566 research outputs found
Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state
G protein-coupled receptors (GPCRs) represent a major pharmaceutical drug target. However, one exception has been the visual photoreceptor rhodopsin, long considered βdifferentβ due to its covalently bound, light-sensitive retinal ligands. Here we demonstrate that, in contrast to prior assumptions, release of the agonist all-trans retinal (ATR) is not an irreversible process. Instead, during decay of the active species, ATR can rebind any rhodopsin remaining in an active-like conformation, and this active-like state can transiently persist even after agonist dissociation. These insights demonstrate rhodopsin behaves like other diffusible ligand-binding GPCRs and raise the possibility of treating rhodopsin by pharmaceutical agents
Baseline Needs Assessment for a Hospital-Based Violence Intervention Program 1-Year Pilot
The objectives of the present study were to measure and describe the baseline participant needs of a hospital-based violence intervention 1-year pilot program, assess differences in expected hospital revenue based on changes in health insurance coverage resulting from program implementation and discuss the programβs limitations. Methods: Between September 2020 and September 2021 Encompass Omaha enrolled 36 participants. A content analysis of 1199 progress notes detailing points of contact with participants was performed to determine goal status. Goals were categorized and goal status was defined as met, in process, dropped, or participant refusal. Results: The most frequently identified needs were help obtaining short-term disability assistance or completing FMLA paperwork (86.11%), immediate financial aid (86.11%), legal aid (83.33%), access to food (83.3%), and navigating medical issues other than the primary reason for hospitalization (83.33%). Conclusions: Meeting the participantsβ short-term needs is critical for maintaining their engagement in the long-term. Further, differences in expected hospital revenue for pilot participants compared with a control group were examined, and this analysis found a reduction in medical and facility costs for program participants. The pilot stage highlighted how complex the needs and treatment of victims of violence are. As the program grows and its staff become more knowledgeable about social work, treatment, and resource access processes, the program will continue to improve
Perceptions of violence in justice-involved youth
Background Youth are tragically affected by violence. Justice-involved youth are at elevated risk for the effects of violence, as incarceration serves as a risk factor. The objective of this study is to explore the risks and needs of justice-involved youth and identify channels for future hospital-based programming. Methods Four weekly focus groups were conducted by a credible messenger at the Douglas County Youth Center with former participants of Dusk 2 Dawn, a youth violence prevention program delivered at the Douglas County Youth Center. Eight participants were prompted with preset interview questions. All focus groups were recorded and transcribed by a professional transcription service. A thematic analysis was performed by 2 independent coders to identify themes using Dedoose software. Results The 3 most frequently occurring themes involved topics on protection, identified 40 times; family, identified 36 times; and the challenge of overcoming violence, identified 31 times. These themes often overlapped with one another, demonstrating the complexity of youth violence. Conclusion Providing a safe and judgement-free space for the youth to discuss issues of violence was beneficial for 3 reasons: (1) inclusion of youth perspectives allows violence prevention programs to be tailored to specific needs, (2) participants were able to deeply reflect on violence in their own lives and consider steps toward positive change, and (3) open communication encourages trust building and collaborative prevention efforts between the hospital and community
Infrared spectroscopy of phytochrome and model pigments
Fourier-transform infrared difference spectra between the red-absorbing and far-red-absorbing forms of oat phytochrome have been measured in H2O and 2H2O. The difference spectra are compared with infrared spectra of model compounds, i.e. the (5Z,10Z,15Z)- and (5Z,10Z,15E)-isomers of 2,3,7,8,12,13,17,18-octaethyl-bilindion (Et8-bilindion), 2,3-dihydro-2,3,7,8,12,13,17,18-octaethyl-bilindion (H2Et8-bilindion), and protonated H2Et8-bilindion in various solvents. The spectra of the model compounds show that only for the protonated forms can clear differences between the two isomers be detected. Since considerable differences are present between the spectra of Et8-bilindion and H2Et8-bilindion, it is concluded that only the latter compound can serve as a model system of phytochrome. The 2H2O effect on the difference spectrum of phytochrome supports the view that the chromophore in red-absorbing phytochrome is protonated and suggests, in addition, that it is also protonated in far-red-absorbing phytochrome. The spectra show that protonated carboxyl groups are influenced. The small amplitudes in the difference spectra exclude major changes of protein secondary structure
Entropic Tension in Crowded Membranes
Unlike their model membrane counterparts, biological membranes are richly
decorated with a heterogeneous assembly of membrane proteins. These proteins
are so tightly packed that their excluded area interactions can alter the free
energy landscape controlling the conformational transitions suffered by such
proteins. For membrane channels, this effect can alter the critical membrane
tension at which they undergo a transition from a closed to an open state, and
therefore influence protein function \emph{in vivo}. Despite their obvious
importance, crowding phenomena in membranes are much less well studied than in
the cytoplasm.
