1,721,001 research outputs found
A fit-less approach to the elasticity of the handles in optical tweezers experiments
Full text linkThe elastic properties of the double-stranded DNA handles used in optical
tweezers experiments on biomolecules are customarily modeled by an extensible
worm-like chain model. Fitting such model to experimental data however is no
trivial task, as the function depends on four parameters in a highly non-linear
fashion. We hereby propose a method to bypass the fitting procedure and obtain
an empirical force vs. extension curve that accurately reproduce the elasticity
of the handles.Comment: 5 pages, 6 figure
From folding to function: complex macromolecular reactions unraveled one-by-one with optical tweezers
No full textSingle-molecule manipulation with optical tweezers has uncovered macromolecular behaviour hidden to other experimental techniques. Recent instrumental improvements have made it possible to expand the range of systems accessible to optical tweezers. Beyond focusing on the folding and structural changes of isolated single molecules, optical tweezers studies have evolved into unraveling the basic principles of complex molecular processes such as co-translational folding on the ribosome, kinase activation dynamics, ligand-receptor binding, chaperone-assisted protein folding, and even dynamics of intrinsically disordered proteins (IDPs). In this mini-review, we illustrate the methodological principles of optical tweezers before highlighting recent advances in studying complex protein conformational dynamics - from protein synthesis to physiological function - as well as emerging future issues that are beginning to be addressed with novel approaches
Combining enhanced sampling and deep learning dimensionality reduction for the study of the heat shock protein B8 and its pathological mutant K141E
No full textThe biological functions of proteins closely depend on their conformational dynamics. This aspect is especially relevant for intrinsically disordered proteins (IDP) for which structural ensembles often offer more useful representations than individual conformations. Here we employ extensive enhanced sampling temperature replica-exchange atomistic simulations (TREMD) and deep learning dimensionality reduction to study the conformational ensembles of the human heat shock protein B8 and its pathological mutant K141E, for which no experimental 3D structures are available. First, we combined homology modelling with TREMD to generate high-dimensional data sets of 3D structures. Then, we employed a recently developed machine learning based post-processing algorithm, EncoderMap, to project the large conformational data sets into meaningful two-dimensional maps that helped us interpret the data and extract the most significant conformations adopted by both proteins during TREMD. These studies provide the first 3D structural characterization of HSPB8 and reveal the effects of the pathogenic K141E mutation on its conformational ensembles. In particular, this missense mutation appears to increase the compactness of the protein and its structural variability, at the same time rearranging the hydrophobic patches exposed on the protein surface. These results offer the possibility of rationalizing the pathogenic effects of the K141E mutation in terms of conformational changes
Step-up photophobic responses of the unicellular alga Haematococcus pluvialis and their interpretation in terms of photoreceptive apparatus characteristics
No full textThis paper reports a systematic study of the step-up photophobic responses exhibited by the unicellular alga Haematococcus pluvialis when stimulated unidirectionally or bidirectionally. The stimulus-response curves, obtained at four different wavelengths, are interpreted in terms of the structure of the photoreceptive apparatus. In addition, an indirect method to obtain information about the stigma and photoreceptor(s) is reported. This method is based on the interpolation of experimental with simulated data. Our results confirm the widely accepted view that the photoreceptors are located in the stigma region, and suggest the presence of two photoreceptors
AXOTOMY AFFECTS DENSITY BUT NOT PROPERTIES OF POTASSIUM LEAK CHANNELS, IN THE LEECH AP NEURONS
No full textAxotomy affects density but not properties of potassium leak channels, in the leech AP neuron
Analysis of P element transposase protein-DNA interactions during the early stages of transposition
