Crystals of membrane proteins

Abstract

A polypeptide in crystalline form comprises a G-protein coupled receptor (GPCR) with an IC3 loop substituted by an amino acid residue sequence of lysozyme.

1 . A composition comprising a polypeptide in crystalline form, wherein said polypeptide comprises:
 a G-protein coupled receptor (GPCR) comprising an IC3 loop containing a substitution that comprises the amino acid sequence of a lysozyme.   
     
     
         2 . A composition comprising a fusion protein in crystalline form, wherein said fusion protein comprises, from N-terminus to C-terminus:
 a) a first portion of a G-protein coupled receptor (GPCR), wherein said first portion comprises TM1, TM2, TM3, TM4 and TM5 regions of said GPCR;   b) a domain comprising the amino acid sequence of a lysozyme;   c) a second portion of said GPCR, wherein said second portion comprises TM6 and TM7 regions of said GPCR.   
     
     
         3 . The composition of  claim 2 , wherein said first and second portions of said GPCR comprise the amino acid sequence of a naturally occurring GPCR. 
     
     
         4 . The composition of  claim 1 , wherein said GPCR is a β2-adrenergic receptor. 
     
     
         5 . The composition of  claim 2 , wherein said fusion protein is bound to a ligand for said GPCR. 
     
     
         6 . A composition comprising a polypeptide in crystalline form, wherein said polypeptide comprises, from N-terminus to C-terminus:
 a) a first portion of a G-protein coupled receptor (GPCR), wherein said first portion comprises the amino acid sequence that is N-terminal to the IC3 loop of said GPCR;   b) a domain comprising the amino acid sequence of a lysozyme;   c) a second portion of said GPCR, wherein said second portion comprises the amino acid sequence that is C-terminal to the IC3 loop of said GPCR.   
     
     
         7 . A crystalline fusion protein which comprises a beta-2 adrenergic receptor and lysozyme. 
     
     
         8 . The crystalline fusion protein of  claim 7  wherein the lysozyme is a T4 lysozyme sequence. 
     
     
         9 . The crystalline fusion protein of  claim 7  wherein the beta-2 adrenergic receptor has a E122W mutation. 
     
     
         10 . The crystalline fusion protein of  claim 7  wherein the beta-2 adrenergic receptor has a E122W mutation and wherein the lysozyme is a T4 lysozyme sequence. 
     
     
         11 . The crystalline fusion protein of  claim 7  wherein the lysozyme is T4 lysozyme sequence in place of the third intracellular loop of the beta-2 adrenergic receptor. 
     
     
         12 . The crystalline fusion protein of  claim 7  wherein residues 231 5.70  to 262 6.24  are replaced by residues 2 to 161 of T4 lysozyme, and the beta-2 adrenergic receptor is truncated at position 365. 
     
     
         13 . The crystalline fusion protein of  claim 7  further comprising a ligand non-covalently bound thereto. 
     
     
         14 . The crystalline fusion protein of  claim 13  wherein the ligand is carazolol.