How Nanopore Translocation Experiments Can Measure RNA Unfolding.

Electrokinetic translocation of biomolecules through solid-state nanopores represents a label-free single-molecule technique that may be used to measure biomolecular structure and dynamics. Recent investigations have attempted to distinguish individual transfer RNA (tRNA) species based on the associated pore translocation times, ion-current noise, and blockage currents. By manufacturing sufficiently smaller pores, each tRNA is required to undergo a deformation to translocate. Accordingly, differences in nanopore translocation times and distributions may be used to infer the mechanical properties of individual tRNA molecules. To bridge our understanding of tRNA structural dynamics and nanopore measurements, we apply molecular dynamics simulations using a simplified "structure-based" energetic model. Calculating the free-energy landscape for distinct tRNA species implicates transient unfolding of the terminal RNA helix during nanopore translocation. This provides a structural and energetic framework for interpreting current experiments, which can aid the design of methods for identifying macromolecules using nanopores.

Diffusion of tRNA inside the ribosome is position-dependent.

In recent years, there has been a growing interest to quantify the energy landscape that governs ribosome dynamics. However, in order to quantitatively integrate theoretical predictions and experimental measurements, it is essential that one has a detailed understanding of the associated diffusive properties. Here, all-atom explicit-solvent simulations (50 µs of aggregate sampling) predict that the diffusion coefficient of a tRNA molecule will depend on its position within the ribosome. Specifically, during aa-tRNA accommodation (i.e., the process by which tRNA enters the ribosome), the apparent diffusion coefficient decreases by approximately an order of magnitude. By comparing these to values obtained with an energetically "smooth" model, we show that the observed nonuniform behavior likely arises from electrostatic and solvation interactions between the tRNA and ribosome. These calculations also reveal the hierarchical character of ribosomal energetics, where steric interactions induce a large-scale free-energy barrier, and short-scale roughness determines the rate of diffusive movement across the landscape.

Using SMOG 2 to Simulate Complex Biomolecular Assemblies.

Over the last 20 years, the application of structure-based (Go-like) models has ranged from protein folding with coarse-grained models to all-atom representations of large-scale molecular assemblies. While there are many variants that may be employed, the common feature of these models is that some (or all) of the stabilizing energetic interactions are defined based on the knowledge of a particular experimentally obtained conformation. With the generality of this approach, there was a need for a versatile computational platform for designing and implementing this class of models. To this end, the SMOG 2 software package provides an easy-to-use interface, where the user has full control of the model parameters. This software allows the user to edit XML-formatted files in order to provide definitions of new structure-based models. SMOG 2 reads these "template" files and maps the interactions onto specific structures, which are provided in PDB format. The force field files produced by SMOG 2 may then be used to perform simulations with a variety of popular molecular dynamics suites. In this chapter, we describe some of the key features of the SMOG 2 package, while providing examples and strategies for applying these techniques to complex (often large-scale) molecular assemblies, such as the ribosome.

Nanopore-Based Measurements of Protein Size, Fluctuations, and Conformational Changes.

Proteins are structurally dynamic macromolecules, and it is challenging to quantify the conformational properties of their native state in solution. Nanopores can be efficient tools to study proteins in a solution environment. In this method, an electric field induces electrophoretic and/or electro-osmotic transport of protein molecules through a nanopore slightly larger than the protein molecule. High-bandwidth ion current measurement is used to detect the transit of each protein molecule. First, our measurements reveal a correlation between the mean current blockade amplitude and the radius of gyration for each protein. Next, we find a correlation between the shape of the current signal amplitude distributions and the protein fluctuation as obtained from molecular dynamics simulations. Further, the magnitude of the structural fluctuations, as probed by experiments and simulations, correlates with the ratio of α-helix to ß-sheet content. We highlight the resolution of our measurements by resolving two states of calmodulin, a canonical protein that undergoes a conformational change in response to calcium binding.