蛋白质体系分子动力学模拟的前沿进展-从介科学角度重新审视

doi:10.12034/j.issn.1009-606X.218238

摘要/Abstract

摘要： 蛋白质是生命的物质基础，是生命活动的主要承担者，对蛋白质时空多尺度结构及其控制机制的深入理解是探索生命起源、病理认知及新药开发的基础. 受实验表征手段及时空分辨率的限制，计算机模拟已成为研究蛋白质体系结构及功能的重要手段之一. 由于蛋白质体系模拟所涉及的时间和空间跨度均相当大，因此，准确且快速地描述其时空多尺度结构，从而分析体系的控制机制及相关生理过程，成为分子动力学模拟面临的巨大挑战. 本工作对近半个世纪以来的分子模拟方法，特别是分子动力学方法和相关的增强采样技术在蛋白质体系研究中的应用进行了总结，综述了近年来分子动力学的理论模型和算法的发展，并介绍了这些方法在结构化蛋白质的天然结构与构象变化、固有无序蛋白质的动态结构及其结合底物的动力学过程及分子机理、分子伴侣及病毒等蛋白质复合物体系中的研究成果；汇总了高性能计算的飞速发展所带动的分子动力学模拟软件的变革，拓展了蛋白质模拟的时空尺度，重点阐述了大规模高性能分子动力学模拟在蛋白质研究中的应用；最后，基于介科学理论的飞速发展及其在多种复杂体系的成功运用，对未来蛋白质体系的模拟方法和理论研究的趋势进行了思考和展望.

关键词: 蛋白质, 分子动力学模拟, 时空多尺度结构, 介科学

Abstract: Proteins are the essential parts of living organisms and they participate in virtually every process within cells. An in-depth understanding of the spatiotemporal multi-scale structure and the dominating mechanisms of protein structures would be the basis for scientific exploration of the origin of life, the mechanisms of diseases and the development of new drugs. Due to the limitations of the spatial and temporal resolutions of current experimental methods, computer simulations, especially molecular dynamics simulations, have become one of the most important methods to study the structure and function of protein systems. This article reviewed the progress of molecular simulations and their application in the research of protein systems during the past half century, especially for molecular dynamics simulations and enhanced sampling methods. The time and space involved in protein simulations covers a wide range of scales, which makes it a great challenge to simulate the spatial-temporal multi-scale structures quickly and accurately, or to investigate the physiological process and the underlying dominating mechanisms. Therefore, this article summarized the recent development of the theoretical models and computing algorithms, and their applications in the investigations of the molecular mechanisms of the native structures and structural changes of the structured proteins, the dynamic structure ensemble of intrinsic disordered proteins and the coupled folding and binding with target protein or other biological molecules, protein complex such as molecular chaperonin, virus particle, etc. Furthermore, the evolution of the popular softwares for molecular dynamics simulations driven by the rapid development of high-performance super computers, and their acceleration of the spatial-temporal scales in molecular dynamics simulations of protein systems, were further discussed. At the end of the article, based on the rapid development of mesoscience theory and its successful applications in a variety of complex systems, the future simulation methods and theoretical research of protein systems were prospected.

Key words: Protein, molecular dynamics simulations, spatial-temporal multi-scale structures, mesoscience

任瑛徐骥. 蛋白质体系分子动力学模拟的前沿进展-从介科学角度重新审视[J]. 过程工程学报, 2018, 18(6): 1126-1137.

Ying REN Ji XU. Frontiers of molecular dynamics simulations of protein systems-reexamine from the mesoscience perspective[J]. Chin. J. Process Eng., 2018, 18(6): 1126-1137.

参考文献

[1]. Guo M, Xu Y, Gruebele M. Temperature dependence of protein folding kinetics in living cells[J]. Proceedings of the National Academy of Sciences. 2012,109(44):17863-7.
[2]. Vila-Vic?osa D, Campos SR, Baptista AnM, Machuqueiro M. Reversibility of prion misfolding: Insights from constant-pH molecular dynamics simulations[J]. The Journal of Physical Chemistry B. 2012,116(30):8812-21.
[3]. DeMarco ML, Daggett V. Local environmental effects on the structure of the prion protein[J]. Comptes rendus biologies. 2005,328(10):847-62.
