METHODOLOGY OF PURIFICATION, RECONSTITUTION AND CHARACTERIZATION OF RECEPTORS, SYNTHETIC ANALOGUES OF EPINEPHRINE

Presentation byDEBASHISH CHAKRABARTYM.Sc-3rd SemSLS

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.

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.

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.

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. .

• Affinity purification(eg. Histidine tagged proteins)

- Based upon the affinity of the desired protein towards a particular molecule.

• Ion Exchange Chromatography- Based upon the charge of the desired protein.

• Gel filtration Chromatography- Based upon the size of the protein.

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.

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.

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.

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.

-modified from Kobilka et al. [1987]

Post Translational Modifications1. 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.

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

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.

-Strosberg et al., 1993

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-Packs against Phe 411 and Phe 412 in tm7.

Epinephrine

Isoproterenol(Analogue)

-From wikipedia

-From wikipedia

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.

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.

• The synthetic analogues can be designed on the basis of ligand structure and receptor characterization.

References:-• GE health care- purifying challenging proteins.• Functional reconstitution of β2-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.

THANK YOU