Date :

 12-13,15 October 2007

Place :

 Institute of Physics of Academia Sinica, Taipei

 National Center for Theoretical Sciences (Critical Phenomena and Complex Systems Focus Group)

 Institute of Physics of Academia Sinica (Taipei)

  Miss Chia-Chi Liu (Secretary, Physics Division, NCTS)
 Tel:(886)-2-33665566; Fax:(886)-2-33665565; E-mail: ccliu@phys.ntu.edu.tw

 Miss Shu-Min Yang (Assistant of LSCP, Institute of Physics, Academia Sinica)

 Tel: (886)-2-2782-2467, or (886)-2-27880058 ext. 6012; FAX: (886)-2-2782-2467; E-mail: shumin@phys.sinica.edu.tw

Speakers :

Prof. Bernard Brooks

National Institutes of Health, USA

E-mail: brb@mail.nih.gov

Multi-scale Methods for Macromolecular Systems in Computational Biophysics

    These presentations focus on our recent efforts to develop multi-scale macromolecular modeling and to apply them to problems of biological significance.  One objective in developing multi-scale modeling techniques is to be able to include multiple scale representations within a single study.  By combining scales, you can examine properties that would be difficult or too costly to examine with a single model.  Examples are presented for the following models:

Grid based map objects (EMAP)

Coarse-grained models using elastic normal modes (ENM) methods

Coarse-grained models using Hydrophibic/Hydrophibic/Neutral models (BLN)

Atomic models using a classical force field (CHARMM, Amber,…)

Models employing a quantum mechanical subsystem (CHARMM/Q-Chem,…)

Examples of methods employing these models include:

Structural analysis via single particle electron tomography

Protein-protein docking with grid based methods

Quantitative coupling between global and local dynamics

Chaperonin mediated protein folding

Vibrational free energy partitioning

Pathways interpolation between two ENM structures

Structure and dynamics of lipid bilayers

Examining protein folding in confinement

Examining reaction profiles using QM/MM methods

1. X. Wu and B. R. Brooks, “Modeling of Macromolecular assemblies with map objects,” Proceedings of the 2007 International Conference on Bioinformatics and Computational Biology Vol. II: 411-417 (2007).

2. J. L. S. Milne, X. W. Wu, M. J. Borgnia, J. S. Lengyel, B. R. Brooks, D. Shi, R. N. Perham and S. Subramaniam. "Molecular structure of a 9-MDa icosahedral pyruvate dehydrogenase subcomplex containing the E2 and E3 enzymes using cryoelectron microscopy," J. Biol. Chem., 281 (7): 4364-4370 (2006).

3. W. J. Zheng and B. R. Brooks. "Modeling protein conformational changes by iterative fitting of distance constraints using reoriented normal modes," Biophys. J., 90 (12): 4327-4336 (2006).

4. Zheng, W., Brooks, B.R. and Hummer, G. “Protein conformational transitions explored by mixed elastic network models,” Proteins. 69, 43-57 (2007).

5. J. B. Klauda, B. R. Brooks and R. W. Pastor. "Dynamical motions of lipids and a finite size effect in simulations of bilayers," Journal of Chemical Physics, 125 (14): (2006).

6. H. L. Woodcock, M. Hodoscek, A. T. B. Gilbert, P. M. W. Gill, H. F. Schaefer and B. R. Brooks. "Interfacing Q-chem and CHARMM to perform QM/MM reaction path calculations," Journal of Computational Chemistry, 28 (9): 1485-1502 (2007).

7. H. L. Woodcock, M. Hodoscek and B. R. Brooks. "Exploring SCC-DFTB paths for mapping QM/MM reaction mechanisms," Journal of Physical Chemistry a, 111 (26): 5720-5728 (2007).

8. G. Stan, G. H. Lorimer, D. Thirumalai and B. R. Brooks. "Coupling between allosteric transitions in GroEL and assisted folding of a substrate protein," Proc. Natl. Acad. Sci., 104 (21): 8803-8808 (2007).