[4]. Ellis RJ, Minton AP. Cell biology: join the crowd[J]. Nature. 2003,425(6953):27-8.
[5]. Cheung MS, Klimov D, Thirumalai D. Molecular crowding enhances native state stability and refolding rates of globular proteins[J]. Proceedings of the National Academy of Sciences of the United States of America. 2005,102(13):4753-8.
[6]. Lee EH, Hsin J, Sotomayor M, Comellas G, Schulten K. Discovery Through the Computational Microscope[J]. Structure. 2009,17(10):1295-306.
[7]. Alder B, Wainwright T. Phase transition for a hard sphere system[J]. The Journal of chemical physics. 1957,27(5):1208.
[8]. MacKerell AD, Bashford D, Bellott M, Dunbrack R, Evanseck J, Field MJ, et al. All-atom empirical potential for molecular modeling and dynamics studies of proteins[J]. The journal of physical chemistry B. 1998,102(18):3586-616.
[9]. Cornell WD, Cieplak P, Bayly CI, Gould IR, Merz KM, Ferguson DM, et al. A second generation force field for the simulation of proteins, nucleic acids, and organic molecules[J]. Journal of the American Chemical Society. 1995,117(19):5179-97.
[10]. Ueda Y, Taketomi H, Gō N. Studies on protein folding, unfolding, and fluctuations by computer simulation. II. A. Three‐dimensional lattice model of lysozyme[J]. Biopolymers. 1978,17(6):1531-48.
[11]. Hills RD, Brooks CL. Insights from coarse-grained Gō models for protein folding and dynamics[J]. International journal of molecular sciences. 2009,10(3):889-905.
[12]. Hills R, Brooks C. Insights from Coarse-Grained Gō Models for Protein Folding and Dynamics[J]. International Journal of Molecular Sciences. 2009,10(3):889.
[13]. Marrink SJ, Risselada HJ, Yefimov S, Tieleman DP, de Vries AH. The MARTINI Force Field:? Coarse Grained Model for Biomolecular Simulations[J]. The Journal of Physical Chemistry B. 2007,111(27):7812-24.
[14]. Marrink SJ, Tieleman DP. Perspective on the Martini model[J]. Chemical Society Reviews. 2013,42(16):6801-22.
[15]. Kollman P. Free energy calculations: applications to chemical and biochemical phenomena[J]. Chemical reviews. 1993,93(7):2395-417.
[16]. Christ CD, Mark AE, Van Gunsteren WF. Basic ingredients of free energy calculations: a review[J]. Journal of computational chemistry. 2010,31(8):1569-82.
[17]. Abrams C, Bussi G. Enhanced sampling in molecular dynamics using metadynamics, replica-exchange, and temperature-acceleration[J]. Entropy. 2013,16(1):163-99.
[18]. Bernardi RC, Melo MCR, Schulten K. Enhanced sampling techniques in molecular dynamics simulations of biological systems[J]. Biochimica et biophysica acta. 2015,1850(5):872-7.
[19]. Kirkwood JG. Statistical mechanics of fluid mixtures[J]. The Journal of Chemical Physics. 1935,3(5):300-13.
[20]. Torrie GM, Valleau JP. Nonphysical sampling distributions in Monte Carlo free-energy estimation: Umbrella sampling[J]. Journal of Computational Physics. 1977,23(2):187-99.
[21]. Laio A, Parrinello M. Escaping free-energy minima[J]. Proceedings of the National Academy of Sciences. 2002,99(20):12562-6.
[22]. Barducci A, Bonomi M, Parrinello M. Metadynamics[J]. Wiley Interdisciplinary Reviews: Computational Molecular Science. 2011,1(5):826-43.
[23]. Sugita Y, Okamoto Y. Replica-exchange molecular dynamics method for protein folding[J]. Chemical physics letters. 1999,314(1):141-51.
[24]. Bussi G, Gervasio FL, Laio A, Parrinello M. Free-energy landscape for β hairpin folding from combined parallel tempering and metadynamics[J]. Journal of the American Chemical Society. 2006,128(41):13435-41.
[25]. Chen J. Intrinsically disordered p53 extreme C-terminus binds to S100B (ββ) through “fly-casting”[J]. Journal of the American Chemical Society. 2009,131(6):2088-9.
