The dynamic process of GPCR activation: Insights from the human β 2 AR
|
|
- Mae Lester
- 6 years ago
- Views:
Transcription
1 The structural basis of G protein coupled receptor signaling The dynamic process of GPCR activation: Insights from the human β 2 AR Brian Kobilka Department of Molecular and Cellular Physiology Brian Kobilka Molecular Stanford and Cellular Physiology Stanford University
2 The β 2 AR modulates the activity of multiple signaling pathways Ca 2+ Channel Adenylyl Cyclase Efficacy Basal
3 Agonist binding G protein coupling and nucleotide exchange Activated G protein subunits regulate effector proteins GTP hydrolysis and inactivation of Gα protein GPCR-G Protein Cycle
4 Outline Overview of approaches to characterize GPCR structure GPCR crystallography Mechanistic insights into GPCR-G protein activation Agonist binding G protein coupling and nucleotide exchange Activated G protein subunits regulate effector proteins GTP hydrolysis and inactivation of Gα protein GPCR-G Protein Cycle
5 Approaches to characterizing β 2 AR structure: Cloned DNA Sequence (1986) GAATTCATGCCGCGTTTCTGTGTTGGACAGGGGTGACTTTGTGCC GGATGGCTTCTGTGTGAGAGCGCGCGCGAGTGTGCATGTCGGTGA GCTGGGAGGGTGTGTCTCAGTGTCTATGGCTGTGGTTCGGTATAAG TCTAAGCATGTCTGCCAGGGTGTATTTGTGCCTGTATGTGCGTGCCT CGGTGGGCACTCTCGTTTCCTTCCGAATGTGGGGCAGTGCCGGTG TGCTGCCCTCTGCCTTGAGACCTCAAGCCGCGCAGGCGCCCAGGG CAGGCAGGTAGCGGCCACAGAAGAGCCAAAAGCTCCCGGGTTGG CTGGTAAGCACACCACCTCCAGCTTTAGCCCTCTGGGGCCAGCCA GGGTAGCCGGGAAGCAGTGGTGGCCCGCCCTCCAGGGAGCAGTT GGGCCCCGCCCGGGCCAGCCTCAGGAGAAGGAGGGCGAGGGGA GGGGAGGGAAAGGGGAGGAGTGCCTCGCCCCTTCGCGGCTGCC GGCGTGCCATTGGCCGAAAGTTCCCGTACGTCACGGCGAGGGCA GTTCCCCTAAAGTCCTGTGCACATAACGGGCAGAACGCACTGCGA AGCGGCTTCTTCAGAGCACGGGCTGGAACTGGCAGGCACCGCGA GCCCCTAGCACCCGACAAGCTGAGTGTGCAGGACGAGTCCCCACC ACACCCACACCACAGCCGCTGAATGAGGCTTCCAGGCGTCCGCTC GCGGCCCGCAGAGCCCCGCCGTGGGTCCGCCTGCTGAGGCGCCC CCAGCCAGTGCGCTTACCTGCCAGACTGCGCGCCATGGGGCAACC CGGGAACGGCAGCGCCTTCTTGCTGGCACCCAATAGAAGCCATGC GCCGGACCACGACGTCACGCAGCAAAGGGACGAGGTGTGGGTG GTGGGCATGGGCATCGTCATGTCTCTCATCGTCCTGGCCATCGTGTT TGGCAATGTGCTGGTCATCACAGCCATTGCCAAGTTCGAGCGTCTG CAGACGGTCACCAAC Sequence analysis secondary structure (transmembrane domains) Amino Acid Sequence MGQPGNGSAFLLAPNRSHAPDHDVT QQRDEVWVVGMGIVMSLIVLAIVFGN VLVITAIAKFERLQTVTNYFITSLACADLV MGLAVVPFGAHILMKMWTFGNFWCE FWTSIDVLCVTASIETLCVIAVDRYFAITS PFKYQSLLTKNKARVIILMVWIVSGLTSF LPIQMHWYRATHQEAINCYANETCCDF FTNQAYAIASSIVSFYVPLVIMVFVYSRV FQEAKRQLQKIDKSEGRFHVQNLSQVE QDGRTGHGLRRSSKFCLKEHKALKTLGII MGTFTLCWLPFFIVNIVHVIQDNLIRKE VYILLNWIGYVNSGFNPLIYCRSPDFRIA FQELLCLRRSSLKAYGNGYSSNGNTGEQ SGYHVEQEKENKLLCEDLPGTEDFVGH QGTVPSDNIDSQGRNCSTNDSLL
6 Approaches to characterizing β 2 AR structure: Cloned DNA Sequence (1986) GAATTCATGCCGCGTTTCTGTGTTGGACAGGGGTGACTTTGTGCC GGATGGCTTCTGTGTGAGAGCGCGCGCGAGTGTGCATGTCGGTGA GCTGGGAGGGTGTGTCTCAGTGTCTATGGCTGTGGTTCGGTATAAG TCTAAGCATGTCTGCCAGGGTGTATTTGTGCCTGTATGTGCGTGCCT CGGTGGGCACTCTCGTTTCCTTCCGAATGTGGGGCAGTGCCGGTG TGCTGCCCTCTGCCTTGAGACCTCAAGCCGCGCAGGCGCCCAGGG CAGGCAGGTAGCGGCCACAGAAGAGCCAAAAGCTCCCGGGTTGG CTGGTAAGCACACCACCTCCAGCTTTAGCCCTCTGGGGCCAGCCA GGGTAGCCGGGAAGCAGTGGTGGCCCGCCCTCCAGGGAGCAGTT GGGCCCCGCCCGGGCCAGCCTCAGGAGAAGGAGGGCGAGGGGA GGGGAGGGAAAGGGGAGGAGTGCCTCGCCCCTTCGCGGCTGCC GGCGTGCCATTGGCCGAAAGTTCCCGTACGTCACGGCGAGGGCA GTTCCCCTAAAGTCCTGTGCACATAACGGGCAGAACGCACTGCGA AGCGGCTTCTTCAGAGCACGGGCTGGAACTGGCAGGCACCGCGA GCCCCTAGCACCCGACAAGCTGAGTGTGCAGGACGAGTCCCCACC ACACCCACACCACAGCCGCTGAATGAGGCTTCCAGGCGTCCGCTC GCGGCCCGCAGAGCCCCGCCGTGGGTCCGCCTGCTGAGGCGCCC CCAGCCAGTGCGCTTACCTGCCAGACTGCGCGCCATGGGGCAACC CGGGAACGGCAGCGCCTTCTTGCTGGCACCCAATAGAAGCCATGC GCCGGACCACGACGTCACGCAGCAAAGGGACGAGGTGTGGGTG GTGGGCATGGGCATCGTCATGTCTCTCATCGTCCTGGCCATCGTGTT TGGCAATGTGCTGGTCATCACAGCCATTGCCAAGTTCGAGCGTCTG CAGACGGTCACCAAC Sequence analysis secondary structure (transmembrane domains) Amino Acid Sequence MGQPGNGSAFLLAPNRSHAPDHDVT QQRDEVWVVGMGIVMSLIVLAIVFGN VLVITAIAKFERLQTVTNYFITSLACADLV MGLAVVPFGAHILMKMWTFGNFWCE FWTSIDVLCVTASIETLCVIAVDRYFAITS PFKYQSLLTKNKARVIILMVWIVSGLTSF LPIQMHWYRATHQEAINCYANETCCDF FTNQAYAIASSIVSFYVPLVIMVFVYSRV FQEAKRQLQKIDKSEGRFHVQNLSQVE QDGRTGHGLRRSSKFCLKEHKALKTLGII MGTFTLCWLPFFIVNIVHVIQDNLIRKE VYILLNWIGYVNSGFNPLIYCRSPDFRIA FQELLCLRRSSLKAYGNGYSSNGNTGEQ SGYHVEQEKENKLLCEDLPGTEDFVGH QGTVPSDNIDSQGRNCSTNDSLL
