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Constructing a set of instruments for operation under increased pressure

#2300


Construction of a set of instruments to study energetics of macromolecule stabilization under increased pressure and examination of the influence of ultrahigh pressure on the structural stability and kinetics of protein denaturation/renaturation

Tech Area / Field

  • INS-MEA/Measuring Instruments/Instrumentation
  • BIO-RAD/Radiobiology/Biotechnology
  • BIO-CHM/Biochemistry/Biotechnology
  • PHY-OTH/Other/Physics

Status
3 Approved without Funding

Registration date
31.08.2001

Leading Institute
Institute of Protein Research, Russia, Moscow reg., Puschino

Supporting institutes

  • Institute for High Pressure Physics, Russia, Moscow reg., Troitsk\nTRINITI, Russia, Moscow reg., Troitsk

Collaborators

  • Dow Deutschland GmbH & Co., Germany, Schwalbach

Project summary

The main goal of the project is to construct a set of equipment to study the influence of high pressure on the stability and kinetics of denaturation/renaturation of macromolecules, by employing scanning microcalorimetry and optical methods. The set will include a unique scanning microcalorimeter that operates at pressures reaching 8000atm and it has no analogs in the world. The microcalorimeter will be used to gain necessary thermodynamic information on protein stability in a wide range of external conditions. Data about structural peculiarities of macromolecules under such extreme conditions will be obtained with optical methods. At the moment, data to examine the influence of pressure on the structure and stability of macromolecules are very scarce, and there is no information at all about studies devoted to thermodynamics of the process. Nevertheless, it has become clear that such studies can prove very useful, both for clarifying fundamental problems of molecular biology and biophysics, and for applied purposes, for various requirements in biotechnology.
Another direction of our work is to investigate the affect of ultrahigh pressure in the shock wave front on biological objects. According to our estimates, this pressure can reach 200,000atm. Such an approach seems to be very attractive from the point of view of biotechnological application, because it does not require the construction of large high-pressure chambers, which is a complex task. The above experiments can prove useful for studying the effect of shock waves on the structure of proteins, on the kinetics of extraction of biological materials from cells and tissues and on deuterium exchange in macromolecules. As far as we know, such studies have not been conducted anywhere and they might be useful for the needs of biotechnology.
Expected Results and their Application
The main result of the project would be the construction of equipment and development of the technological basis for wide-scale studies of the influence of ultrahigh pressure on the stability and kinetics of macromolecule denaturation/renaturation.
Firstly, the knowledge of the influence of pressure on denaturation enables evaluation of denaturation and activation volumes for different objects and would allow one to obtain certain statistics, revealing the correlation between the protein structure in the native state and the change in its volume upon denaturation. In its turn, this can be the basis for elaborating a semi-empirical theory, relating structural parameters of macromolecules to denaturation and/or activation volumes.
Secondly, the possible use of ultrahigh pressure in biotechnology is very wide. In particular, high pressure can be used in industrial production of enzymes (industrial drugs and enzymes) and in the food industry. High pressure strongly suppresses aggregation, which gives hope for a higher yield of active products during the renaturation of enzymes. On the other hand, protein denaturation is a key moment in the greatest part of processes in food technology. Denaturation of food proteins within the feedstock results in the formation of a product structure that determines consumer appeal of the product. At the same time, denaturation of proteins of pathogenic microorganisms present in the feedstock provides conditions for sterilization and, consequently, harmlessness and long storage of the food.
Another especially important aspect that can be solved using high-pressure equipment is the clarification of principles of self-organization and structural maintenance of macromolecules from living organisms inhabiting large depths under great pressure (up to 1000atm).
Applied Significance
Taking into account the above, it can be concluded that a detailed and systematic study of thermodynamic stability of proteins under high pressure is required to widen the use of high pressure in scientific research, biotechnology and food technology. To solve the problem, a set of equipment has to be designed that would permit the study of the influence of high pressure on the stability and kinetics of denaturation/renaturation of macromolecules. It is proposed that such a set will include a universal high-pressure controller, a scanning microcalorimeter operating at this pressure and a high-strength optical cell for fluorometry, CD and photometric studies compatible with standard commercial optical instruments. Construction of such a set at the Institute of Protein Research (Russian Academy of Sciences), which will be accessible for a wide community of researchers, can essentially widen the range of solvable problems and will promote the use of this approach. On the other hand, intensive investigations of the influence of high-pressure on the stability of macromolecules and intermolecular interactions would give rise to the need for corresponding commercial equipment. Later, a laboratory model developed upon realization of the project could become a prototype for a commercial laboratory instrument. The proposed development could prove patentable, and the materials could be patented. The constructed equipment could be assigned to a commercial firm for serial production. The set of high-pressure instruments could be used for analysis of solutions at research laboratories, institutes and universities.
Project Realization
The tasks will be solved over the course of 1.5 years by the joint efforts of three research groups from the Institute of Protein Research RAS, the Institute of High Pressure Physics RAS, and the State Research Center of the Russian Federation "Troitsk Institute for Innovation and Fusion Research". The project participants have the required expertise and experience.
Notwithstanding the fact that the majority of participant researchers on the project were involved in weapons development, the project is designed to solve purely peaceful scientific problems. The main goal of the project is development of equipment to contribute to the solution of fundamental and applied problems of biopolymer physics. Russian scientists will work in direct contact with scientists from the collaborator group, and the obtained results will be published in international editions. All this will promote the integration of researchers involved in weapons development into the international scientific community.


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