University of Pannonia Faculty of Information Technology Department of Nanotechnology CREATING OF FLAGELLIN-BASED MOLECULAR OBJECTS Theses of PhD dissertation Anett Sebestyén Environmental Sciences PhD School Supervisor: Dr. Ferenc Vonderviszt Veszprém 2008.
SUMMARY Biosensors and protein-chips usually apply antibodies as recognition elements. However, these proteins are expensive, poorly stable and hard to prepare. It is highly desirable to replace antibodies with other kind of sensing molecules. Flagellin, the subunit protein of bacterial flagellar filaments, is a protein polymerizable to form long filaments. The helical filaments of bacterial flagella are made of several tens of thousands copies of the flagellin protein. Flagellar filaments are self-assembling systems, i.e. under suitable conditions flagellin monomers can spontaneously assemble into filaments with a structure identical to that of the native filaments. Polymerization of flagellin subunits can be controlled easily, and the obtained filaments are resistant to physical and chemical effects, resistant to proteases and their structure is well known at the atomic level. The central portion of the amino acid sequence of flagellin, which forms the outer part of the filament, is highly variable, and can be modified by genetic engineering or chemical treatment without affecting polymerization ability. I have created Ni- and As-binding flagellin mutants by molecular graphics and genetic engineering. These modified flagellins can be used capable of recognizing and binding desired heavy metal ions. Flagellin-based receptors can be produced easily and inexpensively by bacteria, and purified with an ease without lysing the cells, and flagellins, due to their polymerization ability, can be used to build filamentous structures with a very high binding site density on their surface. Moreover I have presented, that the variable D3 domain of flagellin has small size and stable structure makes it a promising protein scaffold for the development of artificial binding proteins. Further these filamentous receptors or the D3 domain alone may serve as basic recognition units of optical sensors to measure heavy metal contamination of fresh waters. 2
NEW SCIENTIFIC RESULTS / THESES 1. In Ni-binding proteins occuring in nature, the coordination of the Ni ions usually performed by imidazole groups of several (2-4) histidine side chains. By computerized graphics and molecular modeling I have established the variable D3 region of the flagellin to identify amino acids having side chains in suitable orientation and distance for constructing metal ion binding centre by replacing the relevant amino acids with histidine. The proposed Ni-binding variants were constructed by site-directed mutagenesis. Quantitative analysis of the Ni-binding ability of Leu209His-Val235His-Lys241His- Ser264His mutant was performed by isothermal titrating microcalorimetry. I have proved, that the dissociation constant of Ni-binding Kd= 5 μm and one Ni ion was bound to one flagellin subunit. 2. In the bacterial arsenic-binding protein (ArsR) can be found a polypetide segment specific binding of arsenic-ion. The amino acid sequence of As-binding motif is the next: SerGlyGluLeuCysValCysAspLeuCysThrAlaLeuAspGln. This segment has strongly streched conformation, distance of ends is close 20 Å. From size of this segment I have found it can be inserted into the Ala 262-Thr273 surface loop region of D3 domain. I have cut the Ala 262-Thr273 region from D3 domain, and I have inserted the As-binding motif by multi-steps PCR. The As-binding flagellin variant also showed an affinity for arsenite in the micromolar range. 3. Investigating the mechanism of filament formation, the hypothesis that the free N- terminal region of filament bind to the C-terminal region of the next subunit I have verified by fluorescence resonance energy transfer method. From examination of truncated flagellin s polymerization I established that the observations are consistent with the coiled-coil model of filament formation, which suggests that the α-helical N- and C- terminal regions of axially adjacent subunits form an interlocking pattern of helical bundles upon polymerization. 3
4. Filamentous structure building Ni-binding variants was immobilized on surface of a sensor chip without dextran matrix and it was examined by SPR method. I have proved, that the mutant filament have very high binding site density on their surface allowing trace level heavy metal ion analysis by optical methods. 5. I have cloned the gene segment encoding the D3 domain of flagellin. The structural stability of isolated D3 was characterized by differential scanning microcalorimetry, and proteolysis by trypsin. The experiments have revealed that isolated D3 has a stable tertiary structure which is highly resistant against proteolytic digestion. Its small size and stable structure makes D3 a promising protein scaffold for the development of artificial binding proteins. 4