7 Approaches to characterizing β 2 AR structure: Sequence analysis secondary structure (transmembrane domains) post-translational modifications glycosylation palmitoylation phosphorylation
8 Approaches to characterizing β 2 AR structure: Sequence analysis secondary structure (transmembrane domains) post-translational modifications Chimeric Receptors and site-directed mutagenesis ligand binding and G protein coupling Ligand binding G protein coupling specificity
9 Approaches to characterizing β 2 AR structure: FLAG Signal Sequence Sequence analysis secondary structure (transmembrane domains) post-translational modifications Chimeric Receptors and Site-directed mutagenesis ligand binding and G protein coupling enhance expression and purification HHHHHH
10 Biochemistry Spectroscopy Crystallography
11 Approaches to characterizing β 2 AR structure: FLAG Signal Sequence Sequence analysis secondary structure (transmembrane domains) post-translational modifications Chimeric Receptors and site-directed mutagenesis ligand binding and G protein coupling enhance expression and purification HHHHHH
12 Approaches to characterizing β 2 AR structure: FLAG Signal Sequence Sequence analysis secondary structure (transmembrane domains) post-translational modifications Chimeric Receptors and site-directed mutagenesis ligand binding and G protein coupling enhance expression and purification Biochemistry unstructured, flexible sequence HHHHHH
13 Approaches to characterizing β 2 AR structure: TM6 Cysteine 265 FLAG Signal Sequence Sequence analysis secondary structure (transmembrane domains) post-translational modifications Chimeric Receptors and site-directed mutagenesis ligand binding and G protein coupling enhance expression and purification Biochemistry unstructured, flexible sequence Spectroscopy (Fluorescence, EPR, NMR) ligand-specific conformational states dynamic, flexible character useful tool for monitoring receptor activity HHHHHH
14 Conformational changes in TM6 in response to norepinepherine: % Change in Intensity Rhodamine Time (sec) Gayathri Swaminath, 2004
15 Conformational changes in TM6 in response to norepinepherine: % Change in Intensity Biphasic changes in intensity over time Rapid t1/2 < 5 sec Slow t1/2 = 70 sec Rhodamine Time (sec) Gayathri Swaminath, 2004
16 Conformational changes in TM6 in response to norepinepherine: Not consistent with single active state A + R AR* % Change in Intensity Biphasic changes in intensity over time Rapid t1/2 < 5 sec Slow t1/2 = 70 sec Rhodamine Time (sec) Gayathri Swaminath, 2004
17 Conformational changes in TM6 in response to norepinepherine: Sequential conformational changes A + R AR + AR* % Change in Intensity Biphasic changes in intensity over time Rapid t1/2 < 5 sec Slow t1/2 = 70 sec Rhodamine Time (sec) Gayathri Swaminath, 2004
18 Fluorescence lifetime experiments Multiple agonist states Ligand-specific conformational states Fluorescein A + R AR + AR* P + R PR + PR # Efficacy AR + Basal AR* AR* PR # Pejman Ghanouni, 2001
19 Insights from spectroscopy studies -fluorescence, NMR, EPR- TM6 Ansgar Philippsen & Ron Dror (D.E. Shaw Research) The β 2 AR is flexible and dynamic TM6 undergoes the largest changes in response to agonists Agonist binding and activation occur through a series of conformational intermediates Agonists and partial agonists stabilize distinct conformational states Agonists alone do not stabilize a single active conformation. Fluorescence spectroscopy aided in identifying optimal conditions for crystallography
20 Outline Overview of approaches to characterize GPCR structure GPCR crystallography Mechanistic insights into GPCR-G protein activation Agonist binding G protein coupling and nucleotide exchange Activated G protein subunits regulate effector proteins GTP hydrolysis and inactivation of Gα protein GPCR-G Protein Cycle
21 Crystals of wild-type β 2 AR Nov 2004 ESRF microfocus beamline ID13, July µm 20Å
22 TM5 Purified protein TM6 Conformationally uniform GPCR High-quality crystal
23 TM5 Purified protein TM6 Conformationally heterogeneous GPCR Poor quality crystal
24 Challenges for crystallography Protein dynamics TM5 TM6
25 Challenges for crystallography Protein dynamics Little polar surface TM5 TM6
26 Challenges for crystallography Protein dynamics Little polar surface Stabilize and increase polar surface Antibodies Protein Engineering
