A5022308851- Receptor Mechanisms and Signaling
- Photoreceptor and optogenetics research
- Retinal Development and Disorders
- Lipid Membrane Structure and Behavior
- Neuropeptides and Animal Physiology
- Advanced Fluorescence Microscopy Techniques
- Protein Structure and Dynamics
- Antimicrobial Peptides and Activities
- RNA and protein synthesis mechanisms
- Influenza Virus Research Studies
- Neuroscience and Neuropharmacology Research
- Neural dynamics and brain function
- Photochromic and Fluorescence Chemistry
- Monoclonal and Polyclonal Antibodies Research
- Fungal and yeast genetics research
- Genomics and Chromatin Dynamics
- Mass Spectrometry Techniques and Applications
- Molecular spectroscopy and chirality
- Yeasts and Rust Fungi Studies
- Circadian rhythm and melatonin
- Neuroendocrine regulation and behavior
- Computational Drug Discovery Methods
- CRISPR and Genetic Engineering
- Cell Adhesion Molecules Research
- Enzyme Structure and Function
University of California, Irvine
2019-2025
University of California System
2023-2024
The California Eye Institute
2020
Case Western Reserve University
2007-2019
Polgenix (United States)
2011-2015
Rutgers, The State University of New Jersey
2008
ActiveSite Pharmaceuticals (United States)
2005-2007
Amgen (United States)
2007
University of Washington
2005
University of Pennsylvania
1999-2004
The changes that lead to activation of G protein-coupled receptors have not been elucidated at the structural level. In this work we report crystal structures both ground state and a photoactivated deprotonated intermediate bovine rhodopsin resolution 4.15 A. state, Schiff base linking chromophore Lys-296 becomes deprotonated, reminiscent protein-activating metarhodopsin II. reveal accompany photoactivation are smaller than previously predicted for II include on cytoplasmic surface possibly...
The M2 proton channel from the influenza A virus is a small protein with single transmembrane helix that associates to form tetramer in vivo. This forms proton-selective ion channels, which are target of drug amantadine. Here, we propose mechanism for pH-dependent association, and amantadine binding M2, based on studies peptide representing segment dodecylphosphocholine micelles. Using analytical ultracentrifugation, find sedimentation curves depend its concentration micellar phase. data...
Abstract G protein-coupled receptors (GPCRs) are cell-surface that respond to various stimuli induce signalling pathways across cell membranes. Recent progress has yielded atomic structures of key intermediates 1,2 and roles for lipids in 3,4 . However, capturing events a wild-type receptor real time, native membrane its downstream effectors, remained elusive. Here we probe the archetypal class A GPCR, rhodopsin, directly from fragments disc membranes using mass spectrometry. We monitor...
The M2 protein from influenza A virus is a 97-residue homotetrameric membrane that functions as proton channel. To determine the features required for assembly of this into its native tetrameric state, was prepared by total synthesis using chemical ligation unprotected peptide segments. Circular dichroism spectroscopy synthetic in dodecylphosphocholine (DPC) micelles indicated approximately 40 residues were an alpha-helical secondary structure. tetramerization full-length compared to...
Significance This study resolves a long-standing question about the ability of humans to perceive near infrared radiation (IR) and identifies mechanism driving human IR vision. A few previous reports our expanded psychophysical studies here reveal that can detect at wavelengths longer than 1,000 nm it as visible light, finding has not received satisfactory physical explanation. We show light activates photoreceptors through nonlinear optical process. also caused photoisomerization purified...
Photoisomerization of the 11-cis-retinal chromophore rod and cone visual pigments to an all-trans-configuration is initiating event for vision in vertebrates. The regeneration 11-cis-retinal, necessary sustained function, endergonic process normally conducted by specialized enzyme systems. However, also can be formed through reverse photoisomerization from all-trans-retinal. A nonvisual opsin known as retinal pigment epithelium (RPE)-retinal G-protein–coupled receptor (RGR) was previously...
Photoreceptors rely on distinct membrane compartments to support their specialized function. Unlike protein localization, identification of critical differences in content has not yet been expanded lipids, due the difficulty isolating domain-specific samples. We have overcome this by using SMA coimmunopurify proteins and native lipids from two regions photoreceptor ROS disks. Each sample's copurified were subjected untargeted lipidomic fatty acid analysis. Extensive between center...