Prof. Chun-Jung Chen

National Synchrotron Radiation Research Center, Hsinchu, TAIWAN

E-mail: cjchen@nsrrc.org.tw

Synchrotron Protein Crystallography on Structural Biology

    Prediction and knowledge of protein functions directly from the corresponding primary gene sequences is one the most tasks and ultimate goals of the genomics and proteomics study.  Three-dimensional structures of biological macromolecules provide valuable information to help us understand their structure-function relationships and mechanisms.  Since the function of a gene product is tightly coupled to its three-dimensional structure, determining the structure, or its folding pattern, may provide important insight into its biochemical function, which, in turn, may help to place it in a particular cellular pathway.  The high-throughput protein structure determination of a great quantity usually presents the success indication in the challenging context of structural genomics studies.  Among those different approaches, protein crystallography has continued to be the dominant method of determining the three-dimensional structures of increasingly larger and more complex molecular problems in the field of structural biology and genomics.  Moreover, in recent years, synchrotron radiation protein crystallography is becoming an increasingly important knowledge in the development of new therapeutic agents a broad range of diseases and structure-based drug designs.  The current status of the protein crystallography facilities at NSRRC and the recent research applications of determined protein structures will be reported and discussed.

Dr. Rita P.-Y. Chen

Institute of Biological Chemistry, Academia Sinica, Taipei, TAIWAN

E-mail: pyc@gate.sinica.edu.tw

Amyloids: Regarding Alzheimer Disease and Prion Diseases

    The common characteristic of prion diseases and Alzheimer’s disease is the formation of amyloid plaque in brain. Amyloid is a special structural form composed of cross β-sheet structure. Compared with the prion protein, the major components of the amyloid in Alzheimer’s disease, Aβ40 and Aβ42 peptides, is much more prone to aggregate into the amyloid. Here, we use Aβ40 peptide as our studying system and try to explore how sequence determines the amyloid formation and why the polypeptide chain tends to associate into amyloid fibril rather than other β-sheet structures. The D-form proline (^DP) has been widely used in designed peptide as the ^DP-G sequence tends to form a type II’ β-turn which is the favorite turn type in the formation of β-hairpin. We substituted each amino acid residue in front of the Gly residue of the Aβ40 peptide to D-form proline individually to create four peptides containing the ^DP-G sequence at different positions. The resulting peptides were synthesized and named S8P, V24P, I32P, and V36P, according to their mutation sites. Interestingly, we found that V24P, I32P, and V36P could still go through a coil-to-β structural conversion. However, this structural conversion is peptide concentration dependent and can be reversed by simple dilution. The formed β–sheet-rich structure is not amyloid fibrils but shows Thioflavin-T (ThT) and Congo-Red (CR) binding ability, uncoupling the ThT and CR binding ability with the amyloid fibril formation.
    Unlike Alzheimer’s disease, prion diseases are transmissible. The amyloids formed in the patients of prion diseases are infectious. It has been known for a long time that there is a “species-barrier” for prion disease transmission among different species and that the barrier is related to the sequence homology between the transmission material and the host. However, it is difficult to estimate and compare the transmission barrier and different results are usually obtained. Here we developed a seed titration method and used synthetic peptides as a simple system to evaluate the sequence-dependent seeding efficiency between hamster and mouse prions[1]. We found that the heterologous seeding efficiency of hamster and mouse prion peptides was four times less than that of homologous seeding. Moreover, residue 139 was not the only residue in determining seeding efficiency. When the seed had Ile at this position, the homology at this position between seed and monomer determined the seeding efficiency. When the seed had Met at this position, homology at residues 109 and 112 determined the seeding efficiency. We argue that the binding surface of the seed is different for different prion peptides, which depends on the conformation of the peptide in its “β-precursor state” (the conformation that allows the peptide to form nuclei). Different amino acid sequences at position 139 lead to different binding surfaces, consistent with different fibril morphologies of the formed amyloid fibrils published previously.

References:

1. L. Y. Lee and R. P. Chen, Quantifying the Sequence-Dependent Species Barrier between Hamster and Mouse Prions, J Am Chem Soc 129, 1644-1652 (2007).

