10 results on '"Michael P. Bokoch"'
Search Results
2. Desmopressin Reverses Overly Rapid Serum Sodium Correction in a Hyponatremic Patient Undergoing Living Donor Liver Transplantation
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Jane S. Yu, Erika L. Brinson, Michael P. Bokoch, and Linda L. Liu
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medicine.medical_specialty ,Sodium ,medicine.medical_treatment ,Chronic Liver Disease and Cirrhosis ,Urology ,chemistry.chemical_element ,Liver transplantation ,Oral and gastrointestinal ,Hemostatics ,End Stage Liver Disease ,03 medical and health sciences ,Liver disease ,0302 clinical medicine ,Clinical Research ,Risk Factors ,030202 anesthesiology ,Living Donors ,Humans ,Medicine ,Deamino Arginine Vasopressin ,Desmopressin ,Transplantation ,Intraoperative Care ,business.industry ,Liver Disease ,General Medicine ,Perioperative ,medicine.disease ,Liver Transplantation ,chemistry ,030211 gastroenterology & hepatology ,Digestive Diseases ,business ,Hyponatremia ,Living donor liver transplantation ,medicine.drug - Abstract
Patients with end-stage liver disease are often hyponatremic due to multiple physiological processes associated with hepatic failure. For severely hyponatremic patients undergoing liver transplantation, intraoperative management of serum sodium concentration ([Na]s) is challenging. [Na]s tends to increase during transplantation by the administration of fluids with higher sodium concentration than the patient's [Na]s. An overly rapid increase in [Na]s (>1 mEq·L·hour) is difficult to avoid and increases the risk of serious perioperative complications. We report the successful use of intravenous desmopressin to reverse an overly rapid rise in [Na]s during living donor liver transplantation.
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- 2018
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3. Entry from the Lipid Bilayer: A Possible Pathway for Inhibition of a Peptide G Protein-Coupled Receptor by a Lipophilic Small Molecule
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Hyunil Jo, William F. DeGrado, Yoga Srinivasan, David E. Shaw, Ron O. Dror, Shaun R. Coughlin, Michael Grabe, James R. Valcourt, Albert C. Pan, Michael P. Bokoch, and Sara Capponi
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0301 basic medicine ,Biochemistry & Molecular Biology ,Stereochemistry ,Protein Conformation ,Pyridines ,Lipid Bilayers ,PAR-1 ,Peptide ,Medical Biochemistry and Metabolomics ,Molecular Dynamics Simulation ,Ligands ,Biochemistry ,Article ,Medicinal and Biomolecular Chemistry ,03 medical and health sciences ,Lactones ,0302 clinical medicine ,Protein structure ,medicine ,Animals ,Humans ,Receptor, PAR-1 ,Binding site ,Receptor ,Lipid bilayer ,G protein-coupled receptor ,Vorapaxar ,chemistry.chemical_classification ,Binding Sites ,Molecular Structure ,Fibroblasts ,Rats ,Transmembrane domain ,030104 developmental biology ,chemistry ,Phosphatidylcholines ,Generic health relevance ,Biochemistry and Cell Biology ,030217 neurology & neurosurgery ,Biotechnology ,medicine.drug ,Protein Binding - Abstract
The pathways that G protein-coupled receptor (GPCR) ligands follow as they bind to or dissociate from their receptors are largely unknown. Protease-activated receptor-1 (PAR1) is a GPCR activated by intramolecular binding of a tethered agonist peptide that is exposed by thrombin cleavage. By contrast, the PAR1 antagonist vorapaxar is a lipophilic drug that binds in a pocket almost entirely occluded from the extracellular solvent. The binding and dissociation pathway of vorapaxar is unknown. Starting with the crystal structure of vorapaxar bound to PAR1, we performed temperature-accelerated molecular dynamics simulations of ligand dissociation. In the majority of simulations, vorapaxar exited the receptor laterally into the lipid bilayer through openings in the transmembrane helix (TM) bundle. Prior to full dissociation, vorapaxar paused in metastable intermediates stabilized by interactions with the receptor and lipid headgroups. Derivatives of vorapaxar with alkyl chains predicted to extend between TM6 and TM7 into the lipid bilayer inhibited PAR1 with apparent on rates similar to that of the parent compound in cell signaling assays. These data are consistent with vorapaxar binding to PAR1 via a pathway that passes between TM6 and TM7 from the lipid bilayer, in agreement with the most consistent pathway observed by molecular dynamics. While there is some evidence of entry of the ligand into rhodopsin and lipid-activated GPCRs from the cell membrane, our study provides the first such evidence for a peptide-activated GPCR and suggests that metastable intermediates along drug binding and dissociation pathways can be stabilized by specific interactions between lipids and the ligand.