Using statistical mechanics results for hard disk liquids, we show that
crowding induces an entropic tension in the membrane, which influences
transitions that alter the projected area and circumference of a membrane
protein. As a specific case study in this effect, we consider the impact of
crowding on the gating properties of bacterial mechanosensitive membrane
channels, which are thought to confer osmoprotection when these cells are
subjected to osmotic shock. We find that crowding can alter the gating energies
by more than in physiological conditions, a substantial fraction of
the total gating energies in some cases.
Given the ubiquity of membrane crowding, the nonspecific nature of excluded
volume interactions, and the fact that the function of many membrane proteins
involve significant conformational changes, this specific case study highlights
a general aspect in the function of membrane proteins.Comment: 20 pages (inclduing supporting information), 4 figures, to appear in
PLoS Comp. Bio
Identifying Ligand Binding Conformations of the Ξ²2-Adrenergic Receptor by Using Its Agonists as Computational Probes
Recently available G-protein coupled receptor (GPCR) structures and biophysical studies suggest that the difference between the effects of various agonists and antagonists cannot be explained by single structures alone, but rather that the conformational ensembles of the proteins need to be considered. Here we use an elastic network model-guided molecular dynamics simulation protocol to generate an ensemble of conformers of a prototypical GPCR, Ξ²2-adrenergic receptor (Ξ²2AR). The resulting conformers are clustered into groups based on the conformations of the ligand binding site, and distinct conformers from each group are assessed for their binding to known agonists of Ξ²2AR. We show that the select ligands bind preferentially to different predicted conformers of Ξ²2AR, and identify a role of Ξ²2AR extracellular region as an allosteric binding site for larger drugs such as salmeterol. Thus, drugs and ligands can be used as "computational probes" to systematically identify protein conformers with likely biological significance. Β© 2012 Isin et al
The Roles of Transmembrane Domain Helix-III during Rhodopsin Photoactivation
Background: Rhodopsin, the prototypic member of G protein-coupled receptors (GPCRs), undergoes isomerization of 11- cis-retinal to all-trans-retinal upon photoactivation. Although the basic mechanism by which rhodopsin is activated is well understood, the roles of whole transmembrane (TM) helix-III during rhodopsin photoactivation in detail are not completely clear.
Principal Findings: We herein use single-cysteine mutagenesis technique to investigate conformational changes in TM helices of rhodopsin upon photoactivation. Specifically, we study changes in accessibility and reactivity of cysteine residues introduced into the TM helix-III of rhodopsin. Twenty-eight single-cysteine mutants of rhodopsin (P107C-R135C) were prepared after substitution of all natural cysteine residues (C140/C167/C185/C222/C264/C316) by alanine. The cysteine mutants were expressed in COS-1 cells and rhodopsin was purified after regeneration with 11-cis-retinal. Cysteine accessibility in these mutants was monitored by reaction with 4, 49-dithiodipyridine (4-PDS) in the dark and after illumination. Most of the mutants except for T108C, G109C, E113C, I133C, and R135C showed no reaction in the dark. Wide
variation in reactivity was observed among cysteines at different positions in the sequence 108β135 after photoactivation. In particular, cysteines at position 115, 119, 121, 129, 131, 132, and 135, facing 11-cis-retinal, reacted with 4-PDS faster than neighboring amino acids. The different reaction rates of mutants with 4-PDS after photoactivation suggest that the amino acids in different positions in helix-III are exposed to aqueous environment to varying degrees. Significance: Accessibility data indicate that an aqueous/hydrophobic boundary in helix-III is near G109 and I133. The lack of reactivity in the dark and the accessibility of cysteine after photoactivation indicate an increase of water/4-PDS accessibility for certain cysteine-mutants at Helix-III during formation of Meta II. We conclude that photoactivation resulted in water-accessible at the chromophore-facing residues of Helix-III.National Institutes of Health (U.S.) (grant GM28289)National Eye Institute (Grant Grant EY11716)National Science Foundation (U.S.) (grant EIA-0225609
Comparative Analysis of the Heptahelical Transmembrane Bundles of G Protein-Coupled Receptors
Background: G protein-coupled receptors represent a large family of eukaryotic membrane proteins, and are involved in almost all physiological processes in humans. Recent advances in the crystallographic study of these receptors enable a detailed comparative analysis of the commonly shared heptahelical transmembrane bundle. Systematic comparison of the bundles from a variety of receptors is indispensable for understanding not only of the structural diversification optimized for the binding of respective ligands but also of the structural conservation required for the common mechanism of activation accompanying the interaction changes among the seven helices. Methodology/Principal Findings: We have examined the bundles of 94 polypeptide chains from almost all available structures of 11 receptors, which we classified into either inactivated chain or activated chain, based on the type of bound ligand. For the inactivated chains, superposition of 200 residue bundles by secondary structure matching demonstrated that the bound ligands share a laterally limited cavity in the extracellular section of the bundle. Furthermore, a distinct feature was found for helix III of bovine rhodopsin, which might have evolved to lower its activity in the presence of 11-cis-retinal, to a level that other receptors could hardly achieve with any currently available ligands. Conclusions/Significance: Systematic analysis described here would be valuable for understanding of the rearrangement o
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