Dr. Yun-Ru Chen

The Genomics Research Center, Academia Sinica, Taipei, TAIWAN

E-mail: yrchen@gate.sinica.edu.tw

Equilibrium Folding and Aggregation of Wild Type and Familial Mutants of Amyloid Beta in Alzheimer’s Disease

    The amyloidogenic protein, amyloid ß peptide (Aß) composed of 40 or 42 amino acids is a critical component in the etiology of the neurodegenerative Alzheimer’s disease.  Aß is prone to aggregate and forms amyloid fibrils progressively both in vitro and in vivo.  To understand the process of amyloidogenesis, it is pivotal to examine the initial stages of the folding process.  We examined the equilibrium folding properties, assembly states and stabilities of the early folding stages of Aß40 and 42.  We found that Aß40 and 42 have different conformations and assembly states upon refolding from their unfolded ensembles.  Aß40 is predominantly an unstable and collapsed monomeric species, whereas, Aß42 populates a stable structured trimer/tetrameric species at concentrations above approximately 12.5 mM.  Thermodynamic analysis showed that the free energy of Aß40 monomer and 42 timer/tetramer are ~1.1 and ~15/22 kcal/mol, respectively while adopting two-state mechanism.  A three-state model for Aβ42 with the presence of monomer is also suggested and may explain the related fibrillization patterns.  Subsequent studies on familiar Aß40 mutants including Artic (E22G), Dutch (E22Q), Italian (E22K), Flemish (A21G), and Iowa (D23N) show that their stability and mechanism are different especially the mutants at the 22 position.  They are more stable than the wild type.  Our results show that the amyloidogenic folded structure of Aß, although considered as natively unfolded protein, is important for the formation of spherical ß oligomeric species.  However, Aß oligomers are not an obligatory intermediate in the process of fibril formation because oligomerization is inhibited at concentrations of urea that have no effect on fibril formation.  The distinct initial folding properties of Aß isomers and the mutants may play an important role in the higher aggregation potential and pathological significance.

Dr. Maciej Dlugosz

Interdisciplinary Centre for Mathematical and Computational Modelling, University of Warsaw, POLAND

E-mail:

1. Constant-pH Molecular Dynamics

    Most biomolecules contain titratable groups i.e., residues which are capable of exchanging protons with their environment. These proton transfer phenomena lead to changes in ionization states of titratable residues and cause modification of charge distribution within molecules. The equilibria of these processes are strongly pH-dependent. Therefore, in the studies of molecular dynamics of biomolecules it is of crucial importance to reliably represent ionization states of titratable groups.

    I will present a method to perform molecular dynamics simulation of solvated molecules which explicitly treats protonation equilibria of their titratable groups at a given pH of the solution. I will also present applications of this constant-pH molecular dynamics algorithm for investigating relationships between protonation and conformation equilibria in small organic compounds.

2. Association of Aminoglycosidic Antibiotics with the Ribosomal A-site.  Brownian Dynamics Simulations

    Aminoglycosidic antibiotics interfere with translation by binding to the tRNA decoding A-site of the small ribosomal subunit. They are positively charged ligands composed of 2 to 4 modified amine sugar rings. Studied antibiotics include neamine, neomycin, ribostamycin and paromomycin. Brownian dynamics methodology was applied to simulate the diffusion and association of these aminoglycosides with the ribosomal A-site. Brownian dynamics simulations provided theoretical estimates of the rates of aminoglycosidic association as well as their reactive trajectories.  I will present the diffusion limited rates of association of aminoglycosides with the ribosomal A-site RNA and their dependence on ionic strength of the surrounding. The influence of structural, electrostatic and hydrodynamic properties of antibiotics on the kinetics of their encounter with the ribosomal RNA will be discussed. The mechanism of diffusion towards RNA leading to the formation of the encounter complex will be also presented.

Prof. Ya-Wei Hsueh

Department of Physics, National Central University, TAIWAN

E-mail: yhsueh@phy.ncu.edu.tw

The Effect of Sterol on Lipid Membranes

    Lipids in biological membranes form domains having distinct physical properties.  Defined mixtures of lipids and sterols are of interest to ascertain the fundamental interactions governing these lipids in the absence of other cell membrane components.  We investigate the effect of sterol on the physical properties of phosphoethanolamine membranes using deuterium nuclear magnetic resonance (2H NMR).  In this talk, I will give a brief introduction on the application of 2H NMR to biological systems. We will show that a small modification in sterol molecule structure has profound effect on lipid organization and phase behavior.