[26]. Dill KA, Ozkan SB, Shell MS, Weikl TR. The protein folding problem[J]. Annual review of biophysics. 2008,37:289.
[27]. Wolynes PG. Evolution, energy landscapes and the paradoxes of protein folding[J]. Biochimie. 2014.
[28]. Compiani M, Capriotti E. Computational and theoretical methods for protein folding[J]. Biochemistry. 2013,52(48):8601-24.
[29]. Dill KA, MacCallum JL. The Protein-Folding Problem, 50 Years On[J]. Science. 2012,338(6110):1042.
[30]. Anfinsen C. Principles that govern the protein folding chains[J]. Science. 1973,181:233-0.
[31]. Rohl CA, Strauss CEM, Misura KMS, Baker D. Protein Structure Prediction Using Rosetta. Methods in Enzymology. 383: Academic Press; 2004. p. 66-93.
[32]. Das R, Baker D. Macromolecular modeling with rosetta[J]. (0066-4154 (Print)).
[33]. Das R, Qian B Fau - Raman S, Raman S Fau - Vernon R, Vernon R Fau - Thompson J, Thompson J Fau - Bradley P, Bradley P Fau - Khare S, et al. Structure prediction for CASP7 targets using extensive all-atom refinement with Rosetta@home[J]. (1097-0134 (Electronic)).
[34]. Levinthal C. How to fold graciously[J]. Mossbauer spectroscopy in biological systems. 1969:22-4.
[35]. Lindorff-Larsen K, Piana S, Dror RO, Shaw DE. How Fast-Folding Proteins Fold[J]. Science. 2011,334(6055):517-20.
[36]. Sohl JL, Jaswal SS, Agard DA. Unfolded conformations of α-lytic protease are more stable than its native state[J]. Nature. 1998,395(6704):817-9.
[37]. Wang Z, Mottonen J, Goldsmith EJ. Kinetically controlled folding of the serpin plasminogen activator inhibitor 1[J]. Biochemistry. 1996,35(51):16443-8.
[38]. Sanchez-Ruiz JM. Protein kinetic stability[J]. Biophysical chemistry. 2010,148(1):1-15.
[39]. Fisher KE, Ruan B, Alexander PA, Wang L, Bryan PN. Mechanism of the kinetically-controlled folding reaction of subtilisin[J]. Biochemistry. 2007,46(3):640-51.
[40]. Schrader TE, Schreier WJ, Cordes T, Koller FO, Babitzki G, Denschlag R, et al. Light-triggered β-hairpin folding and unfolding[J]. Proceedings of the National Academy of Sciences. 2007,104(40):15729-34.
[41]. Puchner EM, Alexandrovich A, Kho AL, Hensen U, Sch?fer LV, Brandmeier B, et al. Mechanoenzymatics of titin kinase[J]. Proceedings of the National Academy of Sciences. 2008,105(36):13385-90.
[42]. Savage B, Saldívar E, Ruggeri ZM. Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor[J]. Cell. 1996,84(2):289-97.
[43]. Matsushita T, Sadler JE. Identification of amino acid residues essential for von Willebrand factor binding to platelet glycoprotein Ib. Charged-to-alanine scanning mutagenesis of the A1 domain of human von Willebrand factor[J]. Journal of Biological Chemistry. 1995,270(22):13406-14.
[44]. Han M, Xu J, Ren Y, Li J. Simulations of flow induced structural transition of the β-switch region of glycoprotein Ibα[J]. Biophysical Chemistry. 2016,209:9-20.
[45]. Wright PE, Dyson HJ. Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm[J]. Journal of molecular biology. 1999,293(2):321-31.
[46]. Pancsa R, Tompa P. Structural disorder in eukaryotes[J]. PLoS one. 2012,7(4):e34687.
[47]. Oldfield CJ, Cheng Y, Cortese MS, Brown CJ, Uversky VN, Dunker AK. Comparing and combining predictors of mostly disordered proteins[J]. Biochemistry. 2005,44(6):1989-2000.
[48]. Uversky VN, Dunker AK. Understanding protein non-folding[J]. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics. 2010,1804(6):1231-64.
