Báo cáo khoa học: F1 – Structure and Function of Motor Proteins pot

F1 – Structure and Function of Motor Proteins F1-001 What single-molecule mechanics can tell us about mitosis? J. Howard Max Planck Institute of Molecular Cell Biology & Genetics, Dresden, Germany. E-mail: howard@mpi-cbg.de Our laboratory is interested in the biochemical and biophysical basis of cell structure. The structure of a cell is determined primarily by its cytoskeleton, which serves as a scaffold to sup- port the plasma membrane, and as a network of tracks along which motor proteins transport sub cellular structures. Our research is therefore focused on the mechanics of the cytoskele- ton, with a particular emphasis on microtubules and microtu- bule-based motors. On one hand, we are interested in the mechanisms by which these proteins work: i.e. how do kinesins and dyneins convert chemical energy derived from the hydrolysis of ATP into mechanical work used to move along or to Abstracts 332 depolymerize microtubules? And on the other hand, we are inter- ested in the roles that microtubules and their motors play in cell morphology and motility. In this regard we are particularly inter- ested in how the dynamic properties of microtubules and motors drive spindle and chromosome movements in mitosis. To address these questions we are combining molecular biology techniques with image processing, modeling, mechanical measurements and single-molecule techniques. F1-002 Control of actin assembly in cell motility M F. Carlier CNRS, Gif-sur-Yvette, France. E-mail: carlier@lebs.cnrs-gif.fr Living cells change shape and move in response to environmen- tal signals. These motile processes play a pivotal role in mor- phogenesis, migration of embryonic and metastatic cells, angiogenesis, synaptic plasticity, immune response and interac- tion of the host cells with pathogens. They are generated by polarized, spatially directed actin assembly. It is the treadmilling (dissipative turnover) of actin filaments, regulated by specific proteins, which is responsible for force and directional move- ment. Two cellular machineries are responsible for spatially directed initiation of actin filaments and they operate in distinct processes, (i) the WASP-Arp2/3 system is at the origin of the formation of a branched filament array; (ii) formins, in associ- ation with profilin, catalyze the rapid processive assembly of non-branched actin filaments. We have combined a biochemical and a biomimetic approach to understand the molecular mecha- nisms of these auto-organized processes. We have reconstituted the sustained actin-based movement of a N-WASP- or formin- functionalized particle in a biochemically controlled medium, which enables measurement of force production in correlation with structure and motility, and we can derive information on the molecular mechanism of movement by single molecule measurements. F1-003 The structure of the myosin VI motor reveals the mechanism of directionality reversal J. Me ´ ne ´ trey 1 , A. Bahloul 1 , C. Yengo 2 , A. Wells 2 , C. Morris 2 , H. L. Sweeney 2 and A. Houdusse 1 1 UMR144 - CNRS, Institut Curie, Paris, France, 2 Departement of Physiology, Pennsylvania School of Medicine, Philadelphia, PA 19104–6085 United States of America. E-mail: anne.houdusse@curie.fr We have solved a 2.4 A ˚ structure of a truncated version of the reverse direction myosin motor, myosin VI that contains the motor domain and binding sites for two calmodulins. Surpris- ingly, the structure reveals only minor differences in the motor domain as compared to plus-end directed myosins, with the exception of two unique inserts. The first insert is near the nuc- leotide-binding pocket, and alters the rates of nucleotide associ- ation and dissociation. The second unique insert forms an integral part of the myosin VI converter domain along with a calmodulin bound to a previously unseen binding motif within the insert. This serves to redirect the effective ‘‘lever arm’’ of myosin VI, which includes a second myosin VI calmodulin bound to an ‘‘IQ motif’’, towards the pointed (–) end of the actin fil- ament. This repositioning largely accounts for the reverse direc- tionality of this class of myosin motors. We propose a model incorporating a kinesin-like uncoupling/docking mechanism to fully explain the movements of myosin VI. F1-004 Regulation and mechanics of myosin V J. R. Sellers Laboratory of Molecular Physiology, National Heart, Lung and Blood Institute, National Institute of Health, Bethesda, MD United States of America. E-mail: sellersj@nhlbi.nih.gov Myosin V is vesicle motor that moves processively on actin filaments. In melanocytes it functions in cooperation with microtubule motors to localize melanosomes to the dendritic tips. Myosin V dimerizes via a coiled coil motif in its tail region to produce a two-headed structure. Each head is composed of a compact motor domain and a long neck formed by the association of calmodulin residues with six tandem IQ motifs. This structure allows the molecule to take 36 nm steps along an actin filament corresponding to the helical pitch of the actin and, in turn, allows the molecule to keep its cargo positioned above the cytoskeleton. We study the mechanics of the processive motor using optical trapping and total internal reflection fluores- cence microscopy (TIRF). These data confirm that the neck of myo- sin V acts as a rigid lever arm and that myosin V moves in a hand- over-hand manner. The enzymatic activity of myosin V is regulated by calcium in vitro. This is coupled to a large conformational change in the molecule. In the absence of calcium myosin V folds into a compact triangular-shaped structure in which the heads bend down and contact the globular tail domain. Under these conditions the molecule migrates with a sedimentation coefficient of 14S. In the presence of calcium or at high ionic strength the molecule opens up to form T- or Y-shaped structures which sediment at 11S. We pro- pose that in the cell, the transition between the open, active state and the folded, inactive state is regulated by binding of receptor or dock- ing proteins to the myosin V globular tail domain. This would not only target the myosin to the proper cargo, but would also lock the myosin into an active form. We are currently studying the interac- tions responsible for the folded, off state of this molecule. F1-005 F-actin modulates myosin conformational states by switch I loop movement B. Kintses 1 , M. Gyimesi 1 , W. Zeng 2 , P. B. Conibear 2 , C. R. Bagshaw 2 and A. Ma ´ lna ´ si-Csizmadia 1 1 Departement of Biochemistry, Eo ¨ tvo ¨ s Lora ´ nd University, Buda- pest, Hungary, 2 Department of Biochemistry, University of Leices- ter, Leicester, United Kingdom. E-mail: amc26@leicester.le.uk Myosin converts chemical energy to mechanical work. It undergoes a large conformational change during the ATPase cycle that results a step on the actin filament. The main question is how the func- tional regions communicate with each other. Another problem concerns the development and relaxation of the mechanical strains during the catalytic cycle and their perturbation by external force. We have extensively characterized the energetic coupling between nucleotide binding, the so-called open/closed transition and actin binding using different single tryptophans located at the relay loop (W501, Dictyostelium sequence), the nucleotide binding region (W129) and the switch 1 region (W239 and W241). The fluores- cence from native actin tryptophan residues is not significantly per- turbed on binding to myosin, although a fluorescence signal is detected as a consequence of a light scatter artifact. ATP, ATPa ˜ S and ADP binding affected less than threefold by actin. The isome- rization detected by W129 clearly precedes the dissociation of actin in the case of ADP and ATPa ˜ S binding. The fluorescence from the conserved W501 residue located at the distal end of the relay helix is very sensitive to the lever arm disposition and the observed fluor- escence emission intensity can be used to estimate the equilibrium constant between the pre and post power-stroke conformations. Actin modulates this equilibrium by no more than twofold. These data suggest that actin activates another process in the mechanism, Abstracts 333 such as switch 1 movement, rather than influencing the switch 2 equilibrium. Consequently, despite actin does not have major effect on nucleotide binding and the