Prof. Kensei Kobayashi

Graduate School of Engineering, Yokohama National University, JAPAN

E-mail: kkensei@ynu.ac.jp

Chemical Evolution from Complex Organics in Space to Life

    Complex organic compounds were found in carbonaceous chondrites and comets, and their roles in the generation of life in the primitive Earth have been discussed.  It is suggested that these organic compounds were originally formed in interstellar dust particles in molecular clouds.  Several laboratory simulation experiments have shown that amino acid precursors can be formed in simulated molecular clouds environments by the action of cosmic rays or ultraviolet light.  When a mixture of methanol (or carbon monoxide), ammonia and water was irradiated with high-energy heavy ions, complex organic compounds with large molecular weights were formed, which gave a wide variety of amino acids after acid hydrolysis.  Thus we can say that complex amino acid precursors can be formed in interstellar environments.

    Amino acids synthesized abiotically were generally racemic mixtures.  Cronin and Pizzarello reported that some amino acids found in carbonaceous chondrites.  One of the proposed mechanisms to give enantiomeric excess of amino acids is asymmetric formation / destruction of amino acids by circular-polarized light from neutron stars.  We irradiated the “complex amino acids precursors” synthesized in simulated interstellar environments with circular-polarized ultraviolet light from a synchrotron.  After acid hydrolysis of the products, enantiomeric excesses of alanine were detected. 

    I will propose a possible scenario of chemical evolution from complex organic compounds in interstellar dust particles to the first life on the Earth or elsewhere.

References:

1. "Formation of Organic Compounds in Simulated Interstellar Media with High Energy Particles," by T. Kasamatsu, T. Kaneko, T. Saito and K. Kobayashi, Bull. Chem. Soc. Jpn., 70, 1021-1026 (1997).

2. "Amino Acid Formation in Gas Mixtures by Particle irradiation," by K. Kobayashi, T. Kaneko, T. Saito and T. Oshima, Origins Life Evol. Biosphere, 28, 155-165 (1998).

3. "Characterization of Complex Organic Compounds Formed in Simulated Planetary Atmospheres," by K. Kobayashi, T. Kaneko and T. Saito, Adv. Space Res., 24, 461-464 (1999).

4. "Formation of Bioorganic Compounds in Simulated Planetary Atmospheres by High Energy Particles or Photons," by K. Kobayashi, H. Masuda, K. Ushio, K. Ohashi, H. Yamanashi, T. Kaneko, J. Takahashi, T. Hosokawa, H. Hashimoto and T. Saito, Adv. Space Res., 27, 207-215 (2001).

5. "Prebiotic Synthesis from CO Atmosphere: Implications for the Origins of Life," by S. Miyakawa, H. Yamanashi, K. Kobayashi, H. J. Cleaves and S. L. Miller, Proc. Nat. Acad. Sci., USA, 99, 14628-14631 (2002).

6. "Pyrolysis of high-molecular-weight complex organics synthesized from simulated interstellar gas mixture irradiated with 3 MeV proton irradiation," by Y. Takano, T. Tsuboi, T. Kaneko, K. Kobayashi, and K. Marumo. Bull. Chem. Soc. Jpn., 77, 779-783 (2004).

7. "Experimental Verification of Photostability for Free and Bound Amino Acids Exposed to g-rays and UV Irradiation, "  by Y. Takano, T. Kaneko, K. Kobayashi, D. Hiroishi, H. Ikeda, Earth Planets Space, 56, 669-704 (2004).

8. " Pyrolysis of Complex Organics Following High-Energy Proton Irradiation of Simple Inorganic Gas Mixture, "　by Y. Takano, K. Marumo, S. Yabashi, T. Kaneko, and K. Kobayashi,  Appl. Phys. Lett., 85, 1633-1635 (2004).