[49]. Demarest SJ, Martinez-Yamout M, Chung J, Chen H, Xu W, Dyson HJ, et al. Mutual synergistic folding in recruitment of CBP/p300 by p160 nuclear receptor coactivators[J]. Nature. 2002,415(6871):549-53.
[50]. Lee CW, Martinez-Yamout MA, Dyson HJ, Wright PE. Structure of the p53 transactivation domain in complex with the nuclear receptor coactivator binding domain of CREB binding protein[J]. Biochemistry. 2010,49(46):9964-71.
[51]. Dyson HJ, Wright PE. Coupling of folding and binding for unstructured proteins[J]. Current opinion in structural biology. 2002,12(1):54-60.
[52]. Kiefhaber T, Bachmann A, Jensen KS. Dynamics and mechanisms of coupled protein folding and binding reactions[J]. Current Opinion in Structural Biology. 2012,22(1):21-9.
[53]. Dyson HJ. Roles of intrinsic disorder in protein–nucleic acid interactions[J]. Molecular BioSystems. 2012,8(1):97-104.
[54]. Wright PE, Dyson HJ. Linking folding and binding[J]. Current opinion in structural biology. 2009,19(1):31-8.
[55]. Huang Y, Liu Z. Kinetic Advantage of Intrinsically Disordered Proteins in Coupled Folding–Binding Process: A Critical Assessment of the “Fly-Casting” Mechanism[J]. Journal of Molecular Biology. 2009,393(5):1143-59.
[56]. Espinoza-Fonseca LM. Reconciling binding mechanisms of intrinsically disordered proteins[J]. Biochemical and biophysical research communications. 2009,382(3):479-82.
[57]. Han M, Xu J, Ren Y, Li J. Simulation of coupled folding and binding of an intrinsically disordered protein in explicit solvent with metadynamics[J]. Journal of Molecular Graphics and Modelling. 2016,68:114-27.
[58]. Wang J, Lu Q, Lu HP. Single-molecule dynamics reveals cooperative binding-folding in protein recognition[J]. PLoS Comput Biol. 2006,2(7):842-52.
[59]. Ganguly D, Zhang W, Chen J. Synergistic folding of two intrinsically disordered proteins: searching for conformational selection[J]. Molecular BioSystems. 2012,8(1):198-209.
[60]. Turjanski AG, Gutkind JS, Best RB, Hummer G. Binding-induced folding of a natively unstructured transcription factor[J]. PLoS Comput Biol. 2008,4(4):e1000060.
[61]. Sugase K, Dyson HJ, Wright PE. Mechanism of coupled folding and binding of an intrinsically disordered protein[J]. Nature. 2007,447(7147):1021-5.
[62]. Ganguly D, Chen J. Atomistic details of the disordered states of KID and pKID. Implications in coupled binding and folding[J]. Journal of the American Chemical Society. 2009,131(14):5214-23.
[63]. Zhang W, Ganguly D, Chen J. Residual structures, conformational fluctuations, and electrostatic interactions in the synergistic folding of two intrinsically disordered proteins[J]. 2012.
[64]. Chen J. Towards the physical basis of how intrinsic disorder mediates protein function[J]. Archives of Biochemistry and Biophysics. 2012,524(2):123-31.
[65]. Rust RR, Baldisseri DM, Weber DJ. Structure of the negative regulatory domain of p53 bound to S100B (ββ)[J]. Nature Structural & Molecular Biology. 2000,7(7):570-4.
[66]. Hoff KG, Avalos JL, Sens K, Wolberger C. Insights into the sirtuin mechanism from ternary complexes containing NAD+ and acetylated peptide[J]. Structure. 2006,14(8):1231-40.
[67]. Lowe ED, Tews I, Cheng KY, Brown NR, Gul S, Noble ME, et al. Specificity determinants of recruitment peptides bound to phospho-CDK2/cyclin A[J]. Biochemistry. 2002,41(52):15625-34.
[68]. Wiewiora RP, Chen S, Beauchamp K, Luo M, Chodera JD. Conformational Dynamics of Histone Lysine Methyltransferases by Millisecond-Timescale Molecular Dynamics on Folding@home[J]. Biophysical Journal. 2017,112(3, Supplement 1):189a.