Abstract Rhodopsin is a prototypical G protein-coupled receptor (GPCR) critical for vertebrate vision. Research on GPCR signaling states has been facilitated using llama-derived nanobodies (Nbs), some of which bind to the intracellular surface allosterically modulate receptor. Extracellularly binding allosteric have also investigated, but structural basis their activity not resolved date. Here, we report library Nbs that extracellular rhodopsin and thermodynamics its activation process....
Rhodopsin (Rho) and cone opsins are essential for detection of light. They respond via photoisomerization, converting their Schiff-base-adducted 11-cis-retinylidene chromophores to the all-trans configuration, eliciting conformational changes activate opsin signaling. Subsequent Schiff-base hydrolysis releases all-trans-retinal, initiating two important cycles that maintain continuous vision – Rho photocycle visual cycle pathway. has been thoroughly studied with photoactivated rhodopsin but...
For sustained vision, photoactivated rhodopsin (Rho*) must undergo hydrolysis and release of all-trans-retinal, producing substrate for the visual cycle apo-opsin available regeneration with 11-cis-retinal. The kinetics this has yet to be described in its native membrane environment. We developed a method consisting simultaneous denaturation chromophore trapping by isopropanol/borohydride, followed exhaustive protein digestion, complete extraction, liquid chromatography-mass spectrometry....
Abstract Delivering ribonucleoproteins (RNPs) for in vivo genome editing is safer than using viruses encoding Cas9 and its respective guide RNA. However, transient RNP activity does not typically lead to optimal outcomes. Here we show that the efficiency of delivering RNPs can be enhanced by cell-penetrating peptides (covalently fused protein or as excipients) lipid nanoparticles (LNPs) encapsulating optimized stability, delivery potency. Specifically, after screening suitable ionizable...
Activation of G-protein-coupled receptors (GPCRs) initiates signal transduction cascades that affect many physiological responses. The worm Caenorhabditis elegans expresses >1000 these along with their cognate heterotrimeric G proteins. Here, we report properties 9-cis-retinal regenerated bovine opsin [(b)isoRho] and human melanopsin [(h)Mo], two light-activated, heterologously expressed GPCRs in the nervous system C. various genetically engineered alterations. Profound transient...
Hybrid structural methods have been used in recent years to understand protein-protein or protein-ligand interactions where high resolution crystallography NMR data on the protein of interest has limited. For G protein-coupled receptors (GPCRs), structures native forms other than rhodopsin not yet achieved; gaps our knowledge filled by creative studies that developed stable multiple means. The neurotransmitter serotonin (5-hydroxytryptamine) is a key GPCR-based signaling molecule affecting...
Noninvasive imaging of visual system components in vivo is critical for understanding the causal mechanisms retinal diseases and developing therapies their treatment. However, ultraviolet light needed to excite endogenous fluorophores that participate metabolic processes retina highly attenuated by anterior segment human eye. In contrast, 2-photon excitation fluorescence with pulsed infrared overcomes this obstacle. Reducing exposure laser radiation remains a major barrier advancing...
The role of the membrane lipid composition and individual Trp residues in conformational rearrangement gramicidin A along folding pathway to its channel conformation has been examined phospholipid bilayers by means previously described size-exclusion high-performance liquid chromatography HPLC-based strategy (Bañó et al. (1991) Biochemistry 30, 886). It demonstrated that chemical influences transition rate peptide from double-stranded dimers β-helical monomers. modification residues, or...
Second-order nonlinear optical imaging of chiral crystals (SONICC), which portrays second-harmonic generation (SHG) by noncentrosymmetric crystals, is emerging as a powerful technique for protein in media opaque to visible light because its high signal-to-noise ratio. Here we report the incorporation both SONICC and two-photon excited fluorescence (TPEF) into one system that allows visualization small ~10 μm their longest dimension. Using this system, then documented an inverse correlation...
G protein-coupled receptors (GPCRs) constitute the largest superfamily of transmembrane signaling proteins; however, only known GPCR crystal structure is that rhodopsin. This disparity reflects difficulty in generating purified samples sufficient quantity and quality. Rhodopsin, light receptor retinal rod neurons, produced large amounts homogeneous quality vertebrate retina. We used transgenic Xenopus laevis to convert these retina cells into bioreactors successfully produce 20 model GPCRs....
New strategies for expression, purification, functional characterization, and structural determination of membrane-spanning G-protein-coupled receptors (GPCRs) are constantly being developed because their importance to human health. Here, we report a Caenorhabditis elegans heterologous expression system able produce milligram amounts native engineered GPCRs. Both bovine opsin [(b)opsin] adenosine A2A subtype receptor [(h)A2AR] expressed in neurons or muscles C. were localized cell membranes....