87 research outputs found
A New Approach to Low Temperature Embedding: Quick Freezing, Freeze-Drying and Direct Infiltration in Lowicryl K4M
Lowicryl resins are most commonly used for low temperature embedding by progressively lowering the temperature during dehydration. Freeze-substitution has also been successfully used with Lowicryl, but both of these techniques generally rely on chemical fixation and prolonged incubations in organic solvents. Freeze-drying may be combined with embedding in Lowicryl K4M. This technique eliminates all chemical fixation and exposure to organic solvents since the samples are quick-frozen, dried in vacuo and directly infiltrated in pure Lowicryl resin. If a primary aldehyde fixation is desired, freeze-drying may be used as an alternative to dehydration with organic solvents. These new approaches may be of significance for histochemistry and immunohistochemistry
Crowding Promotes the Switch from Hairpin to Pseudoknot Conformation in Human Telomerase RNA
Formation of a pseudoknot in the conserved RNA core domain in the
ribonucleoprotein human telomerase is required for function. In vitro
experiments show that the pseudoknot (PK) is in equilibrium with an extended
hairpin (HP) structure. We use molecular simulations of a coarse-grained model,
which reproduces most of the salient features of the experimental melting
profiles of PK and HP, to show that crowding enhances the stability of PK
relative to HP in the wild type and in a mutant associated with dyskeratosis
congenita. In monodisperse suspensions, small crowding particles increase the
stability of compact structures to a greater extent than larger crowders. If
the sizes of crowders in a binary mixture are smaller than the unfolded RNA,
the increase in melting temperature due to the two components is additive. In a
ternary mixture of crowders that are larger than the unfolded RNA, which mimics
the composition of ribosome, large enzyme complexes and proteins in E. coli,
the marginal increase in stability is entirely determined by the smallest
component. We predict that crowding can restore partially telomerase activity
in mutants, which dramatically decrease the PK stability.Comment: File "JACS_MAIN_archive_PDF_from_DOC.pdf" (PDF created from DOC)
contains the main text of the paper File JACS_SI_archive.tex + 7 figures are
the supplementary inf
Influence of Nanoparticle Size and Shape on Oligomer Formation of an Amyloidogenic Peptide
Understanding the influence of macromolecular crowding and nanoparticles on
the formation of in-register -sheets, the primary structural component
of amyloid fibrils, is a first step towards describing \emph{in vivo} protein
aggregation and interactions between synthetic materials and proteins. Using
all atom molecular simulations in implicit solvent we illustrate the effects of
nanoparticle size, shape, and volume fraction on oligomer formation of an
amyloidogenic peptide from the transthyretin protein. Surprisingly, we find
that inert spherical crowding particles destabilize in-register -sheets
formed by dimers while stabilizing -sheets comprised of trimers and
tetramers. As the radius of the nanoparticle increases crowding effects
decrease, implying smaller crowding particles have the largest influence on the
earliest amyloid species. We explain these results using a theory based on the
depletion effect. Finally, we show that spherocylindrical crowders destabilize
the ordered -sheet dimer to a greater extent than spherical crowders,
which underscores the influence of nanoparticle shape on protein aggregation
A Didactic Model of Macromolecular Crowding Effects on Protein Folding
A didactic model is presented to illustrate how the effect of macromolecular crowding on protein folding and association is modeled using current analytical theory and discrete molecular dynamics. While analytical treatments of crowding may consider the effect as a potential of average force acting to compress a polypeptide chain into a compact state, the use of simulations enables the presence of crowding reagents to be treated explicitly. Using an analytically solvable toy model for protein folding, an approximate statistical thermodynamic method is directly compared to simulation in order to gauge the effectiveness of current analytical crowding descriptions. Both methodologies are in quantitative agreement under most conditions, indication that both current theory and simulation methods are capable of recapitulating aspects of protein folding even by utilizing a simplistic protein model
Effects of macromolecular crowding on intracellular diffusion from a single particle perspective
Compared to biochemical reactions taking place in relatively well-defined aqueous solutions in vitro, the corresponding reactions happening in vivo occur in extremely complex environments containing only 60–70% water by volume, with the remainder consisting of an undefined array of bio-molecules. In a biological setting, such extremely complex and volume-occupied solution environments are termed ‘crowded’. Through a range of intermolecular forces and pseudo-forces, this complex background environment may cause biochemical reactions to behave differently to their in vitro counterparts. In this review, we seek to highlight how the complex background environment of the cell can affect the diffusion of substances within it. Engaging the subject from the perspective of a single particle’s motion, we place the focus of our review on two areas: (1) experimental procedures for conducting single particle tracking experiments within cells along with methods for extracting information from these experiments; (2) theoretical factors affecting the translational diffusion of single molecules within crowded two-dimensional membrane and three-dimensional solution environments. We conclude by discussing a number of recent publications relating to intracellular diffusion in light of the reviewed material
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