equilibriums of the pre- and post- power-stroke the fluxes of the kinetic routes change fundamentally by actin binding: in the absence of actin phosphate release precedes the closed-open transition while in ternary complex the main route is that products releases follow the power stroke. F1-006 Calmodulin and calmodulin-like protein as light chains for myosin-10: Specificity and role in protein stabilization leading to prolonged function E. E. Strehler, A. J. Caride, A. S. Mauer and R. D. Bennett Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, United States of America. E-mail: strehler.emanuel@mayo.edu Vertebrate myosin-10 (MYO10) is involved in filopodial motility, phagocytosis, and microtubule-F-actin interactions. Each MYO10 heavy chain contains three IQ motifs, which are light chain binding sites. Previous work has shown that the first two IQ domains of MYO10 bind calmodulin (CaM) whereas IQ3 alternatively binds calmodulin-like protein (CLP). We performed stopped-flow experi- ments with fluorescent derivatives of CaM (TA-CaM) and CLP (TA-CLP) to analyze their binding to the IQ3 peptide. TA-CaM bound to IQ3 in the absence and in the presence of 100 lm Ca 2+ . In the absence of Ca 2+ the binding was faster, albeit with lower affinity, suggesting an even faster dissociation rate. In the presence of Ca 2+ , the time course of the TA-CaM-IQ3 reaction was best described by an exponential function. CLP was able to reduce the amplitude of this exponential in the presence, but not in the absence, of Ca 2+ . These results suggest a novel IQ domain interac- tion, in which Ca 2+ regulates binding of CaM to IQ3 by modula- ting competition with CLP. In HeLa cells over-expressing CLP, endogenous MYO10 was strongly upregulated. Likewise, transfec- tion with GFP-MYO10 resulted in increased fluorescence in cells that co-expressed CLP or excess CaM. MYO10 upregulation resul- ted in an increase in the size and number of filopodia. Cells expres- sing CLP displayed increased motility as indicated by their shortened wound-healing time compared to control cells lacking CLP. CLP-dependent upregulation of MYO10 was due to increased protein stability. CLP expression in specific epithelial cells may thus prolong MYO10 function in conditions of elevated intracellular Ca 2+ and limiting CaM. Acknowledgment: Suppor- ted by grants from the Susan G. Komen Breast Cancer Foundation (EES) and the American Heart Association (AJC). F1-007P Crystal structure of the portal protein from bacteriophage SPP1 and model for DNA translocation A. A. Lebedev 1 , M. H. Krause 2 , A. Vagin 1 , E. V. Orlova 3 , E. J. Dodson 1 , P. Tavares 4 and A. A. Antson 1 1 YSBL, Chemistry Department, York University, York, United Kingdom, 2 Max-Planck Institut fu ¨ r Molekulare Genetik, Berlin, Germany, 3 Birkbeck College, Department of Crystallography, Uni- versity of London, London, United Kingdom, 4 Unite ´ de Virologie Mole ´ culaire et Structurale, Gif-sur-Yvette, France. E-mail: fred@ysbl.york.ac.uk The mechanism of DNA translocation into a viral procapsid remains one of the most intriguing questions of viral particle assembly [1]. Tailed bacteriophages and herpes viruses have a specialized vertex for double-stranded DNA (dsDNA) entry into the procapsid during viral chromosome packaging [2, 3]. The main component of this specific doorway is the portal pro- tein, a circular oligomer with a central tunnel through which the DNA transfer occurs. Together with viral ATPase (termi- nase) the portal protein forms a molecular motor that is able to translocate DNA against high internal pressure [4]. We determined the X-ray structure of the SPP1 portal protein (gp6) in its 13-subunit oligomeric form where the tunnel residue segments, not visible in the previously determined structures of bacteriophage phi-29 portal protein [5, 6], are well defined and form a DNA-transfer arm. Our X-ray and electron microscopy data suggest that DNA translocation is driven by a novel mechanism involving mechanical movements of arms along the inner walls of the tunnel. We propose a model for DNA trans- location where such movements propagate around the double helix of DNA similar to a ‘‘Mexican wave’’ moving across a stadium. References 1. Hendrix RW. Proc Natl Acad Sci USA 1978; 75: 4779–47783. 2. Bazinet C, King, J. Annu Rev Microbiol 1985; 39: 109–129. 3. Droge A, Tavares P. J Mol Biol 2000; 296: 103–115. 4. Smith DE, et al. Nature 2001; 413: 748–752. 5. Simpson AA, et al. Nature 2000; 408: 745–750. 6. Guasch A, et al. J Mol Biol 2002; 315: 663–676. F1-008P Unraveling the catalytic mechanism of the bacteriophage T7 gene 4 helicase D. J. Crampton, S. Mukherjee, A. van Oijen and C. C. Richardson Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA. E-mail: dcrampton@hms.harvard.edu The gene 4 helicase of bacteriophage T7 is a molecular motor that unwinds double-stranded DNA using the energy derived from the hydrolysis of deoxynucleoside 5-triphosphates. Here we present data pertaining to four essential activities of the heli- case that act cooperatively to unwind DNA. (i) Oligomer For- mation. The T7 gene 4 helicase forms both heptamers and hexamers dependent upon the presence of nucleoside di- or tri- phosphates respectively. We find that the heptamer cannot bind DNA but rather, in the presence of single-stranded DNA, hep- tamer converts to hexamer when both nucleoside di- and tri- phosphates are present together. This conversion between oligomers is regulated by histidine 465 through differentiation of the absence or presence of a c-phosphate on the bound nuc- leotide. (ii) DNA Binding. The loop comprised of residues 466– 475 has been postulated to be the major site for the binding of single-stranded DNA. Lysines 471 and 473 of this loop were altered to determine their contribution to the overall binding of single-stranded DNA. (iii) dTTP Hydrolysis. We find that all subunits of the hexamer are active in the hydrolysis of dTTP. Changing the catalytic base glutamate 343 to glutamine creates a non-catalytic subunit that responds to dTTP in a manner sim- ilar to the effect of non-hydrolyzable analog b,cc-methylene dTTP on wild-type helicase. The presence of a single non-cata- lytic subunit per hexameric unit abolishes all dTTPase activity. (iv) Translocation. We are developing methods of observing movement along DNA by the gene 4 helicase using single-mole- cule fluorescence microscopy. Abstracts 334 F1-009P Myosin VI in chromaffin cells M. Dominik 1 , L. Kiljanek 2 , and M. J. Redowicz 1 1 Laboratory of Cell Motility, Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland, 2 Laboratory of Confocal Microscopy, Nencki Institute of Experi- mental Biology, Warsaw, Poland. E-mail: mdominik@nencki.gov.pl Myosin VI (MVI), the member of one of at least 18 different myosin families, is ubiquitously expressed in multicellular eukary- otes. It is an unusual actin-based motor since it walks towards the minus end of actin filaments, in the opposite direction to other myosins. MVI is involved in subcellular transport of mem- brane-containing structures, it has been found to be engaged in clathrin-based endocytosis and cell spreading and migration. Here, we aimed at testing the involvement of MVI in chromaffin granule trafficking within chromaffin cells. MVI, but not myosin V, has been found in vesicular fractions isolated from bovine adrenal medulla and rat pheochromocytoma cells (PC12). MVI is on the apical side of the vesicle and its association with the gran- ule seems to be very tight as the stripping with high salt concen- tration or high pH does not remove it from the granule surface; MVI is stripped from the granules only after addition of Triton X100. MVI colocalizes with dopamine-b-hydroxylase (DbH), chromaffin granules marker, both in PC12 cells and primary cell culture of adrenal medulla. It has been observed that MVI and DbH were still found on the same granules after their isolation from PC12 cells, confirming strong association of MVI with the granules. Five minute stimulation of PC12 cells with 56 mm KCl seems to affect MVI localization as the fluorescence intensity cor- responding to MVI was enhanced within the perinuclear area only in stimulated cells. These preliminary data seem to indicate the important role of MVI in secretory cells. F1-010P Enzyme kinetics above denaturation temperature M. Gyimesi, Z. Simon and A. Ma ´ lna ´ si-Csizmadia Department of Biochemistry, Eo ¨ tvo ¨ s Lora ´ nd