Self-Assembly Dynamics of Linear Virus-like Particles: Theory and - PowerPoint PPT Presentation

Self-Assembly Dynamics of Linear Virus-like Particles: Theory and Experiment TMV Model 200 nm VLP Paul van der Schoot www.virology.wisc.edu/virusworld

Collaborators Experiments Theory Armando Renko Daniela Melle Willem Hernandez-Garcia de Vries Kraft Punter Kegel Funding:

Simple viruses www.apsnet.org/edcenter/intropp/lessons/viruses/pages/tobaccomosaic.aspx CCMV TMV www.vcbio.science.ru.nl/en/fesem/tem/ 100 nm ss RNA coat protein ss RNA coat protein (3000 nts) (180 copies) (6400 nts) (2100 copies) deskeng.com/articles/aaahnd.htm www.sweetpics.site/t/tobacco-mosaic-virus-model.html

Random templated assembly? stacking  - 5 k B T binding  - 10 k B T enthalpy competition!  - 20 k B T T = 263 K [dT q ] = 0.25 mM [G]/[dT q ]= 40 templated theory theory assembly self-assembly Janssen et al. JACS 131 (2009), 1222. Jabbari et al. Macromol. 43 (2010), 5833.

Directional self-assembly of TMV PMV, CYMV, PRSV, TRV… 6400 nt OAS = packaging signal W 18 kDa 3’ 5’ coat protein cap lockwasher 34 cp bilayer disk OAS growing allosteric helical binding virus “threading” Lebeurier et al. PNAS 74 (1977), 149. http://www.rsc.org/ej/CS/2001 Chandrika et al. Virol 273 (2000) 198 Caspar Biophys J 32 (1980) 103 Koch et al. Beilstein J. Nanotechnol. 7 (2016) , 613.

Engineering directional assembly g < 0 → templated assembly random binding templated interaction → co -operativity assembly → self-assembly 𝜁 < 0 𝜁 < 0 directional → suppresses self-assembly switching templated assembly → co -operativity allostery ℎ > 0           mass action → * S exp g / P P P equilibrium statistical    T  P ( n ) stoichiometry → / distribution P mechanics        co-operativity → exp h rate        non-equilibrium k ( n ) k ( n ) exp[ g ( h ) ] P ( n , t )   n , 1 equations distribution Punter et al. J Phys Chem B 120 (2016), 6286.

The advantages of zipping… ∗ 𝑇 = 𝜚 𝑄 /𝜚 𝑄 mass action intermediates are suppressed 1.05 stoichiometry    T  / P sharp transition 1.02 1.00 co-operativity  0.98 longer template q binding affinity increases co-operativity Kraft et al. Biophys J 102 (2012), 2845. Punter et al. J Phys Chem B 120 (2016), 6286.

Zipper dynamics excess template q = 51 𝜏 = 0.007 𝑇 = 𝑓 2 overshoot! excess protein undershoot! overshoot! 𝜐 ≡ 𝑙 + 𝜚 𝑄 t Punter et al. J Phys Chem B 120 (2016), 6286.

Our model triblock coat protein C-S n -B TMV coat protein template binding basic block B: K 12 protein silk-like block S n : interaction (GAGAGAGQ) n template shielding protein 𝑜 ≥ 10 binding interaction 2.5 kbp 2.5 kbp dsDNA shielding dsDNA collagen-like block C: hydrophilic random coil (400 amino acids) 300 nm 200 nm Garcia-Hernandez et al. Nature Nano 9 (2014), 698.

Comparison with experiments λ = 0.101 λ = 0.134 C-S 10 -B C-S 14 -B theory: q = 417 C-S 10 -B C-S 14 -B TMV 500 nm ϵ + g (k B T) −17 −17 −17 h − ϵ (k B T) 6 3 7 k + (min −1 ) 4 × 10 9 4 × 10 9 3 × 10 8 200 nm 2.5 kDa DNA c DNA = 0.65 nM Punter et al. J Phys Chem B 120 (2016), 6286. Kraft et al. Biophys J 102 (2012), 2845.

Things get yet more complicated… polyfluorene: optomechanical proxy genome C-S 10 -B : 50 fraction taut  T = 0.06 m M  T = 0.6 m M Joris Sprakel 1 𝑔 + ≡ zipping 1 + 𝜇 excess protein! Garcia-Hernandez et al. Nature Nano 9 (2014), 698. Langmuir • parasitic self-assembly • co-assembly templates nucleation Cigil et al. J. Am. Chem. Soc. 139 (2017), 4962

Things get yet more complicated… polyfluorene: optomechanical proxy genome C-S 10 -B : Joris Sprakel fraction taut excess protein! FRET efficiency 𝑔 + = 0.1 𝑔 + = 0. 3 𝑔 + = 0.5 𝑔 + = 0.7 self-assembly co-assembly Cigil et al. J. Am. Chem. Soc. 139 (2017), 4962

Langmuir, Zipper & Micelle dynamics… coat protein micelle ⇄ ⇄ OAS Sander Kuipers ⇄ template Langmuir Zipper critical concentrations mass action aggregate sizes co-operativity       e    q 50 0 * 12 * 2 / e T , P , Z P P Z    q 5    e 0 . 2 * 11 M P , L    e * 9 P , M excess protein!

Langmuir, Zipper & Micelle dynamics… fraction packaged templates sites     e 9 k / k M Z  7 e  5 e Sander Kuipers Langmuir  3 e zipper    k L k / 1 Z dimensionless time Kuipers, bachelor thesis (Utrecht U, 2017)

Conclusions • Protein polymers can be designed to mimic coat proteins of linear viruses • Our model triblock protein co-polymer successfully encapsulates DNA • Allostery and directional assembly seem crucial ingredients • The kinetic zipper model describes the time evolution of the encapsulation of DNA • We predict over- & undershooting under conditions of excess DNA • Overshooting under conditions of excess of protein may occur in competition with micellisation