[69]. Hartl FU, Hayer-Hartl M. Molecular Chaperones in the Cytosol: from Nascent Chain to Folded Protein[J]. Science. 2002,295(5561):1852-8.
[70]. Fenton WA, Horwich AL. Chaperonin-mediated protein folding: fate of substrate polypeptide[J]. Quarterly Reviews of Biophysics. 2003,36(02):229-56.
[71]. Xu Z, Horwich AL, Sigler PB. The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex[J]. Nature. 1997,388(6644):741-50.
[72]. Braig K, Otwinowski Z, Hegde R, Boisvert DC, Joachimiak A, Horwich AL, et al. The crystal structure of the bacterial chaperonln GroEL at 2.8 A[J]. Nature. 1994,371(6498):578-86.
[73]. Braig K, Simon M, Furuya F, Hainfeld JF, Horwich AL. A Polypeptide Bound by the Chaperonin groEL is Localized Within a Central Cavity[J]. Proceedings of the National Academy of Sciences. 1993,90(9):3978-82.
[74]. Steinbacher S, Ditzel L. Review: Nucleotide Binding to the Thermoplasma Thermosome: Implications for the Functional Cycle of Group II Chaperonins[J]. Journal of Structural Biology. 2001,135(2):147-56.
[75]. Tang Y-C, Chang H-C, Roeben A, Wischnewski D, Wischnewski N, Kerner MJ, et al. Structural Features of the GroEL-GroES Nano-Cage Required for Rapid Folding of Encapsulated Protein[J]. Cell. 2006,125(5):903-14.
[76]. van der Vaart A, Ma J, Karplus M. The Unfolding Action of GroEL on a Protein Substrate[J]. Biophys J. 2004,87(1):562-73.
[77]. Takagi F, Koga N, Takada S. How protein thermodynamics and folding mechanisms are altered by the chaperonin cage: Molecular simulations[J]. Proceedings of the National Academy of Sciences. 2003,100(20):11367-72.
[78]. Ying R, Jian G, Ji X, Wei G, Li J. Explicit solvent molecular dynamics simulations of chaperonin-assisted rhodanese folding[J]. Particuology. 2009,7(3):220-4.
[79]. 潘章, 陈静, 耿轶钊, 张辉, 覃静宇, 纪青. 驱动蛋白的研究进展[J]. 生命科学研究. 2012,16(4):350-6.
[80]. 刘梅, 徐娜, 阮世龙, 孙学松, 胡健饶. 驱动蛋白及其作用研究进展[J]. 杭州师范大学学报(自然科学版). 2013,12(1):40-4.
[81]. 曹添亮, 韩孟之, 徐骥, 任瑛. 驱动蛋白结构与运动机制[J]. 中国生物化学与分子生物学报. 2016,32(7):734-44.
[82]. 曹添亮. 驱动蛋白动态结构模拟与控制机制研究: 中国科学院大学; 2016.
[83]. Freddolino PL, Arkhipov As Fau - Larson SB, Larson Sb Fau - McPherson A, McPherson A Fau - Schulten K, Schulten K. Molecular dynamics simulations of the complete satellite tobacco mosaic virus[J]. (0969-2126 (Print)).
[84]. Zhao G, Perilla JR, Yufenyuy EL, Meng X, Chen B, Ning J, et al. Mature HIV-1 capsid structure by cryo-electron microscopy and all-atom molecular dynamics[J]. Nature. 2013,497(7451):643-6.
[85]. Xu J, Wang X, He X, Ren Y, Ge W, Li J. Application of the Mole-8.5 supercomputer: Probing the whole influenza virion at the atomic level[J]. Chinese Science Bulletin. 2011,56(20):2114-8.
[86]. Rapaport DC. Self-assembly of polyhedral shells: a molecular dynamics study[J]. (1539-3755 (Print)).
[87]. Rapaport DC. Role of Reversibility in Viral Capsid Growth: A Paradigm for Self-Assembly[J]. Physical Review Letters. 2008,101(18):186101.
[88]. Arkhipov A, Roos Wh Fau - Wuite GJL, Wuite Gj Fau - Schulten K, Schulten K. Elucidating the mechanism behind irreversible deformation of viral capsids[J]. (1542-0086 (Electronic)).