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- 2018
4. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor
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Corey W. Liu, Luciano Mueller, Brian K. Kobilka, R. Scott Prosser, Foon Sun Thian, Michael P. Bokoch, Joseph D. Puglisi, Yaozhong Zou, Rie Nygaard, Hee Jung Choi, Daniel M. Rosenbaum, Tong Sun Kobilka, William I. Weis, Leonardo Pardo, Juan Jose Fung, and Søren G. F. Rasmussen
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Models, Molecular ,Drug Inverse Agonism ,Static Electricity ,Allosteric regulation ,Crystallography, X-Ray ,Ligands ,Methylation ,Article ,Substrate Specificity ,Propanolamines ,03 medical and health sciences ,0302 clinical medicine ,Allosteric Regulation ,Adrenergic beta-2 Receptor Antagonists ,Formoterol Fumarate ,Extracellular ,Humans ,Inverse agonist ,14. Life underwater ,Receptor ,Adrenergic beta-2 Receptor Agonists ,Nuclear Magnetic Resonance, Biomolecular ,030304 developmental biology ,G protein-coupled receptor ,0303 health sciences ,Binding Sites ,Multidisciplinary ,Chemistry ,Lysine ,Transmembrane protein ,Protein Structure, Tertiary ,Biochemistry ,Membrane protein ,Ethanolamines ,Biophysics ,Mutant Proteins ,Receptors, Adrenergic, beta-2 ,Salt bridge ,030217 neurology & neurosurgery - Abstract
G-protein-coupled receptors (GPCRs) mediate the majority of cellular responses to hormones and neurotransmitters, and these membrane proteins are the largest group of therapeutic targets for a broad range of diseases. It is very difficult to obtain high-resolution X-ray crystal structures of GPCRs; little is known about the functional role(s) of the extracellular surface in receptor activation or about the conformational coupling of the extracellular surface to the native ligand-binding pocket. In this study, Bokoch et al. used NMR spectroscopy to investigate ligand-specific conformational changes around a salt bridge linking extracellular loops 2 and 3 of the β2 adrenergic receptor. They found that drugs that bind within the transmembrane core (and exhibit different efficacies towards G-protein activation) stabilize distinct conformations of the extracellular surface. New therapeutic agents that target this diverse surface could function as allosteric modulators with high subtype selectivity. G-protein-coupled receptors (GPCRs) mediate the majority of cellular responses to hormones and neurotransmitters and are the largest group of therapeutic targets for a range of diseases. The extracellular surface (ECS) of GPCRs is diverse and therefore an ideal target for the discovery of subtype-selective drugs. Here, NMR spectroscopy is used to investigate ligand-specific conformational changes around a central structural feature in the ECS of a GPCR. G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs1,2,3,4,5 have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the β2 adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.
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- 2010
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5. Steady-state oxidation of cholesterol catalyzed by cholesterol oxidase in lipid bilayer membranes on platinum electrodes
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James D. Burgess, Anando Devadoss, Mariela S Palencsár, and Michael P. Bokoch
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chemistry.chemical_classification ,Chromatography ,Cyclodextrin ,Cholesterol oxidase ,Cholesterol ,Bilayer ,Inorganic chemistry ,Phospholipid ,Biochemistry ,Analytical Chemistry ,chemistry.chemical_compound ,Membrane ,chemistry ,Monolayer ,Environmental Chemistry ,lipids (amino acids, peptides, and proteins) ,Lipid bilayer ,Spectroscopy - Abstract
Cholesterol oxidase is immobilized in electrode-supported lipid bilayer membranes. Platinum electrodes are initially modified with a self-assembled monolayer of thiolipid. A vesicle fusion method is used to deposit an outer leaflet of phospholipids onto the thiolipid monolayer forming a thiolipid/lipid bilayer membrane on the electrode surface. Cholesterol oxidase spontaneously inserts into the electrode-supported lipid bilayer membrane from solution and is consequently immobilized to the electrode surface. Cholesterol partitions into the membrane from buffer solutions containing cyclodextrin. Cholesterol oxidase catalyzes the oxidation of cholesterol by molecular oxygen, forming hydrogen peroxide as a product. Amperometric detection of hydrogen peroxide for continuous solution flow experiments are presented, where flow was alternated between cholesterol solution and buffer containing no cholesterol. Steady-state anodic currents were observed during exposures of cholesterol solutions ranging in concentration from 10 to 1000 μM. These data are consistent with the Michaelis–Menten kinetic model for oxidation of cholesterol as catalyzed by cholesterol oxidase immobilized in the lipid bilayer membrane. The cholesterol detection limit is below 1 μM for cholesterol solution prepared in buffered cyclodextrin. The response of the electrodes to low density lipoprotein solutions is increased upon addition of cyclodextrin. Evidence for adsorption of low density lipoprotein to the electrode surface is presented.
