This document discusses the methodology for purifying, reconstituting, and characterizing receptors and synthetic analogues of epinephrine. It describes how receptor proteins are isolated from natural sources through cloning or from cell membranes using techniques like ultrasonication and centrifugation. The isolated receptors are then solubilized using detergents and purified using affinity, ion exchange, or size exclusion chromatography. The purified receptors can be reconstituted into detergent micelles, bicelles, or nanodiscs to mimic the cell membrane environment. Characterization of the receptors involves studying their primary structure, post-translational modifications, ligand binding sites, and interactions with G-proteins. Synthetic analogues of epinephrine are also designed to mimic its

1. METHODOLOGY OF PURIFICATION,
RECONSTITUTION AND CHARACTERIZATION OF
RECEPTORS, SYNTHETIC ANALOGUES OF
EPINEPHRINE
Presentation by
DEBASHISH CHAKRABARTY
M.Sc-3rd 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.
• Ion 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
-Packs 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.
• The 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 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.

21. THANK YOU