27 Approaches for GPCR crystallogenesis: Antibodies and protein engineering 2007 Søren Rasmussen Dan Rosenbaum TM5 TM6 TM5 TM6 T4L Fab
28 Approaches for GPCR crystallogenesis: Antibodies and protein engineering Lipid-based media: bicelles and lipidic cubic phase 2007 Søren Rasmussen Dan Rosenbaum TM5 TM6 TM5 TM6 T4L Fab
29 Approaches for GPCR crystallogenesis: Antibodies and protein engineering Lipid-based media: bicelles and lipidic cubic phase 2007 Søren Rasmussen Dan Rosenbaum T4L TM5 TM6 TM5 TM6 TM5 TM6 T4L Fab
30 Inactive-state GPCR structures Antibodies and Protein Engineering T4L Other Approaches Thermostabilization through alanine scanning mutations β 1 AR, Adenosine A2A Tate and Schertler Rhodopsin (native) Schertler (2D crystals) Palczewski and Okada (3D) Ernst and Hofmann (Opsin) Fab T4L Recent GPCR-T4L structures from Kobilka Lab and collaborators Stevens Lab and collaborators M2 Muscarinic M3 Muscarinic δ Opioid µ Opioid PAR1 (Haga and Kobayashi) (Jurgen Wess) (Sebastien Granier) (Shaun Coughlin) Adenosine A2A D3 Dopamine CXCR4 Histamine S1P1 κ-opioid
31 Outline Overview of approaches to characterize GPCR structure GPCR crystallography Mechanistic insights into GPCR-G protein activation Agonist binding G protein coupling and nucleotide exchange Activated G protein subunits regulate effector proteins GTP hydrolysis and inactivation of Gα protein GPCR-G Protein Cycle
32 β 2 AR INACTIVE β 2 AR ACTIVE? Inverse Agonist Agonist (covalent) T4L Fab T4L T4L Dan Rosenbaum Ralph Holl Peter Gmeiner Pipette
33 β 2 AR INACTIVE β 2 AR ACTIVE? Inverse Agonist T4L Agonist (covalent) Agonist alone does not fully stabilize active state β 2 AR β 2 AR + Agonist Fab T4L T4L Pipette
34 β 2 AR INACTIVE β 2 AR ACTIVE? Inverse Agonist T4L Agonist (covalent) Agonist alone does not fully stabilize active state β 2 AR β 2 AR + Agonist Fab T4L T4L Stabilizing protein Pipette
35 β 2 AR INACTIVE β 2 AR ACTIVE? Inverse Agonist T4L Agonist (covalent) Agonist alone does not fully stabilize active state β 2 AR β 2 AR + Agonist Fab T4L T4L Stabilizing protein Nanobody variable domain of a single chain camelid antibody Pipette Jan Steyaert Els Pardon
36 β 2 AR INACTIVE β 2 AR ACTIVE Inverse Agonist Agonist (covalent) Partial Agonist Agonist T4L T4L Fab T4L T4L Nb71 Nb80 Stabilizing protein Nanobody variable domain of a single chain camelid antibody Pipette Jan Steyaert Els Pardon
37 β 2 AR INACTIVE β 2 AR ACTIVE Inverse Agonist Agonist (covalent) Partial Agonist Agonist T4L T4L Fab T4L T4L Nb71 Nb80 β 2 AR-Gs Nanobody variable domain of a single chain camelid antibody Pipette Jan Steyaert Els Pardon
38 Technical contributions to crystallizing the β 2 AR-Gs complex High-affinity agonist BI (1 of ~ 60 screened) Removal of GDP Apyrase Detergent: MNG-3 (long-term storage, aids transition into LCP) New mesophase lipid (7.7 MAG) to accommodate G protein (provided by Martin Caffrey) Nanobody to stabilize G protein complex (Jan Steyaert) Amino Terminal T4 Lysozyme Project guided by data from negative stain single particle EM (Georgios Skiniotis)
39 Microcrystallography GM/CA-CAT at Argonne National Labs β 2 AR-Gs complex Andy Kruse, Brian DeVree, Søren Rasmussen me Roger Sunahara Returning from Argonne with final data set April 2011
40 Gsαβγ β 2 AR
41 Inactive Active β 2 AR-Cz β 2 AR-Gs
42 Active state of β 2 AR Cytoplasmic View 14Å 14Å β 2 AR - Inactive β 2 AR-Gs
43 Extracellular Surface β 2 AR-Cz TM3 Carazolol TM5 S203 D113 S204 S207 N312 W286 TM6 TM7
44 Extracellular Surface β 2 AR-Gs BI TM3 TM5 S203 D113 S204 S207 N312 W286 TM6 TM7
45 Inactive Active
46 Extracellular Surface β 2 AR-Cz β 2 AR-Gs TM3 TM5 S203 D113 S204 S207 N312 2Å TM6 W286 TM7
47 Extracellular TM3 β 2 AR-Inactive TM5 TM7 2Å Pro 211 Ile121 Phe282 Asn 318 Packing of conserved amino acids maintains inactive state. 14Å Cytoplasm TM6
48 Extracellular TM5 TM3 TM3 TM7 TM7 β 2 AR-Inactive β 2 AR-Active 2Å Weak coupling between ligand binding and G protein coupling domains Pro Pro Phe282 Ile121 Ile121 Phe282 Asn 318 Asn 318 Rearrangement of conserved amino acids required to accommodate agonist binding. 14Å Cytoplasm TM6 TM6
49 β 2 AR Gsαβγ Inactive Gα Ras-like domain GDP α-helical domain Interactions between the β 2 AR and Gs promote GDP release.
