MOE Tier 3 Programme

MOETier3-interior

The dynamics of a biological system occurs at multiple time and length scales and are interconnected: from the jiggling of atoms (pico-seconds), the domain movements (micro- to milli-seconds), protein folding and diffusion (milli-second to seconds), binding, assembly, and translocation (seconds to minutes). Experimental knowledge of the motions occurring at these different time scales is mostly deduced by inference from static structures derived from kinetically stable states or from stand-alone kinematic and non-dynamical computational models. There is a critical gap in dynamic data from the inability of current methods to “see” the structure, especially at the nanometer and msec-sec scales.

The MoE Tier 3 programme: An integrated framework to study the dynamics of biological structures is designed to fill this gap, first by developing new experimental methods and computational approaches that can generate knowledge on the missing dynamical steps of fundamentally critical biological systems. Equally important is the second step, to develop a platform that allows for the integration of multi-scale dynamical data generated by modelers and experimentalists. Such a platform is essential for assimilating these disparate data into a conceptual framework. The framework is applied to a model biological system, the problem of infection by the Dengue virus (DENV). DENV infects approximately 100 million people worldwide annually and frequent outbreaks of dengue disease have been detected in Singapore.

An integrated framework to study the dynamics of biological structures

MOETier3-interior-pyramid

Objectives and milestones of the programme

The major achievements reported here highlight successes over the first two years and a half of funding for the programme.  The common thread uniting them is the consistent developments of new methods, both experimental and computational, to study the dynamics of DENV over a range of scales that was not previously accessible.  The report features the core services that have been set up, the methodological advancements that have been made, as well as the new insights that are provided on the DENV infection cycle.

Core technologies to study DENV

  • DENV production
  • SAXS/WAXS platform
  • Imaging platform
  • HDXMS platform

Methodological advancements

  • Better liquid cells for imaging proteins in room temperature liquid water
  • Better probes for electron microscopy
  • Better signal processing techniques for TEM
  • Better techniques for characterizing the ensemble of conformations available to a multiple domain protein
  • Improved techniques for the determination of kinetic parameters of minor states of proteins by NMR
  • Better coarse-grained models to study the dynamics of proteins
  • Improved fitting of small-angle and wide-angle X-ray scattering data to protein structures by explicit water modeling
  • Generation of a reliable, near-atomistic model of the DENV envelope for Molecular Dynamics studies
  • Better methods to study transition paths

Comparison to State of the Art in studying Multiscale Dynamics and Dengue virus

Many experimental groups in the world are working on the different serotypes of the Dengue virus, studying their structures using high-resolution techniques such as X-ray crystallography, NMR, or cryo-EM, their infection mechanisms, as well as their interactions with antibodies as a means to develop vaccine candidates.  Some of the most advanced laboratories in those fields are already directly affiliated to this programme or act as collaborators.   What is unique however in the setting of this programme is a comprehensive integration of these two approaches (experimental and computational), both for methodology developments and for applications.

Economic /societal benefits to Singapore

A possible short title for the programme is “From basic research to bedside”. Namely, the intended impact of the proposal is to improve the wellbeing of patients affected with Dengue Virus related diseases.  It is expected however that efforts put in reaching this goal will have broader impacts.

The programme has brought together a diverse interdisciplinary team of researchers from mathematics, core disciplines in imaging sciences, and virology.   This concentration of diverse talents and expertise provides a unique opportunity to train a new generation of graduate students and postdoctoral fellows.  The team is taking the necessary steps to engage students involved in experimental research to understand the processes by which their data are translated into knowledge and understanding.

