The folding mechanisms of proteins are increasingly being probed through single-molecule experiments in which the protein is immobilized on a surface. Nevertheless, a clear understanding of how the surface might affect folding, and whether or not it changes folding from its bulk behavior, is lacking. In this work, we use molecular dynamics simulations of a model -barrel protein tethered to a surface to systematically investigate how the surface impacts folding. In the bulk, this protein folds in a three-state manner through a compact intermediate state, and its transition state (TS) has a well formed hydrophobic core. Upon tethering, we find that folding rates and stability are impacted differently by the surface, with dependencies on both the length and location of the tether. Significant changes in folding times are observed for tether points that do not alter the folding temperature. Tethering also locally enhances the formation of structure for residues proximal to the tether point. We find that neither the folding mechanism nor the TS of this protein are altered if the tether is in a fully structured or completely unstructured region of the TS. By contrast, tethering in a partially structured region of the TS leads to dramatic changes. For one such tether point, the intermediate present in bulk folding is eliminated, leading to a two-state folding process with a heterogeneous, highly unstructured TS ensemble. These results have implications for both the design of single-molecule experiments and biotechnological applications of tethered proteins. molecular dynamics simulations ͉ protein-surface interactions ͉ single-molecule spectroscopy S ingle-molecule spectroscopy has recently emerged as a powerful technique for watching individual proteins fold (1-3). By attaching donor and acceptor dyes to key residues of a protein, fluorescence resonance energy transfer (FRET), already successful in ensemble folding experiments (4), can be used as a distance probe to monitor individual folding pathways. There are several different experimental techniques for doing FRET-based single-molecule experiments on proteins, each with distinct advantages and challenges. Bulk experiments use a focusing laser beam that monitors folding as proteins diffuse freely through the area illuminated by the laser (5, 6). Although this approach is advantageous in that the protein is allowed to fold in a relatively nondisrupted manner, solution experiments are diffusion limited and cannot examine slower (տ10 ms) phenomena (2). Proteins enclosed in surface-tethered vesicles allow observations on a more spatially localized scale than the bulk but do not allow for the rapid exchange of buffer conditions or the use of extreme denaturing environments (7). By immobilizing them directly on a surface (8), proteins can be observed in both a spatially localized region and over longer time scales than those accessible in diffusion-limited experiments. Despite these advantages, the folding behavior of surface-tethered proteins also may be influenced by ...
Males and females exhibit several differences in brain structure and function. To examine the basis for these sex differences, we investigated the influences of sex hormones and sex chromosomes on brain structure and function in mice. We used the Four Core Genotype (4CG) mice, which can generate both male and female mice with XX or XY sex chromosome complement, allowing the decoupling of sex chromosomes from hormonal milieu. To examine whole brain structure, high-resolution ex vivo MRI was performed, and to assess differences in cognitive function, mice were trained on a radial arm maze. Voxel-wise and volumetric analyses of MRI data uncovered a striking independence of hormonal versus chromosomal influences in 30 sexually dimorphic brain regions. For example, the bed nucleus of the stria terminalis and the parieto-temporal lobe of the cerebral cortex displayed steroid-dependence while the cerebellar cortex, corpus callosum, and olfactory bulbs were influenced by sex chromosomes. Spatial learning and memory demonstrated strict hormone-dependency with no apparent influence of sex chromosomes. Understanding the influences of chromosomes and hormones on brain structure and function is important for understanding sex differences in brain structure and function, an endeavor that has eventual implications for understanding sex biases observed in the prevalence of psychiatric disorders.
The widespread use of the mouse as a model system to study brain development has created the need for noninvasive neuroimaging methods that can be applied to early postnatal mice. The goal of this study was to optimize in vivo three-(3D) and four-dimensional (4D) manganese (Mn)-enhanced MRI (MEMRI) approaches for acquiring and analyzing data from the developing mouse brain. The combination of custom, stage-dependent holders and self-gated (motion-correcting) 3D MRI sequences enabled acquisition of high-resolution (100-µm isotropic), motion artifact-free brain images with a high level of contrast due to Mn-enhancement of numerous brain regions and nuclei. We acquired high-quality longitudinal brain images from two groups of FVB/N strain mice, six mice per group, each mouse imaged on alternate odd or even days (6 3D MEMRI images at each day) covering the developmental stages between postnatal days 1 to 11. The effects of Mn-exposure, anesthesia and MRI were assessed, showing small but significant transient effects on body weight and brain volume, which recovered with time and did not result in significant morphological differences when compared to controls. Metrics derived from deformation-based morphometry (DBM) were used for quantitative analysis of changes in volume, position and signal intensity of a number of brain regions. The cerebellum, a brain region undergoing significant changes in size and patterning at early postnatal stages, was analyzed in detail to demonstrate the spatiotemporal characterization made possible by this new atlas of mouse brain development. These results show that MEMRI is a powerful tool for quantitative analysis of mouse brain development, with great potential for in vivo phenotype analysis in mouse models of neurodevelopmental diseases.
Using neuroimaging technologies to elucidate the relationship between genotype and phenotype and brain and behavior will be a key contribution to biomedical research in the twenty-first century. Among the many methods for analyzing neuroimaging data, image registration deserves particular attention due to its wide range of applications. Finding strategies to register together many images and analyze the differences between them can be a challenge, particularly given that different experimental designs require different registration strategies. Moreover, writing software that can handle different types of image registration pipelines in a flexible, reusable and extensible way can be challenging. In response to this challenge, we have created Pydpiper, a neuroimaging registration toolkit written in Python. Pydpiper is an open-source, freely available software package that provides multiple modules for various image registration applications. Pydpiper offers five key innovations. Specifically: (1) a robust file handling class that allows access to outputs from all stages of registration at any point in the pipeline; (2) the ability of the framework to eliminate duplicate stages; (3) reusable, easy to subclass modules; (4) a development toolkit written for non-developers; (5) four complete applications that run complex image registration pipelines “out-of-the-box.” In this paper, we will discuss both the general Pydpiper framework and the various ways in which component modules can be pieced together to easily create new registration pipelines. This will include a discussion of the core principles motivating code development and a comparison of Pydpiper with other available toolkits. We also provide a comprehensive, line-by-line example to orient users with limited programming knowledge and highlight some of the most useful features of Pydpiper. In addition, we will present the four current applications of the code.
We report the results of a molecular dynamics (MD) study of the effects of confinement and molecular crowding on the thermodynamics and kinetics of folding of a 46 residue off-lattice minimalist β-barrel protein. Crowding was mimicked by restricting the protein to a sphere with a soft well repulsive potential. MD simulations were performed on the protein in an unconfined environment, as well as confined to spheres of two different radii, 5.88σ and 11.76σ. Here, σ is the bond length between two adjacent residues and the radius of gyration of the protein in its native state is 2.87σ. We find that for the larger sphere (11.76σ), the folding and collapse temperatures are virtually unchanged from their bulk values, but the average folding time is decreased by 35%. By contrast, the smaller sphere has a much more significant thermodynamic effect (the folding temperature is raised by 28%) but the average folding time is only decreased by 58%. Confinement is also seen to restrict the conformational space accessible to the protein in its denatured state. In addition, confinement appears to change the folding mechanism for this protein, as long-lived intermediates present in the bulk are both modified and have shorter lifetimes when the protein is confined.
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