50 β 2 AR Gsαβγ Inactive Active
51 β 2 AR α helical domain Gsαβγ Active Inactive
52 Mobility of alpha helical domain confirmed by EM G Ras G αh Nb37 Gerwin Westfield and Georgios Skiniotis, Univ. Michigan
53 Ca 2+ Channel Adenylyl Cyclase Future Directions Efficacy Basal
54 β 2 AR-Gs Team GPCR Workshop, Maui, Dec. 2011
55 Many Thanks Tong Sun Kobilka Bob Lefkowitz and colleagues in the Lefkolab Kobilka lab students, postdoctoral fellows and collaborators ( ) Bill Weis and Roger Sunahara Stanford Søren Rasmussen Foon Sun Thian Tong Sun Kobilka Yaozhong Zou Andrew Kruse Ka Young Chung Jesper Mathiesen Bill Weis β 2 AR-Gs Team University of Michigan Brian DeVree Diane Calinski Gerwin Westfield Georgios Skiniotis Roger Sunahara University of Wisconsin Pil Seok Chae Sam Gellman We will miss Virgil Woods (UCSD), Free University of Brussels Els Pardon Jan Steyaert Trinity College Dublin Joseph Lyons Syed Shah Martin Caffrey Financial Support NIH NINDS and NIGMS Gifts from: Mathers Foundation Lundbeck
Structures of the human! 2 Adrenoceptor ! 2 AR-T4L. ! 2 AR-Fab5. Expression Purification of functional receptor Stabilization!Protein engineering!!
Structures of the human! 2 Adrenoceptor! 2 AR-Fab5! 2 AR-T4L Expression Purification of functional receptor Stabilization!Protein engineering!! 2 AR-Fab complex Lipid-based crystallization Microbeam X-ray
More informationThe Confo body technology, a new platform to enable fragment screening on GPCRs
The Confo body technology, a new platform to enable fragment screening on GPCRs Christel Menet, PhD CSO Miptec, 216 Confo Therapeutics Incorporated in June 215 Located in Brussels, Belgium Confo Therapeutics
More informationScience June 3, 1988 v240 n4857 p1310(7) Page 1
Science June 3, 1988 v240 n4857 p1310(7) Page 1 by Brian K. Kobilka, Tong Sun Kobilka, Kiefer Daniel, John W. Regan, Marc G. Caron and Robert J. Lefkowitz COPYRIGHT 1988 American Association for the Advancement
More informationN-Terminal T4 Lysozyme Fusion Facilitates Crystallization of a G Protein Coupled Receptor
N-Terminal T4 Lysozyme Fusion Facilitates Crystallization of a G Protein Coupled Receptor Yaozhong Zou 1, William I. Weis 1,2, Brian K. Kobilka 1 * 1 Department of Molecular and Cellular Physiology, Stanford
More informationGisselle A. Vélez Ruiz
ALLOSTERIC REGULATION OF G S ON AGONIST, ANTAGONIST AND INVERSE AGONIST BINDING TO THE β 2 AR by Gisselle A. Vélez Ruiz A dissertation submitted in partial fulfillment of the requirements for the degree
More informationLarge, medically important class of proteins starts to yield its secrets 12 July 2012
Large, medically important class of proteins starts to yield its secrets 12 July 2012 More than 50 percent of therapeutic drugs target GPCRs, including allergy and heart medication, drugs that target the
More informationAllosteric Control of Beta2-adrenergic Receptor Function
Allosteric Control of Beta2-adrenergic Receptor Function by Jacob Patrick Mahoney A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Pharmacology)
More informationStructural Genomics of the Human GPCR Protein Family
Structural Genomics of the Human GPCR Protein Family Julius Axelrod Symposium ASPET - April, 2010 Stevens Laboratory The Scripps Research Institute G Protein-Coupled Receptors Thousands of Ligands - Chemical
More informationTime allowed: 2 hours Answer ALL questions in Section A, ALL PARTS of the question in Section B and ONE question from Section C.
UNIVERSITY OF EAST ANGLIA School of Biological Sciences Main Series UG Examination 2017-18 CELL BIOLOGY BIO-5005B Time allowed: 2 hours Answer ALL questions in Section A, ALL PARTS of the question in Section
More informationSUPPLEMENTARY INFORMATION
doi: 10.1038/nature06147 SUPPLEMENTARY INFORMATION Figure S1 The genomic and domain structure of Dscam. The Dscam gene comprises 24 exons, encoding a signal peptide (SP), 10 IgSF domains, 6 fibronectin
More informationENGINEERING THE ANGIOTENSIN II TYPE 1 RECEPTOR FOR STRUCTURAL STUDIES
ENGINEERING THE ANGIOTENSIN II TYPE 1 RECEPTOR FOR STRUCTURAL STUDIES JENNIFER ANN THOMAS THIS DISSERTATION IS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY ST EDMUND S COLLEGE SEPTEMBER 2014 TABLE
More informationA hybrid structural approach to analyze ligand binding by the 5-HT4. receptor
Supplement A hybrid structural approach to analyze ligand binding by the 5-HT4 receptor Pius S. Padayatti 1**, Liwen Wang 2**, Sayan Gupta 3, Tivadar Orban 4, Wenyu Sun 1, David Salom 1, Steve Jordan 5,
More informationIntroduction to Proteins
Introduction to Proteins Lecture 4 Module I: Molecular Structure & Metabolism Molecular Cell Biology Core Course (GSND5200) Matthew Neiditch - Room E450U ICPH matthew.neiditch@umdnj.edu What is a protein?
