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The mass of the purified ACh BP from Lymnaea has been determined by mass-spectrometry.The glycosylated form has a mass of about 24720 Da and the de-glycosylated form of about 23832 Da.
The dimensional structure of ACh BP was solved by X-ray crystallography at 2.7Aresolution (current Rfa~t~r = 27.9 %, R,ree = 30.0 %). The high-resolution crystal structure of ACh BP, along with biochemical and pharmacological data, supports the extrapolation of ACh BP as a good mimic of ligand-binding domains of ligand-gated ion channels including n ACh R, 5-HT3R, GABAA,~R and GIy R.2, 4, 6 or 8;preferably in which said solubility-determining amino acids comprise solvent accessible regions in the crystal structure according to Figure 10. The protein of any one of claims 7 to 14 comprising an amino acid sequence having at least 40% amino acid identity to the amino acid sequence 20-223 of any one of SEQ ID Nos. The protein of claim 16, in which said solubility determining amino acids (a) comprise amino acids Asp(36), Asp(68), Glu(115), Arg(137), Asp(143), Asp(148), Glu(150), Arg(167), Arg(189), Glu(215) of SEQ ID No.2, wherein Asp may be exchanged for Glu and vice versa and Lys may be exchanged for Arg and vice versa. The protein of any one of claims 7 to 20 which is capable of binding a ligand of an acetylcholine receptor, in which at least one of the amino acid sequences Trp(101) - Tyr(T08), Trp(162) - His(164) and Tyr(204) - Tyr(211) of SEQ ID No. or (3) a space group of P21 and a unit cell of dimensions of a=121.1. The biological and structural properties of these proteins are disclosed, as is the amino acid and nucleotide sequence.2, 4, 6 or 8, in which the ligand binding amino acids have been replaced with the corresponding amino acids of a ligand-gated receptor. The protein of any one of claims 7 to 15, in which said solubility-determining amino acids (a) comprise hydrophilic amino acids (Asp, Glu, Arg, Lys) from the sequences 20-44, 73-81, 86-92, 112-120, 135-152, 166-189, 196-20, 209-213, and/or 219-227 of SEQ ID No. 2 have been exchanged with the corresponding sequence of the acetylcholine receptor. A method for the production of a water-soluble ligand-gated receptor or a corresponding ligand-binding domain or for improving the water solubility and accessibility to crystallization of such a receptor or domain, said method comprising altering the amino acid sequence of the extracellular domain of a ligand-gated receptor by way of substituting, adding, deleting or modifying at least one amino acid at a position corresponding to an amino acid determining or contributing to the water-solubility of the protein of any one of claims 1 to 21. The method of claim 22 or 23, wherein at least one amino acid is altered to the corresponding amino acid of the amino acid sequence depicted in any one of SEQ ID Nos. The recombinant DNA molecules, and portions thereof, are useful for isolating homologues of the DNA molecules, identifying and isolating genomic equivalents of the DNA molecules, and identifying, detecting or isolating mutant forms of the DNA molecules.Each of the documents cited herein (including any manufacturer's specifications, instructions, etc.) are hereby incorporated herein by reference; however, there is no admission that any document cited is indeed prior art as to the present invention._7 BACKGROUND OF THE INVENTIONThe communication in the central nervous system (CNS) occurs through a complex interaction of electrical and chemical signals.Molecules bearing chemical information are called neurotransmitters.
The chemical information is converted in electric currents on the post-synaptic membrane, which is specialised in recognising and binding neurotransmitters by means of protein receptors.
It has been found according to the invention that acetylcholine-binding proteins (ACh BP) of certain molluscs show a surprising structural similarity with the channel-coupled receptors on the one hand and have interesting physical properties, such as water-solubility, on the other hand.
The molluscan ACh BPs are capable of forming multimers, especially pentamers, and of binding specific toxins such as a-bungarotoxin.
The ACh Rs are the best studied of the ligand-gated receptors; for a review, see Arias, Brain Research Reviews, 25 (1997)133-191 and Arias, Neurochem. The above-defined technical problem is solved by the present invention by providing the embodiments characterized in the claims.
Accordingly, in one aspect the present invention relates to a water-soluble protein derived from a mollusc being capable of binding a ligand of a ligand-gated receptor.
2, 4, 6 or 8, or to a an equivalent amino acid, preferably in which said solubility-determining amino acids comprise solvent accessible regions in the crystal structure according to Figure 10. The method of any one of claims 22 to 25 further comprising (a) culturing a host cell transfected with and capable of expressing a polynucleotide comprising a nucleotide sequence encoding the altered amino acid sequence; and optionally (b) recovering said water-soluble ligand-gated receptor or corresponding ligand-binding domain from the culture. The polynucleotide(s) of claim 32 which comprise(s) (a) a nucleotide sequence having at least 15 continuous nucleotides of the nucleotide sequence depicted in any one of SEQ ID Nos. (2) a space group of P42212 and a unit cell of dimensions of a=b=141.6. Using a recombinant expression system functional DNA molecules encoding the water-soluble ligand-binding proteins as well as chimeras have been functionally produced.