1PT143 THERMODYNAMIC PARAMETERS ASSOCIATED WITH THE GroEL-GroES BINDING(The 50th Annual Meeting of the Biophysical Society of Japan)
1PT143 GroEL-GroES複合体形成の熱力学的解析(日本生物物理学会第50回年会(2012年度))

Toshio Takenaka, Takashi Nakamura, Jin Chen, Kunihiro Kuwajima
2012 Seibutsu Butsuri  
Ikeguchii (tSoko universio" Non-heme fenitin (Ftn) from Escherichia eoli fonms a spherical shell-shape with 41312 symmetry of24 subunits. It stores Fe(I[1) as fenihydrite mineral within the protein she]]. Ftn dissoeiates jnto i2 dirners at acidic pH, and it is able to reassemble into the native 24-mcr when pH is returned to a neutral one. The cireular dichroism (CD) spectrum and smatt angle X-ray scattering (SAXS) have shown that the acid-dissoeiated Ftn dimer maintains native-]ike secondary
more » ... e-]ike secondary and tertiaiy structures, To crarify the assembly mechanisrn of apo-Ftn the reassemb]y of acid-dissociated apo-Ftn dimers was initiated by stopped-flow mixing with appropriate buffers and monitored by synchrotron radiation at SPring-8 (Beamline 4SXU) and a photon-counting two-dimensionat detector PILATUS. The reassembly rate depended on pH; jt was too rapid to measure at pH 6.4 and slowed dewn at higher pHs. At pH 8, we suceeeded to observe an overall change from the initial scattering {dentical to that of acid-dissociated dimer to the final scattering identica] to the native apo-Ftn. The process consisted of at least two kinetic phases. A native-like radius gyration was attained in the faster phase and the zero-angle scattering intensity increased in both the fast and sFow phases. The rate ofthe fast phase depended on the protein concentration,suggestingthatthisphasecorrespondstotheassemb]yofdirners. The tendency of the amyloid fibri] formation of a peptide depends on its sequence. Recently. several programs which can pTedict amy]oidogenicity of a given sequence have been presented. A]though these progrrams pTedict well the amytoidegenicity ofindividual sequence, they do not provide information about the dynamics of the corresponding peptide in so]ution. Thus, in our study, we investigated dynarnics of vafious peptidcs and correlated them with their amyloidogenicity, We previously reported that B2 -rnicroglobulin (B2m) is digested by Achromobueter protease I inte 9 fragments (denoted as Kl-K9). Among them, K3 and K6 were found to form amyloid fibrils, eonsistent with the prediction by Aggrescan, one of the prediction algorithms for amylediogenie seguences, We investigated the urea-concentration-dependent transverse relaxatien rates (R 2 ) of K2, K3, K5, K6, K7 and K9. It was found that nonamy]oidogenic peptides showed only slight increase in R 2 as the urea concentration increased, indicating expansion of the peptide chain with an enhanced solvation, K3 a]so showed the same dependency at high urea eoncentrations, However, at lower urea concentrations, it showed an abrupt increase in R2 at the amyloidogenic ponion of the sequence, indicating a formation of a hydrophobic ctusteT. From these results, we propose that an amyloidogenicity is Tetated te the ability of hydrophobie cluster fonnation. The prediction a]gorithm is assumed to identify specifically such sequences. The tetradecameric chapeTonin GTeEL forms a complex with its co-chaperonin GroES and medjates the refolding of substrate proteins in the cell. Despite a large body of studies to elucidate the undertying rnechanism of the ehaperone-assistedrefo1clingofsubstrateproteins,thethermodynamicpararnetersassociate with the GroEL-GroES binding have not yet been determined. We therefore combined NMR spectroscopy and isothermal titration ca]orimetry (ITC) to quantitatively estimate the GroEL-GroES binding constant in se]ution. In 2-D HSQC NMR measurements, by monitoring amide hydrogen signal intensities of cross peaks in a mobile loop region of 15N labeled GroES as a function of concentration of sing]e-ring of GroEL {SRI), we obtained the equilibrium binding constants between GreES and SRI at difVbrent temperatures in 20 mM Tris-HCI , IOO mM KCI, 10 mM MgC12. and 3 mM ADP (pH 7.5), and the values were compared with those obtained by ITC under the conditions. 