Full text linkP elements are a family of transposable elements found in Drosophila that move by using a cut-and-paste mechanism and that encode a transposase protein that uses GTP as a cofactor for transposition. Here we used atomic force microscopy to visualize the initial interaction of transposase protein with P element DNA. The transposase first binds to one of the two P element ends, in the presence or absence of GTP, prior to synapsis. In the absence of GTP, these complexes remain stable but do not proceed to synapsis. In the presence of GTP or nonhydrolyzable GTP analogs, synapsis happens rapidly, whereas DNA cleavage is slow. Both atomic force microscopy and standard biochemical methods have been used to show that the P element transposase exists as a pre-formed tetramer that initially binds to either one of the two P element ends in the absence of GTP prior to synapsis. This initial single end binding may explain some of the aberrant P element-induced rearrangements observed in vivo, such as hybrid end insertion. The allosteric effect of GTP in promoting synapsis by P element transposase may be to orient a second site-specific DNA binding domain in the tetramer allowing recognition of a second high affinity transposase-binding site at the other transposon end
Guanosine triphosphate acts as a cofactor to promote assembly of initial P-element transposase-DNA synaptic complexes
No full textP transposable elements in Drosophila are members of a larger class of mobile elements that move using a cut-and-paste mechanism. P-element transposase uses guanosine triphosphate (GTP) as a cofactor for transposition. Here, we use atomic force microscopy (AFM) to visualize protein-DNA complexes formed during the initial stages of P-element transposition. These studies reveal that GTP acts to promote assembly of the first detectable noncovalent precleavage synaptic complex. This initial complex then randomly and independently cleaves each P-element end. These data show that GTP acts to promote protein-DNA assembly, and may explain why P-element excision often leads to unidirectional deletions
Replication of mitochondrial DNA occurs by strand displacement with alternative light-strand origins, not via a strand-coupled mechanism
Full text linkThe established strand-displacement model for mammalian mitochondrial DNA (mtDNA) replication has recently been questioned in light of new data using two-dimensional (2D) agarose gel electrophoresis. It has been proposed that a synchronous, strand-coupled mode of replication occurs in tissues, thereby casting doubt on the general validity of the "orthodox," or strand-displacement model. We have examined mtDNA replicative intermediates from mouse liver using atomic force microscopy and 2D agarose gel electrophoresis in order to resolve this issue. The data provide evidence for only the orthodox, strand-displacement mode of replication and reveal the presence of additional, alternative origins of lagging light-strand mtDNA synthesis. The conditions used for 2D agarose gel analysis are favorable for branch migration of asymmetrically replicating nascent strands. These data reconcile the original displacement mode of replication with the data obtained from 2D gel analyses
Direct observation of the three-state folding of a single protein molecule
No full textWe used force-measuring optical tweezers to induce complete mechanical unfolding and refolding of individual Escherichia-coli ribonuclease H (RNase H) molecules. The protein unfolds in a two-state manner and refolds through an intermediate that correlates with the transient molten globule-like intermediate observed in bulk studies. This intermediate displays unusual mechanical compliance and unfolds at substantially lower forces than the native state. In a narrow range of forces, the molecule hops between the unfolded and intermediate states in real time. Occasionally, hopping was observed to stop as the molecule crossed the folding barrier directly from the intermediate, demonstrating that the intermediate is on-pathway. These studies allow us to map the energy landscape of RNase H
A simple method for preparing calibration standards for the three working axes of scanning probe microscope piezo scanners
No full textA method for preparing samples suitable for calibrating scanning probe microscopes (SPM) and for eliminating any distortions in images is described. Samples consist of polystyrene particles organized in monolayers and bilayers with hexagonal-ordered domains. The monolayer is not uniform, but is characterized by areas without particles. These discontinuities allow the measurement of the thickness of the monolayer in order to calibrate the z axes, while the lattice constant of the domains can be used as a calibration standard for the x and y axes. The nondeformability of the particles after the deposition on the substrate has been studied by an optical microscope, equipped for interferometric measurements, scanning force microscopy, and scanning tunneling microscopy. The use of these standards directly as substrates for samples is proposed to correct the distortions in the SPM images. (C) 1996 American Institute of Physics
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