Publications

  1.    Ng, X.W.; Bag, N.; Wohland, T. Characterization of Lipid and Cell Membrane Organization by the Fluorescence Correlation Spectroscopy Diffusion Law, CHIMIA 69 (3), 112-119.
  2.      Jan W Krieger, Anand P Singh, Nirmalya Bag, Christoph S Garbe, Timothy E Saunders, Joerg Langowski & Thorsten Wohland, Imaging fluorescence (cross-) correlation spectroscopy in live cells and organisms, Nature Protocols in press.
  3.      Nirmalya Bag, Shuangru Huang, and Thorsten Wohland, Plasma Membrane Organization of Epidermal Growth Factor Receptor in Resting and Ligand-Bound States, Biophys. J. in press.
  4.      Tay, M. Y. F., Saw, W. G., Zhao, Y., Chan, W. K. K., Singh, D, Chong, Y., Forwood, J. K., Ooi, E. E., Grüber, G., Lescar, J., Luo, D., and Vasudevan, S. G. (2015) The C-terminal 50 amino acid residues of Dengue NS3 protein are important for NS3-NS5 interaction and viral replication. J. Biol. Chem. 290, 2379-2394.
  5.     Saw, W. G., Tria, G., Grüber, A., Manimekalai, M. S. S., Zhao, Y., Chandramohan, A., Anand, G. S., Matsui, T., Weiss, T., Vasudevan, S. and Grüber, G. (2015) Structural insight and flexibility features of NS5 proteins from all four serotypes of Dengue virus in solution. Acta Crystallogr. D, D71, doi:10.1107/S1399004715017721.
  6.      Krishnamurthy, S., Tulsian, N., Chandramohan, A. and Anand, G.S. (2015) Parallel Allostery by cAMP and PDE Coordinates Activation and Termination Phases in cAMP Signaling Biophysical J. Sep 15;109(6):1251-63. doi: 10.1016/j.bpj.2015.06.067. Epub 2015 Aug 11.
  7.      Krishnamurthy, S., Moorthy, B.S., Lim XX, Lim XS, Bharatham, K, Tulsian, N, Mihalek, I and Anand, G.S. (2014) Active Site Coupling in PDE:PKA Complexes Promotes Resetting of Mammalian cAMP Signaling Biophysical J. Sep 16;107(6):1426-40. doi: 10.1016/j.bpj.2014.07.050.
  8.     Krishnamurthy S, Veesler D, Khayat R, Snijder J, Huang RK, Heck AJ , Johnson JE and Anand, G.S. (2014) Distinguishing Direct Binding Interactions from Allosteric Effects in the Protease – HK97 Prohead I d Domain Complex by Amide H/D Exchange Mass Spectrometry Bacteriophage 4:4, e959816.
  9.      Veesler D, Khayat R, Krishnamurthy S, Snijder J, Huang RK, Heck AJ, Anand, G.S., Johnson JE (2014) Architecture of a dsDNA Viral Capsid in Complex with Its Maturation Protease. Structure. 2014 Feb 4;22(2):230-7. doi: 10.1016/j.str.2013.11.007. Epub 2013 Dec 19.
  10.   Gupta G, Lim L, Song J. NMR and MD Studies Reveal That the Isolated Dengue NS3 Protease Is an Intrinsically Disordered Chymotrypsin Fold Which Absolutely Requests NS2B for Correct Folding and Functional Dynamics. PLoS One. 2015 Aug 10;10(8):e0134823.
  11.   Lim L, Shi J, Mu Y, Song J. Dynamically-driven enhancement of the catalytic machinery of the SARS 3C-like protease by the S284-T285-I286/A mutations on the extra domain. PLoS One. 2014 Jul 18;9(7):e101941.
  12.   Lim J, Xiao T, Fan J, Yang D. An off-pathway folding intermediate of an acyl carrier protein domain coexists with the folded and unfolded states under native conditions. Angew Chem Int Ed Engl. 2014, 53(9):2358-61.
  13.   Zhou Y, Yang D. Effects of J couplings and unobservable minor states on kinetics parameters extracted from CEST data. J Magn Reson. 2014, 249C:118-125.
  14.   Z. Fan, H. Ji and Z. Shen, Dual Gramian analysis: duality principle and unitary extension principle, Math Comp, 85, 239-270, 2014.
  15.   Q.  Jiang, Z. Shen, Tight wavelet frames in low dimensions with canonical filters, J. of Approx. Theory, 2015 accepted.
  16.   Z. Fan, A. Heinecke, Z. Shen, Duality for frames, Journal of Fourier Analysis and Applications, accepted, 2015.
  17.   Cai et al, Cine cone beam CT reconstruction using low-rank matrix factorization: algorithm and a proof-of-principle study, IEEE Transactions on Medical Imaging, 33(8), 2014.
  18.   Jiang and Shen, Tight wavelet frames in low dimensions with canonical filters, J. of Approx. Theory, 196 (C), 2015.
  19.   Fan et al, Dual Gramian analysis: duality principle and unitary extension principle, Math Comp, 85, 239-270, 2014.
  20.   Fan et al, Duality for frames, Journal of Fourier Analysis and Applications, accepted.
  21.   Dong et al, Multiscale representation of surfaces by tight wavelet frames with applications to denoising, Applied and Computational Harmonic Analysis, accepted.
  22.   H. Ji, Y. Luo and Z. Shen, Image recovery via geometrically structured approximation. Applied and Computational Harmonic Analysis, accepted.
  23.   Li et al, A re-weighted l2 method for image restoration with Poisson and mixed Poisson-Gaussian noise, Inverse Problem and Imaging, 9(3), 2015.
  24.   Cai, et al, Image restorations: a wavelet frame based model for piecewise smooth functions and beyond, Applied and computational harmonic analysis, accepted.
  25.   Xia et al. Identifying essential pairwise interactions in elastic network model using the alpha shape theory. J. Comp. Chem, 35, 1111 (2014).
  26.   Tong et al. Modeling solution X-ray scattering of biomacromolecules using explicit-solvent simulations and fast Fourier transfer. J. Appl. Cryst. accepted (2015).
  27.  J. Hass and P. Koehl. How round is a protein? Exploring protein structures for globularity using 
conformal mapping, Front. Mol. Biosci., 1, 26 (2014).
  28.   J. Hass and P. Koehl. A metric for genus-zero surfaces, arXiv:1507.00798, [math.DG] (2015).
  29.   Victor A. Kostyuchenko, Pau Ling Chew, Thiam-Seng Ng, Shee-Mei Lok (2014). Near-atomic resolution cryo-EM structure of Dengue serotype 4 virus. Journal of Virology. Vol 88: 477-482.
  30.   Guntur Fibriansah, Joanne L. Tan, Scott A. Smith, Ruklanthi de Alwis, Thiam-Seng Ng, Victor A. Kostyuchenko, Ramesh S. Jadi, Petra Kukkaro, Aravinda M. de Silva, James E. Crowe, Jr. and Shee-Mei Lok. (2015). A highly potent human antibody neutralizes dengue virus serotype 3 by binding across three surface proteins. Nature Communications. Vol 6:6341.
  31.   Guntur Fibriansah, Kristie D. Ibarra, Thiam-Seng Ng, Scott A. Smith, Joanne L. Tan, Xin-Ni Lim, Justin S. G. Ooi, Victor A. Kostyuchenko, Jiaqi Wang, Aravinda M. de Silva, Eva Harris, James E. Crowe, Jr. and Shee-Mei Lok. (2015). A human antibody locks dengue virus E protein dimers and prevents antibody-enhanced severe disease. Science.  (2015) 349:88-91. In press.

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