More informationThe Making of the Fittest: Natural Selection and Adaptation
INTRODUCTION THE ROCK POCKET MOUSE THE BIOCHEMISTRY AND CELL SIGNALING PATHWAY OF MC1R The rock pocket mouse, Chaetodipus intermedius, is a small, nocturnal animal found in the deserts of the southwestern
More informationGα i Activation Assay Kit
A helping hand for your research Product Manual Configuration-specific Monoclonal Antibody Based Gα i Activation Assay Kit Catalog Number 80301 20 assays NewEast Biosciences, Inc 1 Table of Content Product
More informationDNA Binding Domains: Structural Motifs. Effector Domain. Zinc Fingers. Zinc Fingers, continued. Zif268
DNA Binding Domains: Structural Motifs Studies of known transcription factors have found several motifs of protein design to allow sequence-specific binding of DNA. We will cover only three of these motifs:
More informationDEPARTMENT OF BIOCHEMISTRY & MOLECULAR BIOLOGY UNDERGRADUATE POSTER SESSION. Abstract Poster Presentations
DEPARTMENT OF BIOCHEMISTRY & MOLECULAR BIOLOGY UNDERGRADUATE POSTER SESSION Abstract Poster Presentations Thursday, April 1, 2004 Atrium, Biomedical & Physical Sciences Building 3:00 5:00 p.m. Posters
More informationCFSSP: Chou and Fasman Secondary Structure Prediction server
Wide Spectrum, Vol. 1, No. 9, (2013) pp 15-19 CFSSP: Chou and Fasman Secondary Structure Prediction server T. Ashok Kumar Department of Bioinformatics, Noorul Islam College of Arts and Science, Kumaracoil
More informationImmunoglobulins. Harper s biochemistry Chapter 49
Immunoglobulins Harper s biochemistry Chapter 49 Immune system Detects and inactivates foreign molecules, viruses, bacteria and microorganisms Two components with 2 strategies B Lymphocytes (humoral immune
More informationThe Two-Hybrid System
Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine The Two-Hybrid System Carolina Vollert & Peter Uetz Institut für Genetik Forschungszentrum Karlsruhe PO Box 3640 D-76021 Karlsruhe
More informationSupporting Information
Supporting Information Drugs Modulate Interactions Between the First Nucleotide-Binding Domain and the Fourth Cytoplasmic Loop of Human P-glycoprotein Tip W. Loo and David M. Clarke Department of Medicine
More informationIntroduction to Protein Purification
Introduction to Protein Purification 1 Day 1) Introduction to Protein Purification. Input for Purification Protocol Development - Guidelines for Protein Purification Day 2) Sample Preparation before Chromatography
More informationAashish Manglik, M.D., Ph.D. Curriculum Vitae
Aashish Manglik, M.D., Ph.D. Curriculum Vitae Email: amanglik@stanford.edu Beckman Building 279 Campus Drive, Lab B101 Stanford, CA 94305-5461 Research Profile Cellular signaling plays a critical role
More informationPurification: Step 1. Lecture 11 Protein and Peptide Chemistry. Cells: Break them open! Crude Extract
Purification: Step 1 Lecture 11 Protein and Peptide Chemistry Cells: Break them open! Crude Extract Total contents of cell Margaret A. Daugherty Fall 2003 Big Problem: Crude extract is not the natural
More informationPurification: Step 1. Protein and Peptide Chemistry. Lecture 11. Big Problem: Crude extract is not the natural environment. Cells: Break them open!
Lecture 11 Protein and Peptide Chemistry Margaret A. Daugherty Fall 2003 Purification: Step 1 Cells: Break them open! Crude Extract Total contents of cell Big Problem: Crude extract is not the natural
More informationBIRKBECK COLLEGE (University of London)
BIRKBECK COLLEGE (University of London) SCHOOL OF BIOLOGICAL SCIENCES M.Sc. EXAMINATION FOR INTERNAL STUDENTS ON: Postgraduate Certificate in Principles of Protein Structure MSc Structural Molecular Biology
More informationThe Stringent Response
The Stringent Response When amino acids are limiting a response is triggered to shut down a wide range of biosynthetic processes This process is called the Stringent Response It results in the synthesis
More informationCELL BIOLOGY - CLUTCH CH. 7 - GENE EXPRESSION.
!! www.clutchprep.com CONCEPT: CONTROL OF GENE EXPRESSION BASICS Gene expression is the process through which cells selectively to express some genes and not others Every cell in an organism is a clone
More informationBIOCHEMISTRY REVIEW. Overview of Biomolecules. Chapter 13 Protein Synthesis
BIOCHEMISTRY REVIEW Overview of Biomolecules Chapter 13 Protein Synthesis 2 3 4 5 6 7 8 9 10 Are You Getting It?? Which properties are characteristic of the normal genetic code? (multiple answers) a) A
More informationThe mechanism(s) of protein folding. What is meant by mechanism. Experimental approaches
The mechanism(s) of protein folding What is meant by mechanism Computational approaches Experimental approaches Questions: What events occur and in what time sequence when a protein folds Is there a specified
More informationDiscovery on Target. Short Course Preview Deck
Discovery on Target Short Course Preview Deck Targeting of GPCRs with Monoclonal Antibodies Barbara Swanson, Ph.D. Sorrento Therapeutics, Inc. October 2014 GPCR Short Course Overview Introductions Brief
More informationSequence Determinants of a Conformational Switch in a Protein
Sequence Determinants of a Conformational Switch in a Protein Thomas A. Anderson, Matthew H. J. Cordes, and Robert T. Sauer Kyle Skalenko 4/17/09 Introduction Protein folding is guided by the following
More informationRecruitment of Grb2 to surface IgG and IgE provides antigen receptor-intrinsic costimulation to class-switched B cells
SUPPLEMENTARY FIGURES Recruitment of Grb2 to surface IgG and IgE provides antigen receptor-intrinsic costimulation to class-switched B cells Niklas Engels, Lars Morten König, Christina Heemann, Johannes
More informationCell-Extracellular Matrix Interactions
Cell-Extracellular Matrix Interactions Extracellular Matrix (ECM) Organised network outside of the cell s plasma membrane Between cells Composition varies throughout the ECM Continuous sheet of ECM = Basement
More informationHomology Modeling of Mouse orphan G-protein coupled receptors Q99MX9 and G2A
Quality in Primary Care (2016) 24 (2): 49-57 2016 Insight Medical Publishing Group Research Article Homology Modeling of Mouse orphan G-protein Research Article Open Access coupled receptors Q99MX9 and
More informationProtein Folding Problem I400: Introduction to Bioinformatics
Protein Folding Problem I400: Introduction to Bioinformatics November 29, 2004 Protein biomolecule, macromolecule more than 50% of the dry weight of cells is proteins polymer of amino acids connected into
More informationChallenges to measuring intracellular Ca 2+ Calmodulin: nature s Ca 2+ sensor
Calcium Signals in Biological Systems Lecture 3 (2/9/0) Measuring intracellular Ca 2+ signals II: Genetically encoded Ca 2+ sensors Henry M. Colecraft, Ph.D. Challenges to measuring intracellular Ca 2+
More informationMCB110 FINAL. Dec 13, Your name and student ID
MCB110 FINAL Dec 13, 2007 Your name and student ID. QUESTION POINTS 1 (15 points) 2 (15 points) 3 (15 points) 4 (20 points) 5 (25 points) 6 (20 points) 7 (20 points) 8 (20 points) 9 (25 points) 10 (25
More informationA monoclonal antibody for G protein coupled receptor crystallography
A monoclonal antibody for G protein coupled receptor crystallography Peter W Day, Søren G F Rasmussen, Charles Parnot, Juan José Fung, Asna Masood, Tong Sun Kobilka, Xiao-Jie Yao, Hee-Jung Choi, William
More informationAntibody Structure. Antibodies
Antibodies Secreted by B lymphocytes Great diversity and specificity: >10 9 different antibodies; can distinguish between very similar molecules Tag particles for clearance/destruction Protect against
More informationAntibody Structure supports Function
Antibodies Secreted by B lymphocytes Great diversity and specificity: >10 9 different antibodies; can distinguish between very similar molecules Tag particles for clearance/destruction Protect against
More informationBIO 311C Spring Lecture 16 Monday 1 March
BIO 311C Spring 2010 Lecture 16 Monday 1 March Review Primary Structure of a portion of a polypeptide chain backbone of Polypeptide chain R-groups of amino acids Native conformation of a dimeric protein,
More informationHomogeneous GTPγS Assay for High Throughput Screening of GPCRs
Homogeneous GTPγS Assay for High Throughput Screening of GPCRs Gregory Warner, Ph.D.*, Patricia Kasila, and Harry Harney * Enanta Pharmaceuticals Inc., 750 Main Street, Cambridge, MA 02139, 549 Albany
More informationBC 367, Exam 2 November 13, Part I. Multiple Choice (3 pts each)- Please circle the single best answer.
