FP6 priority

1.1.1   Genomics and Biotechnology for Health

1.1.1.1

Title of the proposal

Engineering of biologically important proteins

Institute

Slovak Academy of Sciences, Institute of Molecular Biology
Dubravska cesta 21, 84251 Bratislava, Slovak Republic

Contact

Research subject for a potential FP6 project

Our work is oriented to structural genomics, mainly to crystallization and structure determination of proteins using diffraction methods. We use the structures for study of mechanism of enzyme catalysis and specificity, conformational stability of globular proteins, protein - nucleic acid and protein - protein recognition, molecular modelling, preparation of genetically modified proteins and structure - function relationship. Remarkable results were achieved also in the field of structure determination at atomic resolution. The main objects of the study were microbial ribonucleases, their inhibitors, and amylolytic enzymes. One of our ribonucleases became the object of an intense study due to its high cytotoxic activity against human leukemia cells, comparable to that of onconase (frog ribonuclease). Up to now more than 20 structures have been determined and refined against high resolution data, namely structures of ribonucleases isolated from three strains of Streptomyces aureofaciens, their mutants and complexes with nucleotides and with ribonuclease inhibitor from Bacillus amyloliquefaciens, as well as the structure of glucoamylase isolated from Saccharomycopsis fibuligera and its complex with acarbose. 17 structures have been deposited with Brookhaven Protein Data Bank.Recently the studies were extended to a cytoskeletal protein plectin which plays an important role in cytoskeleton organization and regulation. Owing to large molecular weight of plectin (over 500 kDa) and flexibility of its molecule, individual subdomains are being cloned, expressed and crystallized. The structure of actin binding domain of plectin was determined. The recognition of our work by foreign institutions resulted in an offer to participate with our proteins in the NASA project oriented to crystallization of proteins under microgravity environment in American Space Shuttle Columbia and Atlantis and in an award of grants from National Institute of Health, Copernicus and Howard Hughes Medical Institute.We are ready to collaborate on crystallization and structure determination of any protein.

Recent international cooperation of the research team

European Molecular Biology Laboratory, Hamburg, Germany; Structural Biology Laboratory, Chemistry Department, University of York, York, Great Britain; Vienna Biocenter, University of Vienna, Vienna, Austria; Texas A&M University, College Station, Texas, USA

Proposer´s relevant publications related to the research subject

Sevcik J., Urbanikova L., Dauter Z., Wilson K.S. (1998) Recognition of RNase Sa by the inhibitor barstar: Crystal structure of the complex at 1.7 Å resolution. Acta Cryst D. 54, 954-963
Sevcik J., Solovicova A., Hostinova E., Gasperik J., Wilson K.S., Dauter Z. (1998) Structure of glucoamylase from Saccharomycopsis fibuligera at 1.7 Å resolution. Acta Cryst. D. 54, 854-866
Dvorsky R., Sevcik J., Caves L.S.D., Hubbard R., Verma C.S. (2000) Temperature effects on protein dynamics: a molecular dynamics study of RNase Sa. J.Phys.Chem. B, 104, 10387-10397
Pace C.N., Horn G., Hebert E.J., Bechert J., Shaw K., Urbanikova L., Scholtz J.M., Sevcik J. (2001) Tyrosine Hydrogen Bonds Make a Large Contribution to Protein Stability. J.Mol.Biol. 312, 393-404
Hlinkova V., Urbanikova. L., Krajcikova D., Sevcik J. (2001) Purification, crystallization and preliminary X-ray analysis of two crystal forms of ribonuclease Sa3. Acta Cryst. D57, 737-7739
Urbanikova L., Janda L., Popov A., Wiche G., Sevcik J. (2002) Purification, crystallization and preliminary X-ray analysis of the plectin actin-binding domain. Acta Cryst. D58, accepted