19th-banner-rev.gif

Book of Abstracts: Albany 2011

category image Albany 2011
Conversation 17
June 14-18 2011
©Adenine Press (2010)

Computer-Aided Pathway to Increasing the Thermostability of Small Proteins

A major task of modern bioengineering is the development of molecules with designated properties (1) such as increased stability, including their thermostability. The augmented thermostability of proteins allows to increase the speed of enzymatic catalysis, as well as the duration of their storage.

A few years ago a possible thermostabilization mechanism of small globular proteins (2) was developed in the Laboratory of Structure and Dynamics of Biomolecular Systems at the Institute of Cell Biophysics (Russian Academy of Sciences). It is based on the alternative hydrogen bonding mechanism between side chains of amino acid residues on protein surface (2). This hypothesis based on experimental data has been essentially supplemented later by modelings of dynamics of for proteins from thermophilic and mesophilic organisms (3, 4).

Our work uses this theory for improving the thermostability of human Peroxiredoxin 6. This protein (5) is a promising antioxidant for burn treatment. The spatial structure of human Peroxiredoxin 6 was reported previously (6) (Fig. 1.) and its homologs from various organisms have been well characterized, including their thermodynamic properties (7). We propose to predict what point mutaions in Peroxiredoxin 6 will increase its themostability using our knowledge about alternative hydrogen bonding, as well as the known structure of human Peroxiredoxin 6 and its known homologs.


Figure 1: Structure of human Peroxiredoxin 6 (pdb entry: 1PRX) and active site location.

Data of alignment of Peroxiredoxin 6 homologs (Fig. 2) give us information about the variable and stable parts of the amino acid sequence of this protein. The most probable sites of mutations reside only in the evolutionary variable areas of amino acid sequence because changes in stable regions can affect the functional properties of the protein. It should be noted that certain homologs of Peroxiredoxin 6 possess higher thermostability in native state in comparison to the human protein. It is important to notice that human and rat Peroxiredoxins have the highest homology (91,5 %, i.e. 19 residues) (Fig. 2.). At the same time, rat protein possesses the greatest thermostability (7) among the homologs studied. Thus, by comparing rat and human Peroxiredoxin 6 amino acid sequences, we can get additional information about the preferred locations of mutations to increase the thermostability of human Peroxiredoxin 6.


Figure 2: Alignment of Peroxiredoxins 6 from various organisms (7). (Note, that active site of all Peroxiredoxin’s 6 (CYS47) is located in stable region of amino acids sequence.)

The structures of native human Peroxiredoxin 6 protein and it homologs have been studied by molecular dynamics (MD) at various temperatures on GPU NVIDIA (8). The MD provides a powerway to follow formation and destruction of hydrogen bonds in all biological macromolecules (9-12). In the case of Peroxiredoxin 6 protein we tested the amount of hydrogen bonds on the surface of protein globules in each frame of an MD-trajectory on the pairs of studied proteins. Solvent was considered both in explicit and implicit models.

From the data of sequence alignment and our MD calculations, we predict four amino acid substitutions, which we believe will lead to increased thermostability of human Peroxiredoxin 6 protein without violating the spatial structure and functional properties. The problem of substrate specificity of Peroxiredoxin 6 will also be discussed.

References

  1. Donald Lee Wise. Encyclopedic handbook of biomaterials and bioengineering: Applications, vol.2. , New York:, Marcel Dekker, 1995.
  2. N. N. Khechinashvili, M. V. Fedorov, A. V. Kabanov, S. Monti, C. Ghio, K. Soda, J. Biomol. Struct. Dyn. 24, 255-262 (2006).
  3. A. V. Kabanov, N. N. Khechinashvili, J. Biomol. Struct. Dyn. 24, 756-756 (2007).
  4. N. N. Khechinashvili, S. A. Volchkov, A. V. Kabanov, G.Barone, Biochim. Biophys. Acta Proteins & Proteomics, 1784 (11), P.1830 92084).
  5. I. V. Peshenko, V. I. Novoselo, V. A. Evdokimov, Y. V. Nikolaev, T. M. Shuvaeva, V. M. Lipkin, E. E. Fesenko, FEBS Lett. 381, 14-19.(1996).
  6. H. J. Choi, S. W. Kang , C. H. Yang, S. G. Rhee, S. E. Ryu, Nat Struct Biol. 5, 400-406 (1998).
  7. M. G. Sharapov, V. I. Novoselov, V. K. Ravin, Mol Biol (Mosk). 43, 505-11 (2009).
  8. http://www.nvidia.com/object/cuda_home_new.html
  9. Z. Gong, Y. Zhao and Y. Xiao, J Biomol Struct Dyn 28, 431-441 (2010).
  10. J. Wiesner, Z. Kriz, K. Kuca, D. Jun and J. Koca, J Biomol Struct Dyn 28, 393-403 (2010).
  11. C. Koshy, M. Parthiban, and R. Sowdhamini. J Biomol Struct Dyn 28, 71-83 (2010).
  12. F. Mehrnejad, and M. Zarei. J Biomol Struct Dyn 27, 551-559 (2010).

Maxim Kondratyev
Artem Kabanov
Alexander Samchenko
Vladislav Komarov
Nikolay Khechinashvili

Institute of Cell Biophysics
Pushchino, Russia

ph : +7 4967 739 404
ph : +7 4967 739 484
fx: +7 4967 330 509
ma-ko@bk.ru
kabanov@mail.ru