities of biological material (biomass)©. Isotopically modified biological material from various objects will be separated on the groups of macromolecules (DNA, proteins), allowing to the macromolecules retain its native and function properties unchanged, the structural and the conformational properties of each of them will be studied together by the methods of NMR, X-ray diffraction , IR-, and CDspectroscopy.

Genetic manipulation with deuterated cells is the subject of special research. In particular I would like to get in touch with the availible methylotrophic objects adapted to growth on salt medum containing 98%(v/v) ²H₂O: (1) - M. flagellatum and (2) - B. methylicum. I plan to use them as gene expressing systems to prepare the individual proteins labelled with deuterium, which now are out of this project.

When all above listed questions will be studied and successfully cleared up, quantitative preparation of deuterated macromolecules will be considered, so that to evaluate technological parameters of large scale isotopic production. Ecological purity of such production and the utilization of wastes will be considered in frames of the complex research.

6. What should we inspect from biological substitution of macromolecules by ²H.

6.1. The role of macromolecules in biological adaptation to ²H₂O. In the case with deuterated macromolecules we should inspect the following types of adaptive mechanisms: 1. adaptation of macromolecular components of cells: It is possible to allocate mainly two kinds of such adaptation: (a). Differences of intracellular concentration of macromolecules; (b). The forming of a new macromolecules in ²H₂O with other conformational properties, which could be replacing the macromolecules of those types synthesized by cells in normal conditions. These strategies of adaptation are possible to name accordinly as quantitative and qualitative strategies; 2. adaptation of microenvironment in wich macromolecules are submerged: The essence of this mechanism is, that the adaptive changes in the structure and conformation of macromolecules are conditioned both by modifications of medium composition and by its structure (osmotic pressure, viscosity, density, рН, concentration of ²H₂O). This mechanism has extremely a large importance and supplements the macromolecular adaptation; 3. adaptation at a functional level: when the change of an overall performance of macromolecules is not connected with a change of a number of macromolecules being available or with the macromolecules of their types. Adaptation in this case could provide the changes by using the already existing macromolecules - according to requirements by this or that metabolic activity.

6.2. The biophysical properties of deuterated macromolecules. In general, when the macromolecules transfer to deuterated medium not only water due to the reaction of an exchange (Н₂О -²Н₂О) dilutes with deuterium, but also occurs a very fast isotopic (¹Н-²Н) - an exchange in hydroxylic, carboxilic, sulfurhydrilic and nitrogen groups of all organic compounds including the nucleic acids and proteins. It is known, that in these conditions only С-²Н bond is not exposed to isotopic exchange and thereof only the species with С-²H type of bonds can be synthesized de novo. It is not excluded, that the effects, observe at adaptation to ²Н₂О are connected with formation in ²Н₂О the molecules with conformations having the other structural and dynamic properties, than conformations, formed with participation of hydrogen, and consequently having other activity and biological properties. So, according to the theory of absolute speeds the break of С-²H-bonds can occur faster, than С-¹H-bonds, mobility of an ion ²H+ is less, than mobility of ¹Н+, the constant of ionization ²Н₂О is a little bit less than ionization constant of ²Н₂О. It is possible to conclude, that the sensitivity of various macromolecules to substitution on ²Н can be varried. From the point of view of physical chemistry, the most sensitive to replacement of ¹Н+ on ²H+ can appear the apparatus of macromolecular biosyntesis and respiration system, those ones, which use high mobility of protons and high speed of break of hydrogen bonds. Thus, a principal feature of the structure of such biologically important compounds as proteins and nucleic acids is the maintenance of their structure by virtue of the participation of many hydrogen bonds. One may expect that the hydrogen bonds formed by of many deuterium will be different in their energy from those formed by proton. The differences in the nuclear mass of hydrogen and deuterium may possibly cause disturbances in the DNA-synthesis, leading to permanent changes in its structure and consequently in the cells genotype. The multiplication which would occur in macromolecules of even a small difference between a proton and a deuteron bond would certainly have the effect upon its structure. The sensitivity of enzyme function and the presumed sensitivity of nucleic acids function (genetic and mitotic) to its structure would lead one to expect a noticeable effect on the metabolic pattern and reproductive behavior of the organism. And the next, the changes in dissociation constants of protein ionizable groups on transfer from water to ²H₂O may perturb the charge state of DNA and proteins. Substitution of ¹H for deuterium also affects the stability and geometry of hydrogen bonds in apparently rather complex way and may, through the changes in the hydrogen bond zero-point vibrational energies, alter the conformational dynamics of hydrogen-bonded structures within the DNA and protein. From that it is possible to assume, that the macromolecules could realize the special mechanisms, which promote the stabilizition of the structure in ²H₂O and somewhat the functional reorganization of their work in ²Н₂О.

7. Conclusion.

The successful adaptation of organisms to high (maximum) concentration of ²H₂O with further their using will open a new avenues of investigation. Fully deuterated essential macromolecules will give promise of important biological, biochemical and medical uses. Modern physical methods of study three-dimentional properties of macromolecules, particularly three dimentional NMR in a combination with powerful crystallography methods, X-ray diffraction, IR-, and CDspectroscopy should cast new light on many obscure problems concerning with the biological introduction of deuterium into molecules of DNA and proteins as well as the structure and the function of macromolecules in the presence of ²H₂O. The variety of these and other aspects of biophysical properties of macromolecules in the presence of ²H₂O remain an interesting task for the future. First, I hope that the structural and the functional studies of macromolecules can provide us to the useful information about a many aspects of the synthesis of deuterated macromolecules and their biophysical behaviour in ²H₂O. Second, the extensive body of available structural data about a cell protection system (at the level of the structure and the functioning of deuterated DNA and enzymes) will also form the basis for a particularly useful model for the study of biological adaptation in aspect of molecular evolution of macromolecules. Finally, I also believe, the research can make a favour the medicine and biotechnology, especially for creating a deuterated analogues of enzymes and DNA having something different properties then the protonated species and working in the presence of ²H₂O.

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§ Here and there under the term “high”it is meant a maximum level of enrichment.

+ To create a high isotopic content of ²H₂O in growth media, heavy water with isotopic purity 99,9 at.%, was employed. The phosphate salts were several times preliminarily crystallyezed in 99,9 at% ²H₂O and dried up in vacuum before using (the true content of deuterium in growth media measured by the method of NMR was nevertheless less smaller to 5-10% after the sterillization then isotopic purity of ²H₂O).

# I plan to use this method further to carry out the adaptation of other strains of bacteria.

Under the term of levels of enrichment it is meant the number of hydrogen atoms (bind with carbon atoms) in molecule could be substituted on deuterim (easly exchangeable on deuterium protons at amino -NH₂ groups, as well as -OH and -COOH groups in molecules were not discussed).

Ä It is meant the maximum content of ²H₂O in medium could be achieved in each experiment (see footnote under a symbol +).

©Previously adapted strains of methylotrophic bacteria, bacills and halophilic bacteria will also be used.