Multi-scale Studies of pH-mediated Viral Capsid Dynamics and Mechanics

智力精英病毒的头体是由一些蛋白质的多个拷贝组成的二十面体外壳。在病毒生命周期中，这些外壳在形状和大小上经历了重大变化——它们组装、成熟并最终释放其遗传物质。在许多病毒中，这一过程涉及ph介导的半径变化，有时包括形状变化，例如HK97，它膨胀并从高度圆形转变为高度多面形状。ph诱导的变化是由可电离侧链的质子化平衡和伴随的束缚离子的损失/增益引起的。虽然结构生物学，通过x射线晶体学和低温电镜，已经提供了稳定的结构状态和偶尔的中间产物的一瞥，但对负责ph诱导变化的特定残基或连接两种稳定状态的能量景观的细节知之甚少。这些研究旨在建立详细的原子水平的途径和自由能景观ph介导的形状/大小变化的病毒衣壳T数从3-7不等。从结构角度表征的三个系统：CCMV （T=3)， N（omega)V （T=4）和HK97 （T=7）将被检查，目的是：i）使用新的恒定pH分子动力学方法推导这些衣壳正常和膨胀状态的可电离残基的pKa值，ii）计算膨胀构象转变途径，iii)通过模拟方法推导pH依赖的自由能景观。由此产生的自由能途径将揭示促进或阻碍膨胀的关键相互作用，并进一步了解这一复杂过程背后的生物物理力。此外，由于人们对病毒衣壳的物理和材料特性越来越感兴趣，并且希望了解病毒生命周期中的这些特性，这将为从生物技术和材料科学到成像和单分子操作的应用提供信息，因此将开发一种新的多尺度计算方法来计算表征病毒衣壳的弹性模量，并将其应用于上述三个系统。这种结合弹性计算和ph依赖性转变途径的研究将阐明在ph介导的结构转变过程中衣壳的弹性特性，并将其与正在进行的单分子实验和有限元模型的发展联系起来。更广泛的影响：上述计算和模型开发将产生有关病毒生命周期中控制病毒衣壳膨胀和病毒衣壳形状变化的力量和过程的重要新信息。此外，计算的ph介导的自由能景观将为生物物理学家和物理病毒学家提供关键残基和相互作用的重要信息。景观采样和恒定pH MD的独特结合将为研究pH介导的构象转变建立新的方法，而一种新的多尺度方法的开发和应用将为单分子测量和有限元建模提供信息和扩展。由此产生的软件和模型将提供给仿真和建模社区。PI将参与学生和博士后研究人员培训讲习班的开发和教学，并对参与该项目的本科生进行研究培训。

Intellectual MeritViral capids are comprised of multiple copies of a few proteins organized as icosahedral shells. These shells undergo significant changes in shape and size during the virus life cycle - as they assemble, mature and ultimately release their genetic material. This process in a number of viruses involves pH-mediated changes in radius, and sometimes shape, e.g., as in HK97, which swells and transforms from a highly rounded to a highly faceted shape. The pH-induced changes arise from protonation equilibrium of ionizable side chains and the concomitant loss/gain of bound ions. While structural biology, via X-ray crystallography and cryo-EM, has provided glimpses of stable structural states and occasionally intermediates, little is known in detail about specific residues that are responsible for the pH-induced changes or the energetic landscape that connects the two stable states. These studies are directed toward establishing detailed atomic-level pathways and free energy landscapes for pH-mediated shape/size changes in viral capsids of T number varying from 3-7. Three systems that have been characterized from a structural standpoint: CCMV (T=3), N(omega)V (T=4) and HK97 (T=7) will be examined with the objective of: i) deriving pKa values of ionizable residues for the normal and swollen states of these capsids using novel constant pH molecular dynamics methods, ii) calculating the swelling conformational transition pathway, and iii) deriving the pH-dependent free energy landscapes through simulation methods. The resulting free energy pathways will reveal key interactions that facilitate or hinder swelling and further understanding of the biophysical forces that underlie this complex process. Additionally, driven by growing interest in the physical and material properties of viral capsids, and desire to understand these properties during the virus life cycle, which will inform applications from biotechnology and material science to imaging and single molecule manipulations, a new multi-scale computational method to calculate elastic moduli that characterize viral capsids will be developed and applied to the three systems noted above. This combination elasticity calculations and studies of pH-dependent transition pathways will elucidate the elastic character of the capsid during pH-mediated structural transitions, making connections to ongoing single molecule experiments and development of finite element models.Broader Impact:The calculations and model development described above will yield significant new information about the forces and processes that control virus capsid swelling and shape changes of viral capsids during the virus life-cycle. Furthermore, the calculated pH-mediated free energy landscapes will provide vital information on lynchpin residues and interactions for biophysicists and physical virologists. The unique combination of landscape sampling and constant pH MD will establish new approaches to study pH-mediated conformational transitions, and the development and application of a novel multi-scale approach will inform and extend single molecule measurements and finite element modeling. The resultant software and models will be made available to the simulation and modeling community.The PI will engage in the development and teaching of training workshops for students and postdoctoral researchers and also conduct research training for undergraduate students working on this project.