PUBLICATIONS, PRESENTATIONS SEBESTYÉN, A., MUSKOTÁL, A., VÉGH B.M., & VONDERVISZT, F. The Hypervariable D3 Domain of Salmonella Flagellin Is an Autonomous Folding Unit. Protein and Peptide Letters, 15, 54-57. (2008). SEBESTYÉN, A, VÉGH, B.M., SZEKRÉNYES, Á., KURUNCZI, S., VONDERVISZT, F. Nehézfém-kötő flagellin alapú receptorok. Biokémia XXX/4. (2006). GUGOLYA, Z., MUSKOTÁL, A., SEBESTYÉN, A., DIÓSZEGHY, Z. & VONDERVISZT, F. Interaction of the disordered terminal regions of flagellin upon flagellar filament formation. FEBS Letters, 535, 66-70. (2003). MUSKOTÁL, A., KIRÁLY, R., SEBESTYÉN, A., GUGOLYA, Z.,VÉGH B.M. VONDERVISZT, F. Interaction of FliS flagellar chaperone with flagellin. FEBS Letters, Vol. 580, 3916-3920 (2006). FECZKÓ, T., MUSKOTÁL, A., GÁL, L., SZÉPVÖLGYI, J., SEBESTYÉN, A., VONDERVISZT, F. Synthesis of Ni-Zn ferrite nanoparticles in radiofrequency thermal plasma reactor and their use for purification of histidine-tagged proteins, J. Nanoparticle Research, (accepted for publication). SEBESTYÉN, A., MUSKOTÁL, A., GYIMESI, G., VONDERVISZT, F., BÁRSONY, I.: Ni- and As-binding flagellin-based receptors. E-MRS Spring Meeting, Strasbourg (France, 2007). KURUNCZI, S., NAGY, N., TÓTH, A. L., SEBESTYÉN, A., VONDERVISZT, F., BÁRSONY, I.: Immobilization of protein filaments on surfaces for optical sensing. E-MRS Spring Meeting, Strasbourg (France, 2007). PAP, A. E., KURUNCZI, S., SEBESTYÉN, A., TÓTH, A. L., VONDERVISZT, F., BÁRSONY, I.: Immobilization of protein segments into porous silicon for biosensor application. E-MRS Spring Meeting, Strasbourg (France, 2007). SEBESTYÉN, A., MUSKOTÁL, A., SZEKRÉNYES, Á., GYIMESI, G., KURUNCZI, S., VONDERVISZT, F.: Nehézfém-kötő flagellin alapú receptorok Műszaki Kémiai Napok 07 Veszprém (Hungary, 2007). 5
SEBESTYÉN, A., MUSKOTÁL, A., SZEKRÉNYES, Á., GYIMESI, G., KURUNCZI, S. ÉS VONDERVISZT, F.: Nehézfém-kötő flagellin alapú receptorok Nanobiológia Miniszimpózium, Pécs (Hungary, 2006). best poster award SEBESTYÉN, A., GUGOLYA, Z., JAKAB, G., DIÓSZEGHY, Z., ZÁVODSZKY, P. & VONDERVISZT, F.: The FliH component of the flagellar export apparatus is a multizinc enzyme with phospholipase activity. 30th FEBS Congress - 9th IUBMB Conference, Budapest (Hungary, 2005). SEBESTYÉN, A., GUGOLYA, Z., JAKAB, G., MUSKOTÁL, A., DIÓSZEGHY, Z., ZÁVODSZKY, P., VONDERVISZT, F.: A flagellumspecifikus exportrendszer FliH komponense foszfolipáz aktivitással rendelkező multi-cink fehérje. A Magyar Biofizikai Társaság XXII. Kongresszusa. Debrecen (Hungary, 2005). KURUNCZI, S., TÓTH, A. L., SEBESTYÉN, A., VONDERVISZT, F., BÁRSONY, I.: Immobilization of protein filaments on surfaces for optical sensing. Hungarian Nanotechnolgy Symposium. Budapest (Hungary, 2005). MUSKOTÁL, A., KIRÁLY, R., SEBESTYÉN A., VÉGH B., VONDERVISZT F.: A flagellinspecifikus FliS dajkafehérje jellemzése. A Magyar Biokémiai Egyesület Molekuláris Biológiai Szakosztályának 8. Munkaértekezlete. Tihany (Hungary, 2003). MUSKOTÁL, A., SEBESTYÉN, A.: A flagellinmolekula rendezetlen terminális régióinak szerepe az alegységek kölcsönhatásaiban, ELTE, OTDK Konferencia (2003). 3 rd prize MUSKOTÁL, A., SEBESTYÉN, A., GUGOLYA, Z., DIÓSZEGHY, Z., VONDERVISZT, F.: A flagellinmolekula rendezetlen terminális régióinak szerepe az alegységek kölcsönhatásaiban. A Magyar Biokémiai Egyesület Molekuláris Biológiai Szakosztályának 7. Munkaértekezlete. Keszthely, (Hungary, 2002). VONDERVISZT, F., MUSKOTÁL, A., SEBESTYÉN, A., GUGOLYA, Z., DIÓSZEGHY Z.: Interaction of the disordered terminal regions of flagellin upon filament formation. XIV. International Biophysics Congress, Buenos Aires (Argentina, 2002). MUSKOTÁL A., SEBESTYÉN A., VONDERVISZT F.: A flagellinmolekula rendezetlen terminális régióinak szerepe az alegységek kölcsönhatásaiban. A Magyar Biofizikai Társaság Molekuláris Biofizika Szekciójának szegedi szekcióülése (2002). 6
MUSKOTÁL A., SEBESTYÉN A.: A flagellinmolekula rendezetlen terminális régióinak szerepe az alegységek kölcsönhatásaiban, VIII. Országos Felsőoktatási Környezettudományi Diákkonferencia, Veszprém (2002) Ministry of Environment and Water s prize MUSKOTÁL A., SEBESTYÉN A.: A flagellinmolekula rendezetlen terminális régióinak szerepe az alegységek kölcsönhatásaiban, VE, TDK Konferencia (2001) 2 nd prize 7