University, Budapest, Hungary. E-mail: gyimesimate@cerberus.elte.hu Wide range of temperature dependence of enzyme kinetics meas- urements enables more precise determination of reaction energetics and accurate separation of reaction steps. We have developed a novel temperature-jump/stopped flow method to measure enzyme transient kinetics at high temperature even above denaturation temperature of enzymes. This method enables us to increase the temperature in a millisecond time scale parallel with mixing of the reactants. Heat denaturation normally occurs in a second time scale. If the temperature is increased above the enzyme denatura- tion temperature, essentially all of the events can be followed that are faster than heat denaturation reaction. We have tested the new method on myosin ATPase and GFP folding/unfolding reactions. F1-011P Changes of inter-subunit contacts in growing filaments during salt-induced polymerization of actin A. Galinska-Rakoczy, B. Wawro and H. Strzelecka-Golaszewska Laboratory of Structural Muscle Proteins, Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland. E-mail: galinska@nencki.gov.pl Polymerization/depolymerization of actin underlies numerous motile events in eukaryotic cells. It is well established that conformational changes in G-actin generated by exchange of its tightly bound Ca for Mg accelerate the nucleation reaction but do not influence substantially the rate of filament growth during salt-induced polymerization of actin. We used N, N’’-1,4-phenyl- enebismaleimide (PBM) to probe the conformation of F-actin at various stages of filament growth during polymerization of CaATP-, MgATP-, and MgADP-G-actin. Effects of the type of polymerizing salt, filament stabilization by phalloidin, and pro- teolytic cleavage of actin at Gly-42 within D-loop were also investigated. The earlier described accumulation of ’upper dimer ’ (UD), a product of cross-linking neighboring protomers from two long-pitch F-actin strands, concomitant with disappearance of initially formed ‘‘lower dimmers’’ (LD) with subunits in a non-filamentous, antiparallel orientation, was observed under all conditions except for a polymer of cleaved actin that required phalloidin binding to yield UD. The increase in yield of UD clo- sely followed filament assembly from MgATP-G-actin, whereas it lagged behind polymerization of CaATP- and MgADP-G-actin. Phalloidin accelerated the filament assembly but did not eliminate the delay in UD formation. These results, along with changes in morphology of growing filaments visualized by electron micros- copy, suggest that the tightly bound Mg and initially bound ATP not only accelerate the nucleation reaction but also promote a rearrangement of F-actin structure that eliminates LD’s from growing filaments. They also confirm the suggested role of D-loop in stabilization of both the longitudinal subdomain 2/1 contacts and of the cross-strand contacts in F-actin. F1-012P The dynein light chain binds to a non-coiled- coil tail domain of myosin-Va that includes an alternatively spliced exon coding for three amino acid residues Z. Ho ´ di 1 ,A.Ne ´ meth 1 , E. Kova ´ cs 1 , C. Hete ´ nyi 1 , A. Bodor 2 , A. Perczel 2 and L. Nyitray 1 1 Deptartment of Biochemistry, Eo ¨ tvo ¨ s Lora ´ nd University, Buda- pest, Hungary, 2 Department of Organic Chemistry, Eo ¨ tvo ¨ s Lora ´ nd University, Budapest, Hungary. E-mail: nyitray@cerberus.elte.hu Class V myosins are involved in short-range intracellular trans- port along actin filaments. Of the three mammalian myosin-V heavy chain genes, mutations of MYO5A are responsible for the dilute phenotype and Griscelli syndrome type1 in mice and humans respectively. A dynein light chain (DLC) has been identi- fied as a tail domain light chain of myosin-Va (myo5a). DLC may function as a cargo-binding and/or regulatory subunit of both motor proteins. Our goal was to identify and characterize the binding site of DLC on myo5a. Various fragments of myo5a tail and DLC were expressed in E. coli and human cells. Forma- tion of their complex was analyzed by pull-down assays, gel fil- tration, and spectroscopic methods. DLC was found to bind as a homodimer to a 12 residues segment (Pro1282-Thr1293) locali- zed between the medial and distal coiled-coil predicted domains of the tail. The binding region contains 3 residues, coded by the alternatively spliced exon B that is essential for DLC binding. Using CD spectroscopy, we demonstrate that binding of DLC to the intrinsically unstructured DLC binding domain (DBD) stabil- izes the neighboring coiled-coil domain. NMR spectroscopy and molecular docking simulations show that a short synthetic pep- tide of DBD binds to a surface groove on DLC, as has been found with other known binding partners of DLC. We hypothes- ize that the second binding site of the DLC dimer is either occu- pied by the DBD of the other heavy chain, or by a cargo, or interacts with other regulatory proteins. Acknowledgment: Supported by OTKA T43746 Abstracts 335 F1-013P Mutagenic analysis of the HsdR motor subunit of type IC restriction modification enzyme EcoR124I E. Sisakova and M. Weiserova Laboratory of Molecular Genetics of Bacteria, Division of Cell and Molecular Microbiology, Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic. E-mail: sisak@biomed.cas.cz Enzyme EcoR124I belongs to the IC family of restriction modifi- cation enzymes, intelligent molecular motors. It is able to detect the methylation status of its DNA target sequence and respond with alternative activities, methylation or translocation of DNA. While bound to its target site, it translocates DNA towards itself simultaneously in both directions (500bp/sec). It uses the free energy associated with ATP hydrolysis to translocate DNA so that DNA cleavage occurs remote from the asymmetric recogni- tion site. The enzyme EcoR124I is multifunctional, multi-subunit enzyme, composed of three different subunits, which are encoded by the genes hsdR, hsdM and hsdS. Products of all three genes are required for DNA cleavage, producing the endonuclease. HsdR subunit is a multifunctional motor protein, which has been shown to posses ATPase, helicase and restriction activity. To provide a fully functional molecular motor, which can never cleave DNA, the amino acid motif X, the active site of the endo- nuclease domain of the HsdR subunit, was subjected to site- directed mutagenesis. The complementation analysis proved that the substitutions D151A, E165A, E165D, E165H, K167A in the HsdR subunit fully removed the restriction activity in vivo of the EcoR124I enzyme. The mutant subunits were separately overpro- duced, purified and mixed with purified methylase to reconstitute the EcoR124I endonuclease in vitro. As a substrate for DNA cleavage in vitro we used the plasmid pCFD30 containing a sin- gle site for EcoR124I. The test of restriction activity showed that reconstituted endonucleases were not able to cleave covalently closed plasmid DNA to linear DNA in contrast to the wild-type enzyme. F1-014P Dielectric and fluoroscopic study on the dynamic effects of myosin-S1 with/without ATP on the hyper-mobile water around actin filaments M. Suzuki 1 , M. P. Siddique 1 , T. Miyazaki 1 , J. Mogami 1 , E. Katayama 2 , T. Kodama 3 and T. Q. P. Uyeda 4 1 Laboratory of Physicochemistry of Biomolecular Systems, Depart- ment of Materials Science and Engineering, Tohoku University, Sendai, Japan, 2 Division of Fine Morphology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan, 3 Molecular Enzymology, Department of Bioscience and Bioinfor- matics, Kyushu Institute of Technology, Ihzuka, Japan, 4 Gene Function Research Center, AIST, Tsukuba, Japan. E-mail: msuzuki@material.tohoku.ac.jp Using high resolution microwave dielectric spectroscopy, we have recently shown hyper-mobile water molecules around actin fila- ments (F-actin) which have a much higher rotational mobility than that of bulk water [1] and its marked volume increase with- out significant change in the ordinary hydration shell when the myosin motor-domain (S1) binds to F-actin [2]. Here, we report that (i) hyper-mobile water around actin filaments has been detected with the fluoroscopic technique based on the fact that the fluorescence intensity of fluorophore molecules, such as ribo- flavin and pyrene-derivatives in caotropic salt aqueous solutions was in clear correlation with those viscosity B-coefficients, (ii) the fluorescence intensity from pyrenyl actin markedly increased when myosin-S1 binds to actin filaments, which is consistent with the dielectric result (BBRC, 2004), (iii) in the solution of acto-S1 fused chimera protein and skeletal actin copolymers the fluores- cent intensity from pyrenyl-group (linked at Cys374 of actin SD-I) increased in the presence of ATP indicating a marked decrease of hyper-mobile water around actin filaments. An important implication of the present result is that the S1/F-actin interaction forms an asymmetric field of viscosity-gradient along the filament axis, which drives the unidirectional sliding of S1 hydrolyzing ATP by means of thermal brownian movements. References 1. Kabir SR. et al. Biophys J 2003; 85: 3154–3161. 