A5043616114- Methane Hydrates and Related Phenomena
- Earthquake Detection and Analysis
- Receptor Mechanisms and Signaling
- Seismic Waves and Analysis
- Ionosphere and magnetosphere dynamics
- Photoreceptor and optogenetics research
- Retinal Development and Disorders
- Neuroscience and Neuropharmacology Research
- Neuropeptides and Animal Physiology
- Light effects on plants
- Photosynthetic Processes and Mechanisms
- Cannabis and Cannabinoid Research
- Geophysics and Gravity Measurements
- Lipid Membrane Structure and Behavior
- Protein Structure and Dynamics
- Electron Spin Resonance Studies
- Pancreatic function and diabetes
- Advanced Fluorescence Microscopy Techniques
- Nitric Oxide and Endothelin Effects
- Neurotransmitter Receptor Influence on Behavior
- Monoclonal and Polyclonal Antibodies Research
- Mass Spectrometry Techniques and Applications
- Cancer, Stress, Anesthesia, and Immune Response
- Plant and animal studies
- Photochemistry and Electron Transfer Studies
Oregon Health & Science University
2014-2023
University of Portland
2012-2016
Stanford University
2001-2015
Vanderbilt University
2002
Howard Hughes Medical Institute
2001
Massachusetts Institute of Technology
1992-1999
University of California, Los Angeles
1996-1999
Medical College of Wisconsin
1999
University of Nebraska–Lincoln
1989-1992
University of California, Santa Cruz
1992
Conformational changes are thought to underlie the activation of heterotrimeric GTP-binding protein (G protein)—coupled receptors. Such in rhodopsin were explored by construction double cysteine mutants, each containing one at cytoplasmic end helix C and various positions F. Magnetic dipolar interactions between spin labels attached these residues revealed their proximity, interaction upon light suggested a rigid body movement helices relative another. Disulfide cross-linking prevented...
An increase in fluorescence is observed upon light activation of bovine rhodopsin. The rate monoexponential (t1/2 = 15.5 min) at 20°C 0.1% lauryl maltoside, pH 6.0, and parallels the decay metarhodopsin II. We show that due to release free retinal, which no longer quenches tryptophan fluorescence. extrinsic reporter group, pyrene maleimide, attached rhodopsin also shows an on illumination. matches This result has been used develop a micromethod, requiring order 1 μg rhodopsin, for...
A fluorescent sensor for NAD + in living cells Roles of cellular nicotinamide adenine dinucleotide (NAD ) metabolism, aging, and disease have garnered much interest, but methods been lacking to measure the amounts cells. Cambronne et al. developed a genetically encoded biosensor that can be used monitor concentrations free various compartments cell (see Perspective by Guarente). Such appear important regulating activity -consuming enzymes such as sirtuins ADP-ribosyltransferases. The authors...
G protein-coupled receptors represent the largest class of drug discovery targets. Drugs that activate are classified as either agonists or partial agonists. To study mechanism whereby these different classes activating ligands modulate receptor function, we directly monitored ligand-induced conformational changes in protein-coupling domain β2 adrenergic receptor. Fluorescence lifetime analysis a reporter fluorophore covalently attached to this revealed that, absence ligands, oscillates...
The majority of extracellular physiologic signaling molecules act by stimulating GTP-binding protein (G-protein)-coupled receptors (GPCRs). To monitor directly the formation active state a prototypical GPCR, we devised method to site specifically attach fluorescein an endogenous cysteine (Cys-265) at cytoplasmic end transmembrane 6 (TM6) β 2 adrenergic receptor (β AR), adjacent G-protein-coupling domain. We demonstrate that this tag reports agonist-induced conformational changes in receptor,...
Thirty consecutive single cysteine substitution mutants in the amino acids Q225-I256 of bovine rhodopsin have been prepared and modified with a sulfhydryl specific nitroxide reagent. This sequence includes E-F interhelical loop, transducin interaction site. The accessibilities attached nitroxides to collisions hydrophilic hydrophobic paramagnetic probes solution were determined, electron resonance spectra analyzed terms side chain mobility, both dark after photoactivation. Accessibility cata...
Small conductance Ca(2+)-activated potassium channels (SK channels) are coassembled complexes of pore-forming SK alpha subunits and calmodulin. We proposed a model for channel activation in which Ca2+ binding to calmodulin induces conformational rearrangements the that result gating. now report fluorescence measurements indicate changes subunit after subunit-calmodulin complex. Two-hybrid experiments showed Ca(2+)-independent interaction with requires only C-terminal domain is mediated by...