Name BC 367, Exam 2 November 13, 2008 Part I. Multiple Choice (3 pts each)- Please circle the single best answer. 1. The enzyme pyruvate dehydrogenase catalyzes the following reaction. What kind of enzyme
More informationAntibodies to Challenging Receptor Targets through NGS and Cell-Based Antibody Phage Panning. Mark Tornetta
Antibodies to Challenging Receptor Targets through NGS and Cell-Based Antibody Phage Panning Mark Tornetta Points to convey regarding receptor targets Antigen diversity- cells and engineered ECDs Be creative
More informationChapter 14 Regulation of Transcription
Chapter 14 Regulation of Transcription Cis-acting sequences Distance-independent cis-acting elements Dissecting regulatory elements Transcription factors Overview transcriptional regulation Transcription
More informationThe Central Dogma. DNA makes RNA makes Proteins
The Central Dogma DNA makes RNA makes Proteins TRANSCRIPTION DNA RNA transcript RNA polymerase RNA PROCESSING Exon RNA transcript (pre-) Intron Aminoacyl-tRNA synthetase NUCLEUS CYTOPLASM FORMATION OF
More informationThis article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and
This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution
More informationChapter One. Construction of a Fluorescent α5 Subunit. Elucidation of the unique contribution of the α5 subunit is complicated by several factors
4 Chapter One Construction of a Fluorescent α5 Subunit The significance of the α5 containing nachr receptor (α5* receptor) has been a challenging question for researchers since its characterization by
More informationSupplementary Information
Supplementary Information Supplementary Figure 1 Force-distance curve-based (FD-based) AFM. (a) To contour the surface of the sample FD-based AFM approaches and retracts the tip of the AFM cantilever pixel-by-pixel.
More informationProtein analysis. Dr. Mamoun Ahram Summer semester, Resources This lecture Campbell and Farrell s Biochemistry, Chapters 5
Protein analysis Dr. Mamoun Ahram Summer semester, 2015-2016 Resources This lecture Campbell and Farrell s Biochemistry, Chapters 5 Bases of protein separation Proteins can be purified on the basis Solubility
More informationSome Perspectives from the Office of Science
Some Perspectives from the Office of Science Board on Physics and Astronomy 26 April 2013 Dr. Patricia M. Dehmer Deputy Director for Science Programs, Office of Science U.S. Department of Energy www.science.energy.gov
More informationThe opioid receptors belong to the Class A subfamily of G-Protein Coupled Receptors
POOLE, ELIZABETH B., M.S. Construction of a μ-opioid Receptor Model: Identification of the Opioid Alkaloid Binding Pocket. (2009) Directed by Dr. Patricia H. Reggio. 46pp. Morphine and other analgesics
More informationProspector Custom Peptide Libraries
Prospector Custom Peptide Libraries Prospector custom peptide libraries allow the rapid screening of many peptides for bioreactivity. Overlapping peptide libraries can be used to investigate immune responses
More informationHomology Modelling. Thomas Holberg Blicher NNF Center for Protein Research University of Copenhagen
Homology Modelling Thomas Holberg Blicher NNF Center for Protein Research University of Copenhagen Why are Protein Structures so Interesting? They provide a detailed picture of interesting biological features,
More informationSignal Amplification or Organization?