1PTI44 Local and Global Structura] Ana]ysis of Denatured Protein by CW and pulsed ESR Jun Abei, Munehito Arai2, Satoshi Takahashi:, Seigo Yamauehii, Yasunori Ohbai (ilMRAM, iGrad, Sch, Art and SL'i, Uhiv. Tbkyo) Details of structural inforrnation of unfolded proteins are limited despite the active investigations in the past. As a structura] medel, a random coil has been assumed. HoweveT, recent researches have revealed that denatured pTotein has residual structures similar to secondary structures: hetices and P sheet[1]. In our study, we analyzed the structure of unfolded protein based on electron spin resonance (ESR) methods (CW-ESR and pulsed ESR) with the site-directed spin labeling. We intredueed the spin ]abels to 22 and 55th residue to rneasure the distance between the he]ices H2 and H3 in the B domain of protein A (BDPA). ESR signals were meagured for the unfolding state by adding guanidinium ch]oride (GdmCl) as denaturant in the foIded and unfolded states. CW-ESR reveals that the rotationa] correlation time in the folded state (GdmCl O M) is faster than that in the unfolded state (GdrnC[5 M) for single labeled BDPA. This result indicates that the motion ofmain chain in denatured protein beeome faster, For doubly labeled BDPA, we found that the well-defined H2-H3 distance for the folded state. In contrast, a broad distribution of the H2-H3 distance is observed for unfolded BDPA. We wi1] compare the obtained distance distributien with that obtained by numerical ealculation which is based on assumption of the random coil behaviors. [1] Shortle, D. and Ackerman, M,S" Science, 293, 4S7-489 {2001) IPT146 SVS;,JCOfiopuadimaMGvaOneut ln yitro analysis ofthe early stage of aggregatioll of tau protein Teikichi Ikura, Nobutoshi Ito (Mbdical Research institute, Tblp,o Adledical and Dentai Uhiversie') Tau protein is essentiat to assembly of microtubules, which mainly consist ef two types of tubulins. Hyperphosphorylation oftau protein abolishes its ability to bind tubulin and promote microtubule assernbly, When it is released from tubulin, phosphory]ated tau protein aggregates into paired helical filaments, which are characterized as the neuropathological hallmarks of Alzheimer's disease, The mechanism of aggregation oftau pretein is still unsolved, though grcat effbrts have been rnade to elucidate it, The early stage of aggregation is especiajly poorly understood because of diencultly of its cletection. Here we synthesized various peptides including rnicTotubu]e binding domains, which showed high aggregation propensity, and then investigated their in-vino aggregation under various conditions by fluorescence and dynamic light scattering measurements. As a result, the tau peptides quickly aggregated into the oligomeric forms even in the ubsence of inducers. Their eritical concentration of unpo]yrnerization was less than 1 micTo-motar in the absence of inducers. Increase in the concentration accelerated the velocity of aggregation. Ionic strength and phosphorylation state a]so affected the ve]ocity of aggregation, The mechanism ofaggregation oftau protein at the early stage will bc discussed on the basis of these results, IPT145 IPT147 MSESitl[diU9Yl"ONanreEfM ¢ geut Muttiscale enhanced sampling simulation of protein interaction Kei Moritsugui, Tohru Teradad'2, Akinori Kiderai'] (iCSRP, RIKEN, ?Grad. Sch, of Agri. and Ltfo Sbi., biniv. of Tblyo, 3G,ud. Slrh. qf' Nlanohiosc'i., lokohama Cio, U}iiu) Protein interaction plays a fundamental ro]e for biologieal processes such as transcription, signal transduction and immune response. Simulating pTotein association dynamics will be chal]enging beyond the conventional computational studies such as predicting protein complex structures and calculating free energy differences via residual mutations at the complex interface, Since protein complex formation involves atornistic interaction and desolvation process, in this study, we aim to achieve a conformational sampling ofprotein interaction on atomistic reso]ution and under physjotogical condition including explicit so]vent using multiscale enhanced sampling (MSES) simulation, TheMSESsimu]ational]owsanenhancedsamplingefthefu11-dimensional,allatommodelinexplicitsolvent,bycoup]ingwiththeaccelerateddynamicsofthe coarse-grained degrees of freedom, Here, this method has been applied to bamase-barster cemplex which has been studied in extensive experiments and computations.Thesamplingoftherelativeconfigurationofthetwoproteinshas been enhanced via coup]ing to a Ca-atom coarse-grained model which moves fieety around the complex structure. The interaction proccss at the interface has been characterized by formation of the hydrogen bonds and deselvation in atomic detail, Thc MSES has atso been applied to the sarne system with a site-pi:-omtimvuyes"aj7fKomnttt7:-m(Fvarervntee eopresuCorre]ation of the dynamics of Prmicroglebulin fragments with their amyloidogenicity -S93-NII-Electronic
doi:10.2142/biophys.52.s93_1 fatcat:gk2mrqwd25drrgfqzqc43vhdkm