Circular dichroism (CD) spectroscopy is a well-established technique for studying the secondary structures, dynamics, folding pathways, and interactions of soluble proteins, and is complementary to ...the high resolution but generally static structures produced by X-ray crystallography, NMR spectroscopy, and cryo electron microscopy. CD spectroscopy has special relevance for the study of membrane proteins, which are difficult to crystallise and largely ignored in structural genomics projects. However, the requirement for membrane proteins to be embedded in amphipathic environments such as membranes, lipid vesicles, detergent micelles, bicelles, oriented bilayers, or nanodiscs, in order for them to be soluble or dispersed in solution whilst maintaining their structure and function, necessitates the use of different experimental and analytical approaches than those employed for soluble proteins. This review discusses specialised methods for collecting and analysing membrane protein CD data, highlighting where protocols for soluble and membrane proteins diverge.
Circular dichroism (CD) spectroscopy is a widely-used method in biochemistry, structural biology and pharmaceutical chemistry. More than 24 000 papers published in the past decade have included CD ...characterisations of proteins; many of those studies have also included other complementary chemical, biophysical, and computational chemistry methods. This tutorial review describes the background to the technique of CD spectroscopy and good practice methods for high quality data collection. It specifically focuses on both established and new methods and tools available for experimental design and interpretation, data processing, visualisation, analysis, validation, archiving, and accession, including tools developed to enhance the complementarity of this method with other structural and chemical biology studies.
Circular dichroism (CD) spectroscopy is a widely‐used method for characterizing the secondary structures of proteins. The well‐established and highly used analysis website, DichroWeb (located at: ...http://dichroweb.cryst.bbk.ac.uk/html/home.shtml) enables the facile quantitative determination of helix, sheet, and other secondary structure contents of proteins based on their CD spectra. DichroWeb includes a range of reference datasets and algorithms, plus graphical and quantitative methods for determining the quality of the analyses produced. This article describes the current website content, usage and accessibility, as well as the many upgraded features now present in this highly popular tool that was originally created nearly two decades ago.
•Instrumentation developments are enabling an enhanced range of measurements.•The PCDDB provides spectra and metadata for traceability and methods development.•New software developments are enabling ...novel types of analyses.•CD spectroscopy can now examine more types of proteins with important biological roles.•CD spectroscopy is being used in many interdisciplinary and structural biology applications.
Circular dichroism (CD) spectroscopy has been used widely in structural biology for literally a half century, primarily to examine the secondary structure, folding, and interactions of proteins in solution. With recent developments in instrumentation, it is now possible to apply CD to many additional types of sample environments, including oriented membranes, films, and dehydrated samples. In addition, developments in bioinformatics have made validated CD spectra and metadata available for novel analysis methods on additional types of samples such as membrane proteins, intrinsically disordered proteins, multiple fold types, and multicomponent, macromolecular complexes. New software has also enabled increased inter-operability of CD with other structural biology methodologies, contributing to their use in joint studies of protein structures at various levels of organization.
The human ovary contains a fixed number of non-growing follicles (NGFs) established before birth that decline with increasing age culminating in the menopause at 50-51 years. The objective of this ...study is to model the age-related population of NGFs in the human ovary from conception to menopause. Data were taken from eight separate quantitative histological studies (n = 325) in which NGF populations at known ages from seven weeks post conception to 51 years (median 32 years) were calculated. The data set was fitted to 20 peak function models, with the results ranked by obtained r2 correlation coefficient. The highest ranked model was chosen. Our model matches the log-adjusted NGF population from conception to menopause to a five-parameter asymmetric double Gaussian cumulative (ADC) curve (r2 = 0.81). When restricted to ages up to 25 years, the ADC curve has r2 = 0.95. We estimate that for 95% of women by the age of 30 years only 12% of their maximum pre-birth NGF population is present and by the age of 40 years only 3% remains. Furthermore, we found that the rate of NGF recruitment towards maturation for most women increases from birth until approximately age 14 years then decreases towards the menopause. To our knowledge, this is the first model of ovarian reserve from conception to menopause. This model allows us to estimate the number of NGFs present in the ovary at any given age, suggests that 81% of the variance in NGF populations is due to age alone, and shows for the first time, to our knowledge, that the rate of NGF recruitment increases from birth to age 14 years then declines with age until menopause. An increased understanding of the dynamics of human ovarian reserve will provide a more scientific basis for fertility counselling for both healthy women and those who have survived gonadotoxic cancer treatments.