A recent proposal for the formation of functionally active rhodopsin has placed critical importance on a movement one its transmembrane helices (Farrens, D. L., Altenbach, C., Yang, K., Hubbell, W. and Khorana, H. G. (1996) Science 274, 768-770). We investigated this hypothesis using series eight mutants containing single reactive cysteine residues in region (helix F) where was previously detected. The were studied two ways, by measuring their reactivity to cysteine-specific reagent...
Studying the interplay between protein structure and function remains a daunting task. Especially lacking are methods for measuring structural changes in real time. Here we report our most recent improvements to method that can be used address such challenges. This method, which now call tryptophan-induced quenching (TrIQ), provides straightforward, sensitive, inexpensive way questions of conformational dynamics short-range interactions. Importantly, TrIQ only occurs over relatively short...
We present a novel method for mapping proximity within proteins. The exploits the quenching of fluorescent label bimane by nearby Trp residues. In studies T4 lysozyme we show that this effect appears to be distance dependent and orientation specific. Specifically, proximal residue can reduce fluorescence intensity up 500% induce complicated decay kinetics. Replacing neighboring with phenylalanine removes these spectral perturbations. advantages using protein structural include low amount...
Conformational changes enable the photoreceptor rhodopsin to couple with and activate G-protein transducin. Here we demonstrate a key interaction between these proteins occurs C terminus of transducin α-subunit (GTα) hydrophobic cleft in cytoplasmic face exposed during receptor activation. We mapped this by labeling mutants fluorescent probe bimane then assessed how binding peptide analogue GTα (containing tryptophan quenching group) affected their fluorescence. From other assays, conclude...
We show that the photoreceptor rhodopsin (Rh) can exist in membrane as a dimer or multimer using luminescence resonance energy transfer and FRET methods. Our approach looked for interactions between Rh molecules reconstituted into asolectin liposomes. The low receptor density used measurements ensured minimal crowding artifactual association. fluorescently labeled were fully functional, measured by their ability to activate G protein transducin. revealed distance of 47–50 Å molecules....
Identifying neurons that have functional opioid receptors is fundamental for the understanding of cellular, synaptic and systems actions opioids. Current techniques are limited to post hoc analyses fixed tissues. Here we developed a fluorescent probe, naltrexamine-acylimidazole (NAI), label based on chemical approach termed 'traceless affinity labeling'. In this approach, high antagonist naltrexamine used as guide molecule transferring reaction acylimidazole at receptor. This generates dye...
To probe proximity relationships between different amino acids in the interhelical loops cytoplasmic domain of rhodopsin, we are using a general approach which two cysteine residues introduced at locations. Here report on characteristics one such mutant that contains naturally occuring 316 near end helix G and second position 65 (H65C), A. The protein after expression COS-1 cells reconstitution with 11-cis-retinal can be bound to anti-rhodopsin antibody 1D4-Sepharose pH 6 form cysteines free...
The cytoplasmic interhelical E-F loop in rhodopsin is a part of the region that interacts with G-protein transducin and kinase during signal transduction. In extending previous work on systematic single cysteine substitutions amino acids C-D loop, we have now replaced, one at time, Q225-I256 by cysteines. All mutants formed characteristic chromophore 11-cis-retinal. While most bleached normally, L226C, showed abnormal bleaching behavior. A study alkylation N-ethylmaleimide dark low...
The rhodopsin crystal structure reveals that intradiscal loop E-2 covers the 11-cis-retinal, creating a “retinal plug.” Recently, we noticed ends of are linked by an ion pair between residues Arg-177 and Asp-190, near highly conserved disulfide bond. This appears biologically significant; it is in almost all vertebrate opsins may occur other G-protein-coupled receptors. We report here Arg-177/Asp-190 critical for folding stability dark state rhodopsin. find mutants regenerate with retinal...
Tryptophan-induced quenching of fluorophores (TrIQ) uses intramolecular fluorescence to assess distances in proteins too small (<15 Å) be easily probed by traditional Forster resonance energy transfer methods. A powerful aspect TrIQ is its ability obtain an ultrafast snapshot a protein conformation, identifying "static quenching" (contact between the Trp and probe at moment light excitation). Here we report new advances this site-directed labeling (SDFL) approach, gleaned from recent studies...