Signal Amplification or Organization? In the past, the biochemistry and in vitro systems emphasized enzymatic activities. Amplification. Today, in vivo studies demonstrate the importance of organization
More informationNature Structural & Molecular Biology: doi: /nsmb.3018
Supplementary Figure 1 Validation of genetic complementation assay in Bmal1 / Per2 Luc fibroblasts. (a) Only Bmal1, not Bmal2, rescues circadian rhythms from cells. Cells expressing various Bmal constructs
More informationa) JOURNAL OF BIOLOGICAL CHEMISTRY b) PNAS c) NATURE
a) JOURNAL OF BIOLOGICAL CHEMISTRY b) c) d) ........................ JOURNAL OF BIOLOGICAL CHEMISTRY MOLECULAR PHARMACOLOGY TRENDS IN PHARMACOLOGICAL S AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY
More informationThe Green Fluorescent Protein. w.chem.uwec.edu/chem412_s99/ppt/green.ppt
The Green Fluorescent Protein w.chem.uwec.edu/chem412_s99/ppt/green.ppt www.chem.uwec.edu/chem412_s99/ppt/green.ppt Protein (gene) is from a jellyfish: Aequorea victoria www.chem.uwec.edu/chem412_s99/ppt/green.ppt
More informationProtein-Protein Interactions I
Biochemistry 412 Protein-Protein Interactions I March 23, 2007 Macromolecular Recognition by Proteins Protein folding is a process governed by intramolecular recognition. Protein-protein association is
More informationNewsletter Issue 7 One-STrEP Analysis of Protein:Protein-Interactions
www.iba-biotagnology.com Newsletter Issue 7 One-STrEP Analysis of Protein:Protein-Interactions Strep-tag and One-STrEP-tag PPI Analysis with the co-precipitation/ mass spectrometry approach 3 Background
More informationConformational properties of enzymes
Conformational properties of enzymes; Physics of enzyme substrate interactions; Electronic conformational interactions, cooperative properties of enzymes Mitesh Shrestha Conformational properties of enzymes
More informationRegulatory Consideration for the Characterization of HOS in Biotechnology Products
Regulatory Consideration for the Characterization of HOS in Biotechnology Products Maria Teresa Gutierrez Lugo, Ph.D. OBP/CDER/FDA 5 th International Symposium on Higher Order Structure of Protein Therapeutics
More informationINSIGHTS FROM FLUORESCENCE RESONANCE ENERGY TRANSFER STUDIES
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 18, pp. 13895 13905, May 4, 2007 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Structure and Conformational
More informationZool 3200: Cell Biology Exam 3 3/6/15
Name: Trask Zool 3200: Cell Biology Exam 3 3/6/15 Answer each of the following questions in the space provided; circle the correct answer or answers for each multiple choice question and circle either
More informationPRACTICAL EVOLUTION: HARNESSING VARIATION AND SELECTION IN RATIONAL DRUG DISCOVERY. Robin Pals Rylaarsdam Benedictine University, Lisle, IL
PRACTICAL EVOLUTION: HARNESSING VARIATION AND SELECTION IN RATIONAL DRUG DISCOVERY Robin Pals Rylaarsdam Benedictine University, Lisle, IL Evolution in hearts and minds Useful principles of evolution Protein
More informationIntroduction to Genome Biology
Introduction to Genome Biology Sandrine Dudoit, Wolfgang Huber, Robert Gentleman Bioconductor Short Course 2006 Copyright 2006, all rights reserved Outline Cells, chromosomes, and cell division DNA structure
More informationSuppl. Figure 1: RCC1 sequence and sequence alignments. (a) Amino acid
Supplementary Figures Suppl. Figure 1: RCC1 sequence and sequence alignments. (a) Amino acid sequence of Drosophila RCC1. Same colors are for Figure 1 with sequence of β-wedge that interacts with Ran in
More informationTopic 2: Proteins. 2-1 specific proteins can be purified from cell extracts. Molecular Biology and Public Health ( 分子生物学与公共卫生 )
HAPTER20: Techniques of Molecular Biology Molecular Biology and Public Health ( 分子生物学与公共卫生 ) -------Protein manipulation ( 蛋白操作 ) Topic 2: Proteins 1. Protein purification ( 蛋白质纯化 ) 2. Affinity chromatography
More informationChapter 8 Lecture Outline. Transcription, Translation, and Bioinformatics
Chapter 8 Lecture Outline Transcription, Translation, and Bioinformatics Replication, Transcription, Translation n Repetitive processes Build polymers of nucleotides or amino acids n All have 3 major steps
More informationARTICLE. Structural insights into m-opioid receptor activation
ARTICLE doi:10.1038/nature14886 Structural insights into m-opioid receptor activation Weijiao Huang 1 *, Aashish Manglik 1 *, A. J. Venkatakrishnan 1,2,3, Toon Laeremans 4,5, Evan N. Feinberg 1,2,3, Adrian
More informationIn mice: V H ~250 D ~15 J ~5. L 1 -V H1 -L 2 -V H2 -L 3 -V H3 ---L n -V Hn ----D 1 -D 2 -D 3 ---D n ----J 1 -J J n V L ~250 J ~4
Lecture 14 ymes Cont. ymes preferentially stabilize transitions states versus ground states ite directed mutagenesis (Tyr ta synthetase) Transition state analogue inhibitors Design of protein active sites
More informationI. Gene Expression Figure 1: Central Dogma of Molecular Biology
I. Gene Expression Figure 1: Central Dogma of Molecular Biology Central Dogma: Gene Expression: RNA Structure RNA nucleotides contain the pentose sugar Ribose instead of deoxyribose. Contain the bases
More informationProtein Synthesis Notes
Protein Synthesis Notes Protein Synthesis: Overview Transcription: synthesis of mrna under the direction of DNA. Translation: actual synthesis of a polypeptide under the direction of mrna. Transcription
More informationBasic concepts of molecular biology
Basic concepts of molecular biology Gabriella Trucco Email: gabriella.trucco@unimi.it Life The main actors in the chemistry of life are molecules called proteins nucleic acids Proteins: many different
More informationNo growth: Mutant cells cannot grow and divide. Classes of Neurospora crassa. Class I mutants Class II mutants Class III mutants
XPRIMNT Growth: Wild-type cells growing and dividing Minimal medium No growth: Mutant cells cannot grow and divide RSULTS Condition Minimal medium (MM) (control) MM + ornithine MM + citrulline Wild type
More informationProtein Structure/Function Relationships
Protein Structure/Function Relationships W. M. Grogan, Ph.D. OBJECTIVES 1. Describe and cite examples of fibrous and globular proteins. 2. Describe typical tertiary structural motifs found in proteins.