9. "Photochemical Abiotic Synthesis of Amino-Acid Precursors from Simulated Planetary Atmospheres by Vacuum Ultraviolet Light,” by J. Takahashi, H. Masuda, T. Kaneko, K. Kobayashi, T. Saito and T. Hosokawa, J. Appl. Phys., 98, 024907-1-6 (2005).

10. “Asymmetric Synthesis of Amino Acid Precursors in Interstellar Complex Organics by Circularly Polarized Light," by Y. Takano, J. Takahashi, T. Kaneko, and K. Marumo and K. Kobayashi, Earth Planet. Sci. Lett. 254, 106-114 (2007).

Prof. Wei Lee

Department of Physics, Chung-Yuan Christian University, TAIWAN

E-mail: wlee@phys.cycu.edu.tw

Origin of the Hydrocarbons of Carbonaceous Chondrites and Interplanetary Dust Particles

    A 20-year-old mystery surrounding unidentified matter emitting certain wavelengths of the infrared in deep space was solved about one and a half decades ago by some laboratory astrophysicists.  The “unidentified” infrared (known as the UIR) bands are emitted from unexpectedly complex molecules, called polycyclic aromatic hydrocarbons (PAHs), spread throughout space.  In this talk I will provide some basic astrophysics background and introduce such “unexpectedly” large organic molecules, with 20–100 atoms, of astrophysical interest.  Also, implied by the results from a plasma-discharge experiment mimicking the environment of the early solar nebula, the chemical pathway, from interstellar PAHs to the formation of hydrocarbon components ubiquitously found in the solar system materials such as carbonaceous meteorites and interplanetary dust particles, will be suggested.

Dr. Karen Petrosyan

Institute of Physics, Academia Sinica, TAIWAN

E-mail: pkaren@phys.sinica.edu.tw

Protein-Mediated Loops and Phase Transition in Nonthermal Denaturation of DNA

Dr. Brigita Urbanc

Physics Department, Boston University, USA

E-mail: brigita@bu.edu

1. Ab Initio Discrete Molecular Dynamics Approach to Studies of Protein Folding and Assembly

    Understanding the toxicity of amyloidogenic protein aggregates and designing therapeutic approaches require the knowledge of their structure at atomic resolution. While solid state NMR, X-ray diffraction, and other experimental techniques are capable of discerning the protein fibrillar structure, determining the structures of early aggregates, called oligomers, is a challenging experimental task.  Computational studies by all-atom molecular dynamics, which provides a complete description of a protein in the solvent, are typically limited to study folding of smaller proteins or aggregation of a small number of short protein fragments.
    I will present an efficient ab initio computer simulation approach to protein folding and aggregation using discrete molecular dynamics (DMD) in combination two coarse-grained protein models and implicit solvent. This approach involves different complexity levels in both the protein model and the interparticle interactions. Starting from the simplest protein model with minimal interactions, and gradually increasing its complexity, while guided by in vitro findings, one can systematically select the key features of the protein model and interactions that drive protein folding and aggregation. Because the methodology employed in this DMD approach does not require any knowledge of the native or any other state of the protein, it can be applied to study degenerative disorders associated with protein misfolding and aberrant protein aggregation.
    The choice of the coarse-grained model depends on the complexity of the protein and specific questions to be addressed, which are mostly suggested by in vitro findings.  Thus, I will illustrate the DMD approach on amyloid beta-protein associated with Alzheimer's disease. Despite simplifications introduced in the DMD approach, the predicted Abeta conformations are in agreement with existing experimental data. The in silico findings also provide further insights into the structure and dynamics of Abeta folding and oligomer formation that are amenable to in vitro testing.
　