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- 2004
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6. From the journal archives: cyclopropane: induction and recovery with a bang!
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Michael P. Bokoch and Adrian W. Gelb
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Cyclopropanes ,medicine.medical_specialty ,Canada ,business.industry ,Archives ,General Medicine ,History, 20th Century ,United States ,Cyclopropane ,chemistry.chemical_compound ,Anesthesiology and Pain Medicine ,chemistry ,Anesthesia ,Rapid onset ,Laboratory Scientists ,Anesthetics, Inhalation ,Medicine ,Humans ,Periodicals as Topic ,business ,Intensive care medicine - Abstract
Lucas G.H.W. Can Anaesth Soc J 1960; 7: 237-56. To review the history of the early development of cyclopropane Cyclopropane was initially investigated because it was thought to be the toxic element in ethylene. Instead, it turned out to be an excellent anesthetic with very rapid onset and recovery while maintaining stable hemodynamics. Its use was ultimately limited because it was highly explosive. Development required collaboration among laboratory scientists and clinicians in Toronto, Canada, clinicians in Madison, USA, and industry in both countries. The phenomenal success of cyclopropane in over 40 years of clinical use resulted from a lucky, but incorrect, hypothesis that it was a toxic contaminant.
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- 2013
7. Lysine methylation strategies for characterizing protein conformations by NMR
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Ferenc Evanics, Sacha Thierry Larda, Michael P. Bokoch, and R. Scott Prosser
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Carbon Isotopes ,Bioconjugation ,Resolution (mass spectrometry) ,Chemistry ,Stereochemistry ,Reducing agent ,Protein Conformation ,Lysine ,Kinetics ,Proteins ,Methylation ,Hydrogen-Ion Concentration ,Biochemistry ,Folding (chemistry) ,Protein structure ,Organic chemistry ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy - Abstract
In the presence of formaldehyde and a mild reducing agent, reductive methylation is known to achieve near complete dimethylation of protein amino groups under non-denaturing conditions, in aqueous media. Amino methylation of proteins is employed in mass spectrometric, crystallographic, and NMR studies. Where biosynthetic labeling is prohibitive, amino (13)C-methylation provides an attractive option for monitoring folding, kinetics, protein-protein and protein-DNA interactions by NMR. Here, we demonstrate two improvements over traditional (13)C-reductive methylation schemes: (1) By judicious choice of stoichiometry and pH, e-aminos can be preferentially monomethylated. Monomethyl tags are less perturbing and generally exhibit improved resolution over dimethyllysines, and (2) By use of deuterated reducing agents and (13)C-formaldehyde, amino groups can be labeled with (13)CH(2)D tags. Use of deutero-(13)C-formaldehyde affords either (13)CHD(2), or (13)CD(3) probes depending on choice of reducing agent. Making use of (13)C-(2)H scalar couplings, we demonstrate a filtering scheme that eliminates natural abundance (13)C signal.