2. Suzuki M. et al. Biochem Biophys Res Comm 2004; 322: 340– 346. F1-015P Drosophila myosin V: solution kinetics and motile properties J. To ´ th 1,2 , M. Kova ´ cs 1 , F. Wang 1 , L. Nyitray 2 and J. R. Sellers 1 1 Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, MD, United States of America, 2 Bioke ´ miai Tansze ´ k, ELTE, Budapest, Hungary. E-mail: tothj@mail.nih.gov Myosin V is the best characterized vesicle transporter myosin motor in vertebrates but it is unknown whether all members of the myosin V family share a common, evolutionarily conserved, mechanism. To address this question, we performed steady state and transient kinetic measurements on a recombinant Drosophila myosin V S1 fragment. Interestingly, none of the product release steps can be identified as a single rate-limiting step. The ADP release rate constant from acto-S1, which is the bottleneck in the vertebrate myosin V ATPase cycle, appears to be 5-fold higher than the maximal steady-state ATPase activity. The Pi release rate constant from the acto-S1-products complex is even faster, implying that Drosophila myosin V does not follow a myosin II-type kinetic pattern, either. The key features of the ATPase cycle are the low ATP hydrolysis equilibrium constant (0.33) and the high steady-state actin attachment (96% at 5 m actin). The behavior of double-headed Drosophila myosin V in an in vitro motility assay suggests that a single molecule of myosin V cannot take multiple steps on the actin filament while being attached to it. We propose a mechanism whereby the ATP hydrolysis and the so-called weak actin-binding equilibria define the steady-state ATPase rate rather than the product release process. This mech- anism suggests that Drosophila myosin V will not act as a single molecule vesicle transporter, in contrast to vertebrate myosin Vs. However, it may be able to perform processive transport if pre- sent in small ensembles on the surface of its cargo. F1-016P A novel centrosomal protein, Cep57 stabilizes microtubules in vivo and binds FGF-2 in vitro. Y. Yoshitake, N. Tsuruoka and A. Iwai Department of Biochemistry, Kanazawa Medical University, Uchi- nada, Ishikawa Japan. E-mail: yositake@kanazawa-med.ac.jp We have identified a gene encoding a novel protein that binds microtubules (MT) and fibroblast growth factor-2 (FGF-2). It was found as FGF-2 associated protein in the course of studying the function of the 24kDa isoform of FGF-2 in nuclei by yeast two-hybrid screening. This gene shares a sequence similarity with an uncharacterized cDNA reported in GenBank as KIAA0092 and the encoded protein is identical to Cep57, a coiled-coil pro- Abstracts 336 tein in the centrosomes. The occurrence of Cep57 in human cen- trosomes has been recently reported but its function is unknown. GFP-fused protein over-expressed in HeLa cells was predomin- antly observed as filamentous structures and co-localized with MT. Weekly-expressed tagged proteins were co-localized with MT as spindle, spindle pole and MT in the midbody of the mito- tic cells and also localized in the centrosomes of interphase cells. Treatments disturbing the MT network did not have any effect on this protein distribution in the cells. Over-expressed Cep 57 protected the MT structure against those treatments. Therefore, Cep 57 stabilizes MT. Recombinant Cep57 protein bound both isoforms of FGF-2 (24kDa and 18kDa) in vitro. However, the localization patterns of endogenous Cep57 observed by immuno- fluorescent staining using the antibodies differed in both patterns of FGF-2 isoforms. siRNA to Cep57 induced a morphological change (spindle-like shape with a poor cytoskeletal MT network) and decreased cell growth in HeLa cells. Cep57 may work as a MT stabilizing protein in the centrosomes and spindle pole, and then play a role on MT network formation and cell division. F1-017P A novel dynamic model for actin polymerization based on the idea of self-catalysis J. Zhang 1,2 and Y. L. Wang 2 1 Department Biology, Chongqing University of Medical Sciences, Chongqing City, PR China, 2 Key Lab of Biomechanics & Tissue Engineering, Institute of Bioengineering, Chongqing University, Chongqing City, PR China. E-mail: zhangjun1017@sohu.com More and more researches have recently demonstrated that there exist multiple pathways in actin polymerization. In order to eluci- date the macroscopic dynamics of actin assembly, a novel phe- nomenological theory for actin polymerizing under simple thermodynamic conditions has been put forward. The theoretical model, called self-catalytic dynamics, emphasizes that the process of actin polymerizing can be thought of an autocatalytic chain reactions. To set up the quantified kinetic equations group, three basic postulations is employed, which are in good agreement with the fundamental physical rules and published literatures: (i) the actin monomers driven by Brownian motion in solution can interact with each other, and further form actin dimer via non- covalent association under certain molecular colliding conditions; (ii) the formed actin dimer and higher polymer spontaneously own catalyzing ability and can catalyze free actin monomer or dimer changing into actin residues (that refer to the G-actin monomers existing in actin filaments); (iii) the polymerization/ depolymerization processes can go on synchronously at the both ends (generally, the barbed and pointed ends, sometimes, also at the side of the elongating filaments) with different polymeriza- tion/depolymerization rates. According to the analyses and basic enzyme catalysis principles, a scheme of kinetic differential equa- tions can be established with definite initial conditions. By strug- gling out the integral curves of the equations group, we get a series of dynamic functions, via which we can accurately calculate the length distribution of filaments, the concentration of free G-actin and polymerized filaments versus the polymerizing time, and also quantificationally explain the treadmilling phenomenon of actin turnover. F2–Single Molecule Biochemistry and Mechanics F2-001 Recent Advances in Single Molecule Biophysics C. Bustamante Department of Physics, University of California, Berkeley, CA, USA. E-mail: carlos@alice.berkeley.edu I will present our recent results on the packaging of DNA by the connector motor at the base of the head of bacteriophage Ø29. As part of their infection cycle, many viruses must package their newly replicated genomes inside a protein capsid to insure its proper transport and delivery to other host cells. Bacteriophage Ø29 packages its 6.6 mm long double-stranded DNA into a 42 nm dia · 54 nm high capsid via a portal complex that hydro- lyses ATP. This process is remarkable because entropic, electro- static, and bending energies of the DNA must be overcome to package the DNA to near-crystalline density. We have used opti- cal tweezers to pull on single DNA molecules as they are pack- aged, thus demonstrating that the portal complex is a force generating motor. We find that this motor can work against loads of up to 57 picoNewtons on average, making it one of the strongest molecular motors ever reported. Movements of over 5 mm are observed, indicating high processivity. Pauses and slips also occur, particularly at higher forces. We establish the force– velocity relationship of the motor and find that the rate-limiting step of the motor’s cycle is force dependent even at low loads. Interestingly, the packaging rate decreases as the prohead is filled, indicating that an internal pressure builds up due to DNA compression. We estimate that at the end of the packaging the capsid pressure is 6 MegaPascals, corresponding to an internal force of 50 pN acting on the motor. The biological implications of this internal pressure