2. Oligomer Formation of Alzheimer's Amyloid β Proteins Aβ40 and Aβ42

    Alzheimer's disease (AD) is a progressive brain disorder, clinically characterized by the accumulation of extracellular amyloid deposits composed of amyloid β-protein (Aβ), intracellular neurofibrillary tangles, and neuronal loss.  Pathological folding and oligomer formation of Aβ are widely perceived as central to AD. Thus, elucidating Aβ oligomer structure and the mechanisms of their formation is critical for developing therapeutic agents. I will address the differences between the two most common Aβ alloforms, Aβ40 and Aβ42, which oligomerize differently in vitro. I will present a discrete molecular dynamics study of oligomerization of Aβ40 and Aβ42, using an intermediate-resolution protein model with up to four beads per amino acid. This four-bead model incorporates backbone hydrogen bond interactions and amino acid-specific interactions mediated through hydrophobic and hydrophilic elements of the side chains. During the simulations we observe monomer folding and aggregation of monomers into oligomers of variable sizes. Aβ40 forms significantly more dimers than Aβ42, while pentamers are significantly more abundant in Aβ42 relative to Aβ40. Structure analysis reveals a turn centered at Gly37-Gly38 that is present in a folded Aβ42 monomer but not in a folded Aβ40 monomer and is associated with the first contacts that form during monomer folding. Our results suggest that this turn plays an important role in Aβ42 oligomer formation. Aβ pentamers have a globular structure comprising hydrophobic residues within the pentamer's core and hydrophilic N-terminal residues at the pentamer's surface. The N-termini of Aβ40 pentamers are more spatially restricted than Aβ42 pentamers. Aβ40 pentamers form a β-strand structure involving Ala2-Phe4, which is absent in Aβ42 pentamers.  These structural differences imply a different degree of hydrophobic core exposure between pentamers of the two alloforms, with the Aβ42 pentamer's hydrophobic core being more exposed and thus more prone to form larger oligomers.
    I will discuss how the presence of electrostatic interactions (EIs) between pairs of charged amino acids affects Aβ40 and Aβ42 oligomer formation. Our results indicate that EIs promote formation of larger oligomers in both Aβ40 and Aβ42.  Both Aβ40 and Aβ42 display a peak at trimers/tetramers, but Aβ42 displays additional peaks at nonamers and tetradecamers.  EIs thus shift the oligomer size distributions to larger oligomers. Nonetheless, the Aβ40 size distribution remains unimodal, whereas the Aβ42 distribution is trimodal, as observed experimentally. We show that structural differences between Aβ40 and Aβ42 that already appear in the monomer folding, are not affected by EIs. Aβ42 folded structure is characterized by a turn in the C-terminus that is not present in Aβ40. We show that the same C-terminal region is also responsible for the strongest intermolecular contacts in Aβ42 pentamers and larger oligomers. Our results indicate that this C-terminal region plays a key role in the formation of Aβ42 oligomers and the relative importance of this region increases in the presence of EIs. Subsequently, inhibitors targeting the C-terminal region of Aβ42 oligomers may be able to prevent oligomer formation or structurally modify the assemblies to reduce their toxicity.
　

3. Amyloid β Protein Assembly in the Presence of Peptide Inhibitors

    The studies presented in the Talks 1 and 2 suggest that the modeling paradigm presented above can be used to examine the effect of inhibitors on Aβ oligomerization. I will present the application of the DMD approach to study Aβ42 assembly in the presence of C-terminal fragments (CTFs) derived from Aβ42, which have been shown to inhibit Aβ42 neurotoxicity and/or oligomerization in vitro. Consistent with dynamic light scattering (DLS) data, our results show that CTFs associate with Aβ42 to form heterooligomers. Contact map analysis demonstrates that upon heterooligomerization, CTFs induce partial Aβ42 unfolding in the N-terminal region but have little effect on the conformation in the hydrophobic C-terminal region. As the CTF/Aβ42 concentration ratio increases, CTFs ultimately prevent all intermolecular contacts among Aβ42 molecules in heterooligomers. The structures of the Aβ42/CTF heterooligomers are different from those of pure Aβ42 oligomers, providing an explanation to the inhibition of Aβ42 toxicity by CTFs. Examination of the solvent accessible surface area (SASA) per amino acid in Aβ42 within heterooligomers shows that in the presence of CTFs, the N-terminal region of Aβ42, D1-D7, is substantially less exposed to the solvent than the same region in Aβ42 oligomers formed in the absence of CTFs. The solvent exposure of Aβ42 within heterooligomers shows a remarkable resemblance to Aβ40 oligomers formed in the absence of CTFs. These findings suggest that the D1-D7 region in Aβ42 oligomers may be involved in mediating oligomer neurotoxicity.