Synaptic interactions to extract information about wavelength, and thus color, begin in the vertebrate retina with three classes of light-sensitive cells: rod photoreceptors at low light levels, ...multiple types of cone photoreceptors that vary in spectral sensitivity, and intrinsically photosensitive ganglion cells that contain the photopigment melanopsin. When isolated from its neighbors, a photoreceptor confounds photon flux with wavelength and so by itself provides no information about color. The retina has evolved elaborate color opponent circuitry for extracting wavelength information by comparing the activities of different photoreceptor types broadly tuned to different parts of the visible spectrum. We review studies concerning the circuit mechanisms mediating opponent interactions in a range of species, from tetrachromatic fish with diverse color opponent cell types to common dichromatic mammals where cone opponency is restricted to a subset of specialized circuits. Distinct among mammals, primates have reinvented trichromatic color vision using novel strategies to incorporate evolution of an additional photopigment gene into the foveal structure and circuitry that supports high-resolution vision. Color vision is absent at scotopic light levels when only rods are active, but rods interact with cone signals to influence color perception at mesopic light levels. Recent evidence suggests melanopsin-mediated signals, which have been identified as a substrate for setting circadian rhythms, may also influence color perception. We consider circuits that may mediate these interactions. While cone opponency is a relatively simple neural computation, it has been implemented in vertebrates by diverse neural mechanisms that are not yet fully understood.
Light-evoked voltage responses of rod and cone photoreceptor cells in the vertebrate retina must be converted to a train of synaptic vesicle release events for transmission to downstream neurons. ...This review discusses the processes, proteins, and structures that shape this critical early step in vision, focusing on studies from salamander retina with comparisons to other experimental animals. Many mechanisms are conserved across species. In cones, glutamate release is confined to ribbon release sites although rods are also capable of release at non-ribbon sites. The role of non-ribbon release in rods remains unclear. Release from synaptic ribbons in rods and cones involves at least three vesicle pools: a readily releasable pool (RRP) matching the number of membrane-associated vesicles along the ribbon base, a ribbon reserve pool matching the number of additional vesicles on the ribbon, and an enormous cytoplasmic reserve. Vesicle release increases in parallel with Ca
2+
channel activity. While the opening of only a few Ca
2+
channels beneath each ribbon can trigger fusion of a single vesicle, sustained release rates in darkness are governed by the rate at which the RRP can be replenished. The number of vacant release sites, their functional status, and the rate of vesicle delivery in turn govern replenishment. Along with an overview of the mechanisms of exocytosis and endocytosis, we consider specific properties of ribbon-associated proteins and pose a number of remaining questions about this first synapse in the visual system.
Anthracycline-based chemotherapy can result in the development of a cumulative and progressively developing cardiomyopathy. Doxorubicin is one of the most highly prescribed anthracyclines in the ...United States due to its broad spectrum of therapeutic efficacy. Interference with different mitochondrial processes is chief among the molecular and cellular determinants of doxorubicin cardiotoxicity, contributing to the development of cardiomyopathy. The present review provides the basis for the involvement of mitochondrial toxicity in the different functional hallmarks of anthracycline toxicity. Our objective is to understand the molecular determinants of a progressive deterioration of functional integrity of mitochondria that establishes a historic record of past drug treatments (mitochondrial memory) and renders the cancer patient susceptible to subsequent regimens of drug therapy. We focus on the involvement of doxorubicin-induced mitochondrial oxidative stress, disruption of mitochondrial oxidative phosphorylation, and permeability transition, contributing to altered metabolic and redox circuits in cardiac cells, ultimately culminating in disturbances of autophagy/mitophagy fluxes and increased apoptosis. We also suggest some possible pharmacological and nonpharmacological interventions that can reduce mitochondrial damage. Understanding the key role of mitochondria in doxorubicin-induced cardiomyopathy is essential to reduce the barriers that so dramatically limit the clinical success of this essential anticancer chemotherapy.
Voltage‐gated sodium channels are essential for electrical signalling across cell membranes. They exhibit strong selectivities for sodium ions over other cations, enabling the finely tuned cascade of ...events associated with action potentials. This paper describes the ion permeability characteristics and the crystal structure of a prokaryotic sodium channel, showing for the first time the detailed locations of sodium ions in the selectivity filter of a sodium channel. Electrostatic calculations based on the structure are consistent with the relative cation permeability ratios (Na+ ≈ Li+ ≫ K+, Ca2+, Mg2+) measured for these channels. In an E178D selectivity filter mutant constructed to have altered ion selectivities, the sodium ion binding site nearest the extracellular side is missing. Unlike potassium ions in potassium channels, the sodium ions in these channels appear to be hydrated and are associated with side chains of the selectivity filter residues, rather than polypeptide backbones.
Synopsis
Structural localisation of sodium ions passing through the prokaryotic NavM channel selectivity filter explains this channel's strong sodium preference over other cations in electrochemical signalling across cell membranes.
Crystal structures of the NavMs prokaryotic sodium channel indicate the locations of sodium ions in its selectivity filter.
Electrostatic calculations based on the structure are consistent with the relative cation permeability ratios measured for these channels.
An E178D selectivity filter mutant constructed to validate the structure/function relationships has altered ion preference, and the sodium ion binding site nearest the extracellular side is missing.
The sodium ions appear to be hydrated and are associated with side chains of the filter residues.
Structural localisation of sodium ions passing through the prokaryotic NavM channel selectivity filter explains this channel's strong sodium preference over other cations in electrochemical signalling across cell membranes.
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