and the mechano-chemical efficiency of the engine are discussed. F2-002 The complete folding/unfolding trajectory of a protein captured with single molecule force-clamp spectroscopy J. M. Fernandez Biological Sciences, Columbia University, New York, NY 10027, USA. E-mail: jfernandez@columbia.edu A dense network of interconnected proteins and carbohydrates forms the complex mechanical scaffold of living tissues. The recently developed technique of single molecule force spectro- scopy has enabled a detailed analysis of the force-induced confor- mations of these molecules and the determinants of their mechanical stability. These studies provide some of the basic knowledge required to understand the mechanical interactions that define all biological organisms. The application of mechanical force to biological polymers produces conformations that are dif- ferent than those that have been investigated by chemical or ther- mal denaturation, and are inaccessible to conventional methods of measurement such as NMR spectroscopy and X-ray crystallogra- phy. Force-induced conformational transitions are physiologically Abstracts 337 important, and offer novel perspectives on the structure of bio- molecules. Recent developments in single molecule force spectros- copy have enabled study of the full unfolding and refolding pathways of a protein under force-clamp conditions. Mechanical unfolding of a protein placed under a constant stretching force allows us to obtain precise information about the pathway and kinetics of unfolding. Force-quench experiments capture for the first time the full folding pathway of a protein. Due to the force of gravity and the need of living organisms to perform mechan- ical work, mechanical stretching is most likely to have played a role in the evolution of proteins. By contrast, the large changes in temperature or chemical denaturants commonly employed in protein folding studies are not found in living cells. Hence, the mechanical unfolding/folding trajectories captured by force-clamp spectroscopy reflect much more closely the conformations and pathways of proteins in vivo, compared to those obtained by means of thermal or chemical manipulations. This lecture will focus mostly on the use of these novel techniques to study the dynamic changes that proteins undergo in response to a mechan- ical stretching force. References 1. Fernandez JM, Li HB. Force-clamp spectroscopy monitors the folding trajectory of a single protein. Science 2004; 303: 1674– 1678. 2. Carrion-Vazquez M, Li H, Lu H, Marszalek PE, Oberhauser AF, Fernandez JM. The mechanical stability of ubiquitin is linkage dependent. Nature Structural Biology 2003; 10(9): 738– 743. 3. Li HB, Linke WA, Oberhauser AF, Carrion-Vazquez M, Kerkvliet JG, Lu H, Marszalek PE, Fernandez JM. Reverse engineering of the giant muscle protein titin. Nature 2002; 418: 998–1002. F2-003 Tethering single protein concatamers to enable measurement of thermal noise response by Atomic Force Microscopy (AFM) K. Byrne, M. Kawakami, B. Khatri and A. Smith Laboratory of Molecular and Nanoscale Physics, Department of Physics and Astronomy, University of Leeds, Leeds, West Yorkshire, UK. E-mail: phykb2@phys-irc.leeds.ac.uk The thermal noise response of single molecules under tension can be measured using AFM and used to reveal both the conserva- tive and dissipative response of the molecule. This recently devel- oped method has been applied successfully to single sugar molecules tethered via non-specific interactions between the AFM cantilever and the base substrate. In order to apply the method to single protein concatamers we have found that a more durable means of tethering the molecules is required. Single pro- tein molecules can be suspended between a cantilever and sub- strate using non-specific tethering but the tether breaks before sufficient data can be captured to give an accurate noise spec- trum in all cases observed thus far. Tethering molecules through covalent rather than non-specific bonds should enable longer dur- ation experiments to be carried out with ease and has the added advantage that the exact location of the tether point is known. A protocol to tether a modified 5-domain concatamer of I27 between a gold coated cantilever and gold substrate via distinct covalent links is being developed. The gold binding functionality of the C-terminal cysteine residue and the ability of N-terminal histadine residues to bind succinamide will be exploited. The lysine residues have been removed from the concatamer so that succinamide-amine bonds should only form at the N-terminal end of the concatamer. A self assembled monolayer of the short linker molecule Di-thio-bis-succinamidyl-propionate (DSP) on gold will be used to tether the N-terminus. The short linker will interfere only minimally in the thermal noise spectrum molecule of interest. F2-004 Single molecule and single paritcle imaging in solution and in live cells X. Zhuang Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA. E-mail: zhuang@chemistry.harvard.edu Understanding the molecular mechanisms of complex biological processes is one of the major goals in modern biology. As molecular and cell biology get increasingly quantitative, a com- prehensive understanding of biological processes at the molecular level is becoming more readily accessible. However, roadblocks still exist, among which is the challenge that we face in character- izing the complex dynamics of biological processes. The existence of multiple kinetic paths and transient intermediate states often makes these processes difficult to dissect, as individual steps of a multi-step process are typically not synchronized among mole- cules. To tackle this problem, we are exploring optical imaging techniques to monitor, in real-time, the behavior of individual biological molecules and complexes, in vitro and in live cells. In this talk, I will report our recent progress in the following two areas. (i) Molecular mechanisms of viral infection: our single- virus tracking experiments allow us to visualize the viral infection process in real time, dissect individual stages of the viral entry pathway, and obtain a better understanding of the molecular mechanisms governing the influenza infection. (ii) Structural dynamics of RNA and ribonucleoprotein enzymes: our single- molecule studies provide critical insights into the molecular mechanisms governing RNA structural dynamics, and the effects of proteins on the structural dynamics of RNA enzymes. F2-005 Single molecule analysis of protein aggregation and prions by SIFT U. Bertsch 1 , K. F. Winklhofer 2 , T. Hirschberger 3 , J. Bieschke 1 , P. Weber 1 , F. U. Hartl 2 , P. Tavan 3 , J. Tatzelt 2 , H. A. Kretzschmar 1 and A. Giese 1 1 ZNP, University of Munich, Munich, Germany, 2 Department of Cellular Biochemistry, Max-Planck-Institute for Biochemistry, Martinsried, Germany, 3 Department of BioMolecular Optics, University of Munich, Munich, Germany. E-mail: armin.giese@med.uni-muenchen.de Protein aggregation is a key event in a number of diseases such as Alzheimer’s disease, Parkinson’s disease and prion diseases. We present a general method to quantify and characterize pro- tein aggregates by dual-colour scanning for intensely fluorescent targets (SIFT). In addition to high sensitivity, this approach offers a unique opportunity to study co-aggregation processes at the single particle level in complex mixtures containing different types of aggregates. In the case of prion diseases the prion pro- tein (PrPC), a neuronal glycoprotein, undergoes a conformational change from the normal, mainly alpha-helical conformation to a disease-associated, mainly beta-sheeted scrapie-isoform (PrPSc), which forms amyloid aggregates. This conversion, which is cru- cial for disease progression, depends on direct PrPC/PrPSc inter- action. We developed a high-throughput SIFT assay for the identification of drugs, which interfere with this interaction at the molecular level. Screening a library of 10 000 drug-like com- pounds yielded 256 primary hits, 80 of which were confirmed by Abstracts 338 dose–response curves with half-maximal inhibitory effects ranging from 0.3 to 60 lm. Among these, six compounds displayed an inhibitory effect on PrPSc propagation in scrapie-infected N2a cells. Four of these candidate drugs share a N ´ -benzylidene-ben- zohydrazide (NBB) core microstructure. Thus the combination of high-throughput in vitro assay with the