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- 2012
8. Conformational Changes in GPCR Surface and Core Probed by [13C]-Methyl NMR Spectroscopy
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Leonardo Pardo, Rie Nygaard, R. Scott Prosser, Søren G. F. Rasmussen, Luciano Mueller, Michael P. Bokoch, Yaozhong Zou, and Brian K. Kobilka
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Transmembrane domain ,Chemistry ,Stereochemistry ,Allosteric regulation ,Extracellular ,Biophysics ,Inverse agonist ,Nuclear magnetic resonance spectroscopy ,Salt bridge ,Transmembrane protein ,G protein-coupled receptor - Abstract
Recent crystal structures reveal the inactive states of non-rhodopsin G-protein coupled receptors (GPCRs) in beautiful detail. Solution NMR spectroscopy is ideally suited to contribute dynamic information regarding GPCR activation. However, these eukaryotically-expressed membrane proteins remain challenging NMR targets. We apply selective labeling with [13C]methyl probes and two-dimensional NMR to analyze ligand-induced conformational changes in beta2-adrenergic receptor (b2AR).Lysine side chains were labeled with [13C]dimethyl probes to explore conformational changes in the b2AR extracellular surface. Lys305 forms a salt bridge connecting the extracellular end of transmembrane (TM) helix 7 with extracellular loop 2. The Lys305 NMR resonances are sensitive to conformational changes in the receptor extracellular surface. Using NMR, we observe disruption of the Lys305 salt bridge upon receptor activation by agonist. Computational modeling suggests that a lateral displacement of TM7 occurs in concert with an inward motion at the extracellular end of TM6 (thus extending the “global toggle switch” model of Schwartz (2006) Annu. Rev. Pharmacol. Toxicol.) Different conformational changes occur upon inverse agonist binding. Molecular dynamics simulations suggest that a conserved phenylalanine (Phe193) in the orthosteric ligand binding site is key for inverse agonism. Taken as a whole, these results demonstrate conformational coupling between the GPCR extracellular surface and orthosteric ligand binding site within the transmembrane domains (Ahuja (2009) Nat. Struct. Mol. Biol.) This provides rationale for developing allosteric pharmaceuticals targeting the GPCR extracellular surface.Conformational changes within the b2AR transmembrane core are also observed by NMR using selective epsilon-[13CH3] labeling of methionines. While assignments are pending, clear conformational changes are seen with activation or inverse agonist binding. [13C]methyl NMR spectroscopy, in combination with crystal structures and molecular dynamics simulation, provides a dynamic view of the conformational changes intrinsic to GPCR function.
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- 2010
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9. Anti-Brownian ELectrokinetic (ABEL) Trapping of Single High Density Lipoprotein (HDL) Particles
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Samuel Bockenhauer, Michael P. Bokoch, Alexandre Fürstenberg, Roger K. Sunahara, Brian T. DeVree, Brian K. Kobilka, W. E. Moerner, Quan Wang, and Xiao Jie Yao
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Physics::Fluid Dynamics ,Electrokinetic phenomena ,Mathematics::Probability ,Chemistry ,Microfluidics ,Analytical chemistry ,Trapping ,Molecular physics ,Brownian motion - Abstract
The Anti-Brownian ELectrokinetic (ABEL) trap uses voltage feedback to electrokinetically cancel the Brownian motion of single particles in solution in microfluidic geometries. This allows trapping of single high density lipoprotein (HDL) particles for extended observation.
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- 2009
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10. FRET-Based Measurement of GPCR Conformational Changes
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Sébastien Granier, Charles Parnot, Samuel Kim, Juan Jose Fung, and Michael P. Bokoch
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Transmembrane domain ,chemistry.chemical_compound ,Förster resonance energy transfer ,Fluorophore ,Membrane protein ,Chemistry ,Protein domain ,Biophysics ,Arrestin ,sense organs ,G protein-coupled receptor ,Alexa Fluor - Abstract
The C-termini of G protein-coupled receptors (GPCRs) interact with specific kinases and arrestins in an agonist-dependent manner suggesting that conformational changes induced by ligand binding within the transmembrane domains are transmitted to the C-terminus. Forster resonance energy transfer (FRET) can be used to monitor changes in distance between two protein domains if each site can be specifically and efficiently labeled with a donor or acceptor fluorophore. In order to probe GPCR conformational changes, we have developed a FRET technique that uses site-specific donor and acceptor fluorophores introduced by two orthogonal labeling chemistries. Using this strategy, we examined ligand-induced changes in the distance between two labeled sites in the beta(2) adrenoceptor (beta(2)-AR), a well-characterized GPCR model system. The donor fluorophore, LumioGreen, is chelated by a CCPGCC motif [Fluorescein Arsenical Helix or Hairpin binder (FlAsH) site] introduced through mutagenesis. The acceptor fluorophore, Alexa Fluor 568, is attached to a single reactive cysteine (C265). FRET analyses revealed that the average distance between the intracellular end of transmembrane helix (TM) six and the C-terminus of the beta(2)-AR is 62 A. This relatively large distance suggests that the C-terminus is extended and unstructured. Nevertheless, ligand-specific conformational changes were observed (1). The results provide new insight into the structure of the beta(2)-AR C-terminus and ligand-induced conformational changes that may be relevant to arrestin interactions. The FRET labeling technique described herein can be applied to many GPCRs (and other membrane proteins) and is suitable for conformational studies of domains other than the C-terminus.
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- 2009
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