Methodology of purification and characterization of receptors

cell signaling mechanism

1. METHODOLOGY OF PURIFICATION, RECONSTITUTION AND CHARACTERIZATION OF RECEPTORS, SYNTHETIC ANALOGUES OF EPINEPHRINE Presentation by DEBASHISH CHAKRABARTY M.Sc-3 rd Sem SLS

2. Need for ISOLATION AND reconstitution Receptor proteins are present in low levels in biological system. Lipid membrane is complex, heterogeneous and dynamic environment so limits the use of NMR, X-ray diffraction etc. Receptor proteins are trans membrane proteins so can’t be studied in aqueous medium. Easy manipulation in reconstituted system.

3. ISOLATION OF RECEPTORS - Receptor Sources Cloning - 2 methods Natural source Cloning Gene for the receptor Inserted within an expression vector Screening Protein Expression Host Transformation Expression is done by synthesizing fusion protein where tags are used.

4. Cell Disruption Yields a suspension of membrane fragments along with other sub cellular structures. Methods used are:- Ultrasonication . Glass bead milling . Osmotic Shock. Followed by differential centrifugation.

5. Solubilisation The process where membrane proteins are extracted from the lipid membrane by using detergent. Protein detergent complexes are formed and stabilize the proteins. Purification The process to obtain only the desired proteins. All the steps are carried out in presence of detergents. Combination of 2 or 3 purification methods are used. .

6. Affinity purification ( eg . Histidine tagged proteins ) - Based upon the affinity of the desired protein towards a particular molecule. I on Exchange Chromatography - Based upon the charge of the desired protein. Gel filtration Chromatography - Based upon the size of the protein.

7. RECONSTITUTION OF RECEPTORS Detergent and mixed micelles -Most basic strategy. -Prepared from mixture of detergents. Drawbacks Presence of high conc. of free detergent. Micelles are much more disordered. Bicelles Detergent Solubilised receptors are mixed with short and long chain lipids. Lipid bilayer is formed; perimeter stabilised by short chain lipids.

8. Advantages Diameter allows multiple receptor reconstitution . Reduced free detergent concentration. Drawbacks Presence of empty bicelles . Nanodiscs Most novel paradigm for GPCR . Resemble bicelles . Consists of lipid bilayer and MSP.

9. Advantages No high detergent concentration. MSP provide additional labelling sites . Don’t have enclosed topography . Disadvantages No difference in extracellular and intracellular environments . Constraints on the diffusion of receptor proteins.

10. CHARACTERIZATION OF RECEPTORS Most studied receptor is β adrenergic receptor. Primary structure Single polypeptide chain. 400-500 amino acids long. N terminus and C-terminus; 3 extracellular(e), 3 intracellular( i ) stretches and 7 transmembrane domains.

11. -modified from Kobilka et al. [ 1987]

12. Post Translational Modifications 1. N-linked glycosylation 1 or 2 sites within the N-terminus. Absence doesn’t alter ligand binding or signal transduction . 2. Palmitoylation Occurs at a Cys residue present immediately after tm7 domain. Helps in mediating agonist resposne . 3. Disulfide bond Cys 106 and Cys 184 residue involved in disulfide bonding.

13. Ligand Binding Site A pocket within the transmembrane is the place for ligand binding. Asp 113 in tm3 while Ser 204 and 207 in tm5 are crucial for ligand binding. - Strosberg , 1991b

14. Signal Transmission Asp 79 residue present in tm2 is responsible for binding of agonists . Asp 79 and Tyr 316 helps in activation of G protein . Tyr 316 and Asn 312 is prsent in tm7 prevents activation of G protein(antagonists). G protein interaction I3 loop is the main site that interacts with G protein . I2 also has some contribution . Homologus replacement or deletion of the sequence is used for studies.

15. - Strosberg et al., 1993

16. SYNTHETIC ANALOGUES OF EPINEPHRINE Epinephrine is a hormone secreted by adrenal medulla and binds to the adrenergic receptors . Synthetic analogues are chemically synthesized which can mimic its action e.g - Isoproterenol. Epinephrine is a catecholamine so an analogue must contain some of the groups that can interact with the receptor :- N-methyl group - P acks against Phe 411 and Phe 412 in tm7.

17. Epinephrine Isoproterenol ( Analogue ) -From wikipedia -From wikipedia

18. Β -OH group -Forms Hydrogen bond with Asp 113. Aromatic group -Inserted within the tm6 and interaction takes place with Phe 391 and Phe 394. Para and Meta OH groups -Contacts residues within tm3 and tm5. eg .- Thr 118 in tm3 and Ser 204 in tm5.

19. IN BRIEF Receptor proteins can’t be studied within native sites. Needed to be isolated and purified in order to reconstitute in environment that mimics the native site. Characterization helps in studying conformational changes of receptors during ligand interactions. T he synthetic analogues can be designed on the basis of ligand structure and receptor characterization.

20. References:- GE health care- purifying challenging proteins. Functional reconstitution of β 2-adrenergic receptors utilizing self-assembling Nanodisc technology; Andrew J. Leitz1, Timothy H. Bayburt1, Alexander N. Barnakov2, Barry A. Springer2 , and Stephen G. Sligar . Structure, function, and regulation of adrenergic receptors ; A.D . STROSBERG Laboratoire d’ lmmuno-Pharrnacologie Moleculaire , lnstitut Cochin de Genetique Moltculaire , and Universite de Paris VII, Paris, France. Artificial membrane-like environments for in vitro studies of purified G-protein coupled receptors ; Eugene Serebryany , Gefei Alex Zhu, Elsa C.Y. Yan.

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