established cell culture system provides a rapid and efficient method to identify new anti-prion drugs, which corroborates that interaction of PrPC and PrPSc is a crucial molecular step in the propagation of pri- ons. Moreover, SIFT-based screening may facilitate the search for drugs against other diseases linked to protein aggregation. F2-006 Studying protein reaction kinetics by fluorescence correlation spectroscopy in microfluidic mixers P. Galajda 1 , J. Puchalla 2 , R. Riehn 1 and R. H. Austin 1 1 Department of Physics, Princeton University, Princeton, NJ, USA, , 2 Department of Molecular Biology, Princeton University, Princeton, NJ, USA. E-mail: pgalajda@princeton.edu Fluorescence correlation spectroscopy is a powerful method to gather information about the motion and interactions of mole- cules and particles in solution. The autocorrelation of the fluores- cence intensity fluctuation emerging from fluorophores traversing a confined excitation/observation volume is used for analysis. Usually either diffusion or convection dominates on the time- scales of the fluctuations. Measurements lasts for seconds making it inadequate for kinetic studies of most biomolecular reactions. Here we present a method to overcome this limitation. Reactants meet in a microfluidic diffusional mixer. A steady-state flow establishes a direct mapping of the temporal evolution of the reaction to the spatial position along the channel opening a way for kinetic studies. The experimental conditions are set so that we can extract both the characteristics of diffusion and flow from the data. A variety of biological reactions might be studied by the above technique from protein folding to DNA-protein inter- actions. F2-007P Changes in cell morphology, viability, proliferation and cytotoxicity associated with helium–neon laser irradiation of diabetic wounded human skin fibroblasts H. Abrahamse, N. Houreld and D. Hawkins Laser Research Unit, Faculty of Health, University of Johannesburg, Johannesburg, Gauteng South Africa. E-mail: heidi@twr.ac.za Low Level Laser Therapy (LLLT) is a form of phototherapy used to promote wound healing in different clinical conditions. Laser radiation has the capability to inhibit or stimulate cellular activity in the absence of significant heating. Currently, no uni- versally accepted theory has explained the mechanism of laser biostimulation. Although not established, an alternative treat- ment modality for diabetic wound healing includes LLLT. Laser biostimulation of such wounds may be of benefit to patients by reducing healing time. Structural, cellular and genetic events in normal, wounded and unwounded diabetic induced human skin fibroblasts was evaluated after exposing cells to increasing doses of helium–neon (632.8 nm) laser irradiation. Structural changes were evaluated by assessing colony formation, haptotaxis and chemotaxis. Cellular changes were evaluated using cell viability, (ATP), proliferation, (ALP), cytotoxicity (LDH) while the Comet assay evaluated genetic integrity. Morphologically, wounded diabetic fibroblasts responded to single exposure of 5 J/cm 2 with the highest rate of haptotaxis and chemotaxis indicating a stimu- latory effect while 16 J/cm 2 showed the lowest rate indicating an inhibitory effect. Cell viability and proliferation changes indicated a decrease at higher doses while a dose of 5.0 J/cm 2 appeared to stimulate mitochondrial activity, which leads to normalization of cell function with an increase in cell viability. A dose of 5 J/cm 2 showed the highest rate of cell viability and proliferation with a minimal amount of damage to the cell membrane or DNA, sup- porting morphological evidence. In conclusion, LLLT of 5 J/cm 2 stimulates migration, proliferation, and metabolism of wounded fibroblasts to accelerate wound closure. F2-008P Methionine sulfoxide reductase from class A: aminoacids involved in catalysis of the reductase step and in substrate binding S. Boschi-Muller, M. Antoine, A. Gand and G. Branlant Laboratoire MAEM UMR 7567 CNRS-UHP, Universite ´ Henri Poincare ´ Nancy I, vandoeuvre-le ` s-Nancy, France. E-mail: Sandrine.Boschi@maem.uhp-nancy.fr Methionine sulfoxide reductases (Msrs) are ubiquitous enzymes that reduce protein-bound methionine sulfoxide back to Met in the presence of thioredoxine. In vivo, the role of Msrs is des- cribed as essential in protecting cells against oxidative damages, and to play a role in infection of cells by pathogenic bacteria. There exist two structurally-unrelated classes of Msrs, called MsrA and MsrB. MsrA is specific for the S epimer of MetSO, whereas MsrB reduces the R epimer. Both Msrs present a similar catalytic mechanism of sulfoxide reduction by thiols via the sulf- enic acid chemistry. The rate of the reductase step that leads to the formation of the sulfenic acid intermediate is fast. This sup- ports an activation of the catalytic Cys and an increase of the electrophilic character of the sulfur atom of the sulfoxide sub- strate via an acid catalyst from the active site. Both activations should favor the efficiency of the reductase process. Data will be presented which illustrate the role of some amino acids involved in the catalytic mechanism of the reductase step and in the sub- strate recognition of MsrAs, specifically in the formation of the hydrophobic pocket responsible for the methyl group recogni- tion. F2-009P Role of Induced cAMP Early Repressors in B/K Gene Transcription M H. Choi, Y M. Jang, Y S. Jang and O J. Kwon Department of Biochemistry, The Catholic University of Korea, College of Medicine, Seoul, South Korea. E-mail: cmh0912@hanmail.net B/K protein, structurally classified as a member of double C2 domain proteins, is highly expressed in PC12 cells. We previously found that forskolin decreased the expression of both B/K mRNA and protein by PKA-dependent mechanism, and that inducible cAMP early repressor (ICER) might be involved in that process via the interaction with a CRE-like domain that is located in the promoter region of B/K gene. Here, we showed the direct involvement of ICER in the mechanism of forskolin- induced down-regulation of B/K gene expression in PC12 cells. To investigate the role of ICER, we’ve successfully cloned four types of rat ICER (I, Ig, II and IIg). Transient transfection (24 h) of each construct resulted in the successful expression of ICERs, and the expression level was even higher than that induced by 3-h treatment of forskolin, the time when the expres- Abstracts 339 sion of ICER increased to the highest level. In promoter assay, ICER suppressed the promoter activity of B/K in CHO-K1 cells that were cotransfected with the construct containing B/K CRE- like sequence. Inhibition level might not be related with the iso- types of ICER but with the expression level in the cells, suggest- ing the absence of isotype specificity of ICER in the repression of B/K gene transcription. Effect of ICER did not observed in the cells cotransfected with the B/K construct having mutated CRE- like sequence (AC:TG). Direct binding of B/K CRE-like sequence to ICER was also observed in chromatin immunopre- cipitation experiment. These results strongly suggest that ICER may be the principal suppressor molecule in the process of B/K gene expression. F2-010P Comparative structural and conformational studies on two forms of beta lactoglobulin (A and B) upon interaction with lead ion A. Divsalar and A. A. Saboury Laboratory of Biophysical Chemistry, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran. E-mail: divsalar@ibb.ut.ac.ir One of the most widely studied and fascinating proteins of animal origin is Beta lactoglobulin (blg). This polypeptide, with 150–170 amino acids (depending on the species), is an abundant compo- nent of the milk of most animals, although it has not been detec- ted in human milk. Bovine blg consists of 162 amino acid residues and contains two disulfide bonds and a free thiol. Its interaction with a great variety of hydrophobic ligands has led to its inclusion in the lipocalin (transport protein) superfamily. blg is a dimeric native conformation at neutral pH, while the conformation at pH 2 is a monomeric but still native. Thermodynamic studies have been made on the effect of Pb 2+ on the structure of two forms of blg (A and B) in 50 mm sodium chloride at 27 °C using UV spec- trophotometry, circular dichroism (CD) and fluorescence spectros- copy. UV spectrophotometry studies show that the protein