BJ Articles Ahead of Print

Intrinsically disordered proteins (IDPs) often play an important role in protein aggregation. However, it is challenging to determine the structures and interactions that drive the early stages of aggregation because they are transient and obscured in a heterogeneous mixture of disordered states. Even computational methods are limited, because the lack of ordered structure makes it difficult to ensure that the relevant conformations are sampled. We address these challenges by integrating atomistic simulations with high-resolution single-molecule measurements reported previously, using the measurements to help discern which parts of the disordered ensemble of structures in the simulations are most probable while using the simulations to identify residues and interactions that are important for oligomer stability.

Unlike double-stranded (ds)DNA, single-stranded (ss)RNA can be spontaneously packaged into spherical capsids by viral capsid protein because it is a more compact and flexible polymer. Many systematic investigations of this self-assembly process have been carried out using capsid protein from cowpea chlorotic mottle virus (CCMV), with a wide range of sequences and lengths of ssRNA. Among these studies are measurements of the relative packaging efficiencies of these RNAs into spherical capsids. In the present work we address a fundamental issue that has received very little attention, namely the question of the preferred curvature of the capsid formed around different RNA molecules.

Microtubules are multi-stranded polymers in eukaryotic cells that support key cellular functions such as chromosome segregation, motor-based cargo transport, and maintenance of cell polarity. Microtubules self-assemble via “dynamic instability,” where the dynamic plus ends switch stochastically between alternating phases of polymerization and depolymerization. A key question in the field is what are the atomistic origins of this switching, i.e. what is different between the GTP- and GDP-tubulin states that enables microtubule growth and shortening, respectively? More generally, a major challenge in biology is how to connect theoretical frameworks across length-time scales, from atoms to cellular behavior.

Glioblastoma is a primary malignant brain tumor characterized by highly infiltrative glioma cells. Vasculature and white matter tracts are considered to be the preferred and fastest routes for glioma invasion through brain tissue. In this study, we systematically quantified the routes and motility of the U251 human glioblastoma cell line in mouse brain slices by multimodal imaging. Specifically, we used polarization-sensitive optical coherence tomography to delineate nerve fiber tracts while confocal fluorescence microscopy was used to image cell migration and brain vasculature.

Towards the goal of understanding the pathophysiology of mild blast induced traumatic brain injury (bTBI), and identifying the physical forces associated with the primary injury phase, we developed a system that couples a pneumatic blast to a microfluidic channel to precisely and reproducibly deliver shear transients to dissociated human central nervous system (CNS) cells, on a time scale comparable to an explosive blast but with minimal pressure transients. Using fluorescent beads, we have characterized the shear transients experienced by the cells, and demonstrate that the system is capable of accurately and reproducibly delivering uniform shear transients with minimal pressure across the cell culture volume.

DNA damage caused by alkylating chemicals induces an adaptive response in Escherichia coli that increases the tolerance of cells to further damage. Signalling of the response occurs through irreversible methylation of the Ada protein which acts as a DNA repair protein and damage sensor. Methylated Ada induces its own gene expression through a positive feedback loop. However, random fluctuations in the abundance of Ada jeopardize the reliability of the induction signal. I developed a quantitative model to test how gene expression noise and feedback amplification affect the fidelity of the adaptive response.

The development of effective and safe therapies for scar-related ventricular tachycardias (VTs) requires a detailed understanding of the mechanisms underlying the conduction block that initiates electrical reentries associated with these arrhythmias. Conduction block has been often associated with electrophysiological changes that prolong action potential duration (APD) within the border zone (BZ) of chronically infarcted hearts. However, experimental evidence suggests that remodeling processes promoting conduction slowing as opposed to APD prolongation mark the chronic phase.

Ribonucleic acids are one of the most charged polyelectrolytes in nature, and understanding their electrostatics is fundamental to their structure and biological functions. An effective way to characterize the electrostatic field generated by nucleic acids is to quantify interactions between nucleic acids and ions that surround the molecules. These ions form a loosely associated cloud referred as an ion atmosphere. While theoretical and computational studies can describe the ion atmosphere around RNAs, benchmarks are needed to guide the development of these approaches and experiments to date that read out RNA-ion interactions are limited.

Understanding local conformations of DNA at the level of individual nucleic acid bases and base-pairs is important for elucidating molecular processes that depend on DNA sequence. Here we apply linear absorption and circular dichroism (CD) measurements to the study of local DNA conformations, using the guanine base analogue 6-methyl isoxanthopterin (6-MI) as a structural probe. We show that the spectroscopic properties of this probe can provide detailed information about average local base and base-pair conformations as a function of the surrounding DNA sequence.

The specific binding of ligands by proteins and the coupling of this process to conformational changes are fundamental to protein function. We designed a fluorescence-based single-molecule assay and data analysis procedure that allows the simultaneous real-time observation of ligand binding and conformational changes in FeuA. The substrate-binding protein FeuA binds the ligand ferri-bacillibactin and delivers it to the ABC importer FeuBC, which is involved in bacterial iron uptake. The conformational dynamics of FeuA was assessed via Förster resonance energy transfer (FRET), whereas the presence of the ligand was probed by fluorophore quenching.

The specific binding of ligands by proteins and the coupling of this process to conformational changes are fundamental to protein function. We designed a fluorescence-based single-molecule assay and data analysis procedure that allows the simultaneous real-time observation of ligand binding and conformational changes in FeuA. The substrate-binding protein FeuA binds the ligand ferri-bacillibactin and delivers it to the ABC importer FeuBC, which is involved in bacterial iron uptake. The conformational dynamics of FeuA was assessed via Förster resonance energy transfer (FRET), whereas the presence of the ligand was probed by fluorophore quenching.

We use Brownian-Langevin dynamics principles to derive a novel coarse-grained multiscale myofilament model that can describe the thin filament activation process during contraction. The model links atomistic molecular simulations of protein-protein interactions in the thin filament regulatory unit to sarcomere level activation dynamics. We first calculate the molecular interaction energy between tropomyosin and actin surface using Brownian dynamics simulations. This energy profile is then generalized to account for the observed tropomyosin transitions between its regulatory stable states.