More informationBi 8 Lecture 7. Ellen Rothenberg 26 January Reading: Ch. 3, pp ; panel 3-1
Bi 8 Lecture 7 PROTEIN STRUCTURE, Functional analysis, and evolution Ellen Rothenberg 26 January 2016 Reading: Ch. 3, pp. 109-134; panel 3-1 (end with free amine) aromatic, hydrophobic small, hydrophilic
More informationSupplementary Figure 1
Supplementary Figure 1 Supplementary Figure 1. Analysis of unique sequences isolated in Sort 14. Unique sequences are listed next to their frequency of occurrence, in parenthesis. Dithiol resistance is
More informationMacmillan Publishers Limited. All rights reserved
LETTER doi:10.1038/nature10136 Agonist-bound adenosine A 2A receptor structures reveal common features of GPCR activation Guillaume Lebon 1, Tony arne 1, Patricia C. Edwards 1, Kirstie Bennett 2, Christopher
More informationChapter 25: Regulating Eukaryotic Transcription The Ligand Responsive Activators
Chapter 25: Regulating Eukaryotic Transcription The Ligand Responsive Activators At least 5 potential gene expression control points Superfamily of Gene Regulators Activation of gene structure Initiation
More informationTracking Cellular Protein Localization and Movement in Cells with a Flexible Fluorescent Labeling Technology. Chad Zimprich January 2015
Tracking Cellular Protein Localization and Movement in Cells with a Flexible Fluorescent Labeling Technology Chad Zimprich January 2015 Presentation verview HaloTag Fusion Technology Design Functionality
More informationDirect Molecular Evolution of Detergent-Stable G Protein-Coupled Receptors Using Polymer Encapsulated Cells
Direct Molecular Evolution of Detergent-Stable G Protein-Coupled Receptors Using Polymer Encapsulated Cells Daniel J. Scott and Andreas Plückthun Department of Biochemistry, University of Zurich, Winterthurerstrasse
More informationBarbar Lab News December Publications Barbar, E 48, Barbar, E. 284, Barbar E Barbar, E 455,
Barbar Lab News December 2009 The Barbar lab had a very successful year. The highlights include a new NIH RO1 grant awarded for 5 years to study the role of LC8 as a molecular hub that facilitates dimerization
More informationQuick Review of Protein Synthesis
Collin College BIOL. 2401 Quick Review of Protein Synthesis. Proteins and Protein Synthesis Proteins are the molecular units that do most of the work in a cell. They function as molecular catalysts, help
More informationCh Biophysical Chemistry
Ch 247.53. Biophysical Chemistry Nina Rosario L. Rojas 2012-2013 sem 1 Review of Protein Structure Why structure? Primary, secondary, tertiary structure Disulfide bonds scheme 2 STRUCTURE- REGULAR STRUCTURE
More informationSolutions to Problem Set 1
MIT Department of Biology 7.014 Introductory Biology, Spring 004 Question 1 Solutions to 7.014 Problem Set 1 a) Describe the conditions of the atmosphere on prebiotic earth and how these conditions differ
More informationT H E J O U R N A L O F C E L L B I O L O G Y
Supplemental material Thompson et al., http://www.jcb.org/cgi/content/full/jcb.200909067/dc1 T H E J O U R N A L O F C E L L B I O L O G Y Figure S1. Modification-specific antibodies do not detect unmodified
More informationGiri Narasimhan. CAP 5510: Introduction to Bioinformatics. ECS 254; Phone: x3748
CAP 5510: Introduction to Bioinformatics Giri Narasimhan ECS 254; Phone: x3748 giri@cis.fiu.edu www.cis.fiu.edu/~giri/teach/bioinfs07.html 2/19/07 CAP5510 1 HMM for Sequence Alignment 2/19/07 CAP5510 2
More information5.36 Biochemistry Laboratory Spring 2009
MIT OpenCourseWare http://ocw.mit.edu 5.36 Biochemistry Laboratory Spring 2009 For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms. Laboratory Manual for URIECA
More informationUnderstanding the Cellular Mechanism of the Excess Microsporocytes I (EMSI) Gene. Andrew ElBardissi, The Pennsylvania State University
Understanding the Cellular Mechanism of the Excess Microsporocytes I (EMSI) Gene Andrew ElBardissi, The Pennsylvania State University Abstract: Hong Ma, The Pennsylvania State University The Excess Microsporocytes
More informationAli Yaghi. Tamara Wahbeh. Mamoun Ahram
28 Ali Yaghi Tamara Wahbeh Mamoun Ahram This sheet is a continuation of protein purification methods. Isoelectric focusing Separation of proteins based on Isoelectric points(charge),and it is a horizontal
More information466 Asn (N) to Ala (A) Generate beta dimer Interface
Table S1: Amino acid changes to the HexA α-subunit to convert the dimer interface from α to β and to introduce the putative GM2A binding surface from β- onto the α- subunit Residue position (α-numbering)
More informationProtein homology. Antigens & Antibodies I. Administrative issues:
Administrative issues: Recommended text: Goldsby/Kuby Immunology, 6th edition (Note that Innate Immunity is not adequately covered in the 5th edition.) Text book reading assignments are to supplement the
More informationStructure formation and association of biomolecules. Prof. Dr. Martin Zacharias Lehrstuhl für Molekulardynamik (T38) Technische Universität München
Structure formation and association of biomolecules Prof. Dr. Martin Zacharias Lehrstuhl für Molekulardynamik (T38) Technische Universität München Motivation Many biomolecules are chemically synthesized
More informationModule Code: BIO00025H
Examination Candidate Number: Desk Number: BSc and MSc Degree Examinations 2018-9 Department : BIOLOGY Title of Exam: Protein protein recognition Time Allowed: 2 hours Marking Scheme: Total marks available
More informationTranslation Mechanisms
Translation Mechanisms Biology I Hayder A. Giha Translation The translation is the process of protein synthesis, where information in nucleotides sequences of a mrna is translated into amino acids sequence
More informationTranslation. Protein Synthesis
Protein Structure Translation Protein Synthesis Size and Shape Comparison of Proteins Levels of Protein Structure 1 o 2 o 3 o 4 o Amino Acids Peptide Bonds Proteins are formed by creating peptide bonds
More informationSUPPLEMENTARY INFORMATION
SUPPLEMENTARY INFORMATION Contents: Supplementary Figure 1. Additional structural and binding data for designed tuim peptides. Supplementary Figure 2. Subcellular localization patterns of designed tuim
More informationChapter 3 Nucleic Acids, Proteins, and Enzymes
3 Nucleic Acids, Proteins, and Enzymes Chapter 3 Nucleic Acids, Proteins, and Enzymes Key Concepts 3.1 Nucleic Acids Are Informational Macromolecules 3.2 Proteins Are Polymers with Important Structural
More information