absorbance changes due to the increase concentration of ion lead are a biphasic curve for blg-A form and a monophasic sigmoid curve for blg-B form. Far CD studies do not show any change on the secondary structure of blg-A upon interaction with concentra- tions of 15.54 and 35.8 mm of Pb 2+ , while far CD studies show considerable change in the secondary structure of blg-B by increasing the concentration of Pb 2+ . Near CD and fluorescence spectroscopy studies show considerable change in the tertiary structure of Blg-A related to the interaction with lead ion, but these studies do not show any change in the tertiary structure of blg-B. Different behaviors of blg (A and B) upon interaction with lead ion, related to different native structure of both proteins. F2-011P Strong specific binding forces between adhesive extracellular proteoglycan carbohydrates I. Bucior 1 , S. Garcia-Manyes 2 , R. Ros 3 , F. Sanz 2 , D. Anselmetti 3 , M. M. Burger 1 and X. Ferna ` ndez-Busquets 4 1 Friedrich Miescher-Institut, Basel, Switzerland, 2 Department of Physical Chemistry, University of Barcelona, Barcelona, Spain, 3 Experimental Biophysics, University of Bielefeld, Bielefeld, Germany, 4 Laboratory of Nanobioengineering, Barcelona Science Park, University of Barcelona, Barcelona, Spain. E-mail: busquets@qf.ub.es Specific carbohydrate-carbohydrate interactions are rarely repor- ted in biologically relevant situations such as cell recognition. However, carbohydrate structures have immense structural diver- sity, a ubiquitous distribution in vertebrate and invertebrate tis- sues, and are associated with the cell surface, as required of cell recognition molecules. Carbohydrate–carbohydrate interactions are characterized by relatively weak forces which, when multi- merized, can be easily potentiated by orders of magnitude, repre- senting a highly versatile form of cell recognition and adhesion given the extraordinary plasticity of their structures. Sponge cells associate in a species-specific process through multivalent interac- tions of carbohydrate structures on a type of extracellular bifunc- tional proteoglycan molecules termed spongicans. In the marine sponge Microciona prolifera the spongican molecule, Microciona aggregation factor (MAF), self-interacts via a Ca 2+ -dependent interaction. We have dissected MAF into its components, track- ing the individual self-binding units down to the circular core of the sunburst-like molecule and further down to a 200-kDa glycan (g200). Force spectroscopy data and surface plasmon resonance measurements reveal that the carbohydrate self adhesion is highly specific when compared with the binding to other sulfated carbo- hydrates such as chondroitin sulfate. The strength of the binding per surface area between two spongican molecules is comparable to that of focal contacts between vertebrate cells. The results obtained reveal surprisingly high forces and selectivity for this most ancient cell adhesion system, and the existence of poly- morphism between g200 purified from different individuals, thus representing the first indication of specific carbohydrate–carbohy- drate interactions implicated in the discrimination between cells of different individual origin within a single species. Acknowledgments: This work was supported by grant BIO2002-00128 from the Ministerio de Ciencia y Tecnologı ´ a, Spain, that included FEDER funds. F2-012P Scanning probe microscopy as tool to study the stability of azurin in air V. Frascerra, G. Maruccio, V. Arima, L. del Mercato, P. P. Pompa, F. Calabi, R. Cingolani and R. Rinaldi National Nanotechnology Laboratory of INFM, Ingegneria dell’ Innovazione, University of Lecce, Lecce, Italy. E-mail: vanessa.frascerra@unile.it Scanning probe microscopy (SPM) comprises a family of tech- niques that can be used as tool to study biomolecules morphol- ogy and properties on the atomic scale. We focus on the use of the SPM to study the azurin stability in air. Azurin is a metallo- protein involved in electron transfer during denitrification path- way of the bacterium Pseudomonas aeruginosa. Thanks to its electron transfer properties, azurin and its physiological partners could be employed for the implementation of planar biosensors, with enhanced sensitivity and selectivity, exploiting a monolayer of proteins that should act as an optimal surface for the analyte molecules to react with. For this purpose it is fundamental that the immobilized proteins preserve their native conformation and properties, especially in ambient conditions. First, Scanning Tunneling Microscopy (STM) allows us to achieve a very high resolution and to probe the electronic properties of Au (111)- immobilized Azurin, both in buffer solution and in air. Our results demonstrates that immobilized proteins preserve their electron transfer function. Secondly, the force distance mode of atomic force microscopy has been used to measure the interac- tion between tip and SiO2-immobilized azurin by mercaptosi- lanes. The force curves show that the adhesion of the tip to the azurin is weaker than to the SiO2 and silanized surface. The mechanical properties of azurin could be deduced by analyzing these curves and correlated with the native conformation of the protein. Abstracts 340 F2-013P Hydrogen peroxide mediates Rac1 activation of S6K1 J W. Han 1 , Y G. Chung 1 , S. J. Paek 1 , S. H. Park 1 , J S. You 1 , J K. Kang 1 , Y. M. Kim 2 , H. Y. Lee 3 , Y. K. Kim 4 and H. W. Lee 1 1 Lab of Biochem & Mol Biol, College of Pharmacy, Sungkyunk- wan University, Suwon, Gyeonggi-do South Korea, 2 College of Pharmacy, Duksungsung Women’s University, Seoul, South Korea, 3 College of Medicine, Konyang University, Nonsan, Chungcheong- nam-do South Korea, 4 College of Medicine, Kwandong University, Gangneung, Gangwon-do South Korea. E-mail: jhhan551@skku.edu We previously reported that hydrogen peroxide (H 2 O 2 ) mediates mitogen activation of ribosomal protein S6 kinase 1 (S6K1) which plays an important role in cell proliferation and growth. In this study, we investigated a possible role of hydrogen perox- ide as a molecular linker in Rac1 activation of S6K1. Overexpres- sion of recombinant catalase in NIH-3T3 cells led to the drastic inhibition of H 2 O 2 production by PDGF, which was accompan- ied by a decrease in S6K1 activity. Similarly, PDGF activation of S6K1 was significantly inhibited by transient transfection or sta- ble transfection of the cells with a dominant-negative Rac1 (Rac1N17), while overexpression of constitutively active Rac1 (Rac1V12) in the cells led to an increase in basal activity of S6K1. In addition, stable transfection of Rat2 cells with Rac1N17 dramatically attenuated the H 2 O 2 production by PDGF as compared with that in the control cells. In contrast, Rat2 cells stably transfected with Rac1V12 produced high level of H 2 O 2 in the absence of PDGF, comparable to that in the con- trol cells stimulated with PDGF. More importantly, elimination of H 2 O 2 produced in Rat2 cells overexpressing Rac1V12 inhib- ited the Rac1V12 activation of S6K1, indicating the possible role of H 2 O 2 as a mediator in the activation of S6K1 by Rac1. How- ever, H 2 O 2 could be also produced via other pathway which is independent of Rac1 or PI3K, because in Rat2 cells stably trans- fected with Rac1N17, H 2 O 2 could be produced by arsenite which has been shown to be a stimulator of H 2 O 2 production. Taken together, these results suggest that H 2 O 2 plays a pivotal role as a mediator in Rac1 activation of S6K1. F2-014P Reaction of hydroxymethylarginine and endogenous formaldehyde with tetrahydrofolate producing N 5 ,N 10 -methylene tetrahydrofolate coenzyme L. Hulla ´ n 1 , L. Tre ´ zl 2 ,Z.M.Ja ´ szay 3 , M. Tejeda 4 , A. Csiba 5 , J. Bariska 6 and T. Szarvas 7 1 Department of Biochemistry, National Institute of Oncology, Budapest, Hungary, 2 Department of Organic Chemical Technol- ogy, Budapest University of Technology and Economics, Budapest, Hungary, 3 Organic Chemical Technological Research Group, Hun- garian Academy of Science, Budapest, Hungary, 4 Department of Experimental Pharmacology, National Institute of Oncology, Budapest, Hungary, 5 Veterinary and Food Control Station, Buda- pest, Hungary, 6 Central Laboratory, National Institute of Rheuma- tology and Physiotherapy, Budapest, Hungary, 7 Institute of Isotopes Co. Ltd., Budapest, Hungary. E-mail: hullan@oncol.hu The endogenous formaldehyde (E-CH 2 O) means the always exist- ing