[89]. Heller H, Grubmüller H, Schulten K. Molecular dynamics simulation on a parallel computer[J]. Molecular simulation. 1990,5(3-4):133-65.
[90]. Brown D, Clarke JH, Okuda M, Yamazaki T. A domain decomposition parallelization strategy for molecular dynamics simulations on distributed memory machines[J]. Computer Physics Communications. 1993,74(1):67-80.
[91]. Bhandarkar M, Kalé LV, de Sturler E, Hoeflinger J. Adaptive load balancing for MPI programs. Computational Science-ICCS 2001: Springer; 2001. p. 108-17.
[92]. Hess B, Kutzner C, van der Spoel D, Lindahl E. GROMACS 4: Algorithms for highly efficient, load-balanced, and scalable molecular simulation[J]. Journal of Chemical Theory and Computation. 2008,4(3):435-47.
[93]. Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E, Villa E, et al. Scalable molecular dynamics with NAMD[J]. Journal of computational chemistry. 2005,26(16):1781-802.
[94]. Plimpton S. Fast parallel algorithms for short-range molecular dynamics[J]. Journal of computational physics. 1995,117(1):1-19.
[95]. Bowers KJ, Chow E, Xu H, Dror RO, Eastwood MP, Gregersen B, et al., editors. Scalable algorithms for molecular dynamics simulations on commodity clusters. SC 2006 Conference, Proceedings of the ACM/IEEE; 2006: IEEE.
[96]. Larson SM, Snow CD, Shirts M, Pande VS. Folding@ Home and Genome@ Home: Using distributed computing to tackle previously intractable problems in computational biology[J]. arXiv preprint arXiv:09010866. 2009.
[97]. Xu J, Ren Y, Ge W, Yu X, Yang X, Li J. Molecular dynamics simulation of macromolecules using graphics processing unit[J]. Molecular Simulation. 2010,36(14):1131-40.
[98]. Xu J, Ge W, Ren Y, Li J. Implementation of Particle-Mesh Ewald (PME) on graphics processing units[J]. Chin J Comput Phys. 2009.
[99]. Wang X, Ge W, He X, Chen F, Guo L, Li J, editors. Development and application of a HPC system for multi-scale discrete simulation-Mole-8.5. International supercomputing conference Hamburg, Germany; 2010.
[100]. Shaw DE, Deneroff MM, Dror RO, Kuskin JS, Larson RH, Salmon JK, et al. Anton, a special-purpose machine for molecular dynamics simulation[J]. Communications of the ACM. 2008,51(7):91-7.
[101]. Shaw DE, Maragakis P, Lindorff-Larsen K, Piana S, Dror RO, Eastwood MP, et al. Atomic-level characterization of the structural dynamics of proteins[J]. Science. 2010,330(6002):341-6.
[102]. Shaw DE, Grossman JP, Bank JA, Batson B, Butts JA, Chao JC, et al., editors. Anton 2: Raising the Bar for Performance and Programmability in a Special-Purpose Molecular Dynamics Supercomputer. SC14: International Conference for High Performance Computing, Networking, Storage and Analysis; 2014 16-21 Nov. 2014.
[103]. Hu X, Hong L, Dean Smith M, Neusius T, Cheng X, Smith Jeremy C. The dynamics of single protein molecules is non-equilibrium and self-similar over thirteen decades in time[J]. Nature Physics. 2015,12:171.
[104]. Li J. Exploring the Logic and Landscape of the Knowledge System: Multilevel Structures, Each Multiscaled with Complexity at the Mesoscale[J]. Engineering. 2016,2(3):276-85.
[105]. gov.cn/publish/portal0/tab88/info23556.htm. Available from: gov.cn/publish/portal0/tab88/info23556.htm.
[106]. 李静海，胡英，袁权. 探索介尺度科学：从新角度审视老问题[J]. 中国科学 B 辑化学. 2014(3):1-5.
[107]. Li J, Huang W. From Multiscale to Mesoscience: Addressing Mesoscales in Mesoregimes of Different Levels[J]. Annual Review of Chemical & Biomolecular Engineering. 2018,9(1).
[108]. Li J. Exploring the Logic and Landscape of the Knowledge System:Multilevel Structures, Each Multiscaled with Complexity at the Mesoscale[J]. 中国工程科学:英文版. 2016,2(3):276-85.