CH 2 O content of various biological samples. E-CH 2 O is sup- posed to be bound to biomolecules, because it has no toxic effect. N G -hydroxymethyl derivatives of arginine (HMA) synthes- ized by Tre ´ zl and co-workers from CH 2 O and L-arginine may belong to that type of molecules. HMA was found in human blood and urine, and in plants (leaves, fruits and vegetables) in higher concentration than in human samples since HMA is one of the products of photosynthesis. We have proved that the reac- tion of HMA and THF produces – under physiological condi- tions – N 5 ,N 10 -methylene tetrahydrofolate (CH 2 THF), the coenzyme of thymidylate synthase (TS). In recent work 13 C-HMA was synthesized and the reaction of 13 C-HMA and THF was compared to that of 13 C-CH 2 O and THF by 13 C-NMR in the reaction mixture for producing of CH 2 -THF according to the Roberts-method which is used for the determination of TS activities. The two reactions were also compared to the reaction between E-CH 2 O and THF. To test the CH 2 -THF product meas- urement of TS activities was applied using either cytosol of P388 lymphoid leukemia tumor, or TS purified from Lactobacillus ca- sei. Both the NMR spectra and the enzyme assays proved that the reactions produced CH 2 -THF, however, the rate of reaction by free CH 2 O is higher than those of the two other reactions. The results suggest that TS and probably other proteins are the most important molecules in keeping of E-CH 2 O in bound form. Acknowledgment: This work was supported by National Sci- ence Foundation OTKA Grant No. T034245. F2-015P Co-operation of H 2 O 2 -mediated ERK activation with Smad pathway in TGF-b1 induction of p21 WAF1/Cip1 Y. K. Kim 1 , E. K. Lee 2 , J. W. Park 2 , J. Y. Lee 2 , H. Y. Jung 2 , H. J. Kim 2 , J. H. Park 2 , S N. Kim 3 , H. Y. Lee 4 , H. W. Lee 2 and J W. Han 2 1 College of Medicine, Kwandong University, Gangneung, South Korea, 2 College of Pharmacy, Sungkyunkwan University, Suwon, South Korea, 3 Skin Research Institute, Amore-Pacific Corporation, Yong-In, South Korea, 4 College of Medicine, Konyang University, Nonsan, South Korea. E-mail: yksnbk@kwandong.ac.kr Although it has been demonstrated that p21 WAF1/Cip1 could be induced by transforming growth factor-b1 (TGF-b1) in a Smad- dependent manner, the cross-talk of Smad signaling pathway with other signaling pathways still remains poorly understood. In this study, we investigated a possible role of hydrogen peroxide (H 2 O 2 )-ERK pathway in TGF-b1 induction of p21 WAF1/Cip1 in human keratinocytes HaCaT cells. Using pharmacological inhibi- tors specific for MAP kinase family members, we found that ERK, but not JNK or p38, is required for TGF-b1 induction of p21 WAF1/Cip1 . ERK activation by TGF-b1 was significantly attenuated by treatment with N-acetyl-L-cysteine or catalase, indicating that reactive oxygen species (ROS) generated by TGF- b1, mainly H 2 O 2 , stimulates ERK signaling pathway to induce the p21 WAF1/Cip1 expression. In support of this, TGF-b1 stimula- tion caused an increase in intracellular ROS level, which was completely abolished by pre-treatment with catalase. ERK activa- tion does not appear to be associated with nuclear translocation of Smad-3, because ERK inhibition did not affect nuclear trans- location of Smads by TGF-b1, and H 2 O 2 treatment alone did not cause nuclear translocation of Smad-3. On the other hand, ERK inhibition led to the disruption of interaction between Smad-3 and Sp1 induced by TGF-b1, indicating that ERK signa- ling pathway might be necessary for their interaction. Taken together, these results suggest that activation of H 2 O 2 -mediated ERK signaling pathway is required for p21 WAF1/Cip1 expression by TGF-b1 and led us to propose a co-operative model whereby TGF-b1-induced receptor activation stimulates not only a Smad pathway but also a parallel H 2 O 2 -mediated ERK pathway that acts as a key determinant for association between Smads and Sp1 transcription factor. Abstracts 341 [...]... E-mail: myoshida@res.titech.ac.jp The ATP synthase can be thought of as a complex of two motors – the ATP-driven F1 motor and the proton-driven Fo motor – that rotate in opposite directions Our recent findings are follows 1 Direct observation of reaction sequence: Single molecule imaging of F1 motor has provided direct evidence that all of the three catalytic beta subunits cooperate in sequence during... Laboratory of Biochemistry, School of Medicine, University of Patras, Patras, Greece, 2Laboratory of Biochemistry, Department of Biology, University of Patras, Patras, Greece E-mail: konstantinidis.t@med.upatras.gr The function of mutations rdn1A, rdn1T and rdn2 in 18S rRNA of Saccharomyces cerevisiae has been examined They correspond to C1054A, C1054U in helix 34 and G517A in loop 530 of helix 18 of 16S... Control of the direction of rotation of these motors is the basis for the chemotactic response, i.e for the ability of cells, such as E coli, to swim up spatial gradients of chemical attractants I will tell you about motor rotation and its modulation by the cell’s signal transduction pathway F3-004 Three-dimensional structures of the bacterial flagellar rotary motor D R Thomas, N R Francis and D J DeRosier... FliN, forming a complex of two rings, the C ring (FliM and FliN), located in the cytoplasm and the M ring (FliF and FliG) found in the membrane The transmembrane proteins MotA and MotB form the proton channel and are anchored to the peptidoglycan layer thus forming the stator of the motor There are many models for how the motor functions but they have been devised in the absence of structural information... invasion potential (MDA-MB-231) and one with low invasion potential (BT-20), and also normal cells MCF-12A were used The expression of PDGF-R and c-Kit, their ligands, MMP-9 and signal transduction pathways PI3/Akt and MAPK were studied by RT-PCT and western blotting and immunocytochemistry The three receptors are expressed in all the breast cancer cell lines that were studied Exogenous SCF and PDGF-BB... identified by RNase H digestion and primer extension ´ analysis at C1400 and C1411 of the 3-minor domain of 16S rRNA, at positions located in the central loop of domain V, and in helices H42-H44 and H95 of 23S rRNA Specifically, in the central loop of domain V of 23S rRNA, ABA-spermine labels nucleosides U2584 and A2602 Nucleoside A2602 has been sug´ gested to propel the spiral rotation of the tRNA-3 end from... complexes are stable It seems likely that strong and stable, but non-productive interaction of the polymers determinates peroxidase inhibition during the reaction by preventing the access of regular substrates to the active center of the enzyme F3–Rotary Motor Complexes F 3–0 01 The rotary motors in F- and V-type ATPases D Stock and R A Bernal MRC Laboratory of Molecular Biology, Cambridge, UK E-mail: stock@mrc-lmb.cam.ac.uk... obtain paths for ligand binding to heme proteins The truncated hemoglobin, trHbN, from Mycobacterium tuberculosis belongs to the hemoglobin superfamily and may have functional roles in NO detoxification, long-term ligand storage, O2/NO chemistry, and O2 delivery under hypoxic conditions Its tertiary structure is based on a 2-on-2 a helical sandwich rather than the 3-on-3 a helical sandwich of the classical... homeostasis and enable transport processes across membranes While most eubacterial ATPases are of the F-type, some eubacteria and all known archaea have ATPases of the A-type, which are close homologues of V-type ATPases, but are used for ATP synthesis Although V- and A-type ATPases are similar in size and shape to F-type ATPases, only the catalytic subunits and the core of the transmembrane motor share... to a relatively rigid structure of the filament to work as a propeller, the hook has a much higher bending flexibility to function as a universal joint We reported the X-ray crystal structure of a 31 kDa core fragment of FlgE named FlgE31 (residues 7 0–3 63) and an atomic model of the straight hook built by docking the FlgE31 model to a density map obtained by electron cryomicroscopy and image analysis Since . F1 – Structure and Function of Motor Proteins F1- 001 What single-molecule mechanics can tell us about mitosis? J. Howard Max Planck Institute of Molecular. myoshida@res.titech.ac.jp The ATP synthase can be thought of as a complex of two motors – the ATP-driven F1 motor and the proton-driven Fo motor – that rotate in opposite directions.