title: Human neuromodulator SLURP-1: bacterial expression, binding to muscle-type nicotinic acetylcholine receptor, secondary structure, and conformational heterogeneity in solution.
abstract: Human protein SLURP-1 is an endogenous neuromodulator belonging to the Ly-6/uPAR family and acting on nicotinic acetylcholine receptors. In the present work, the gene of SLURP-1 was expressed in E. coli. The bacterial systems engineered for SLURP-1 expression as fused with thioredoxin and secretion with leader peptide STII failed in the production of milligram quantities of the protein. The SLURP-1 was produced with high-yield in the form of inclusion bodies, and different methods of the protein refolding were tested. Milligram quantities of recombinant SLURP-1 and its (15)N-labeled analog were obtained. The recombinant SLURP-1 competed with (125)I-α-bungarotoxin for binding to muscle-type Torpedo californica nAChR at micromolar concentrations, indicating a partial overlap in the binding sites for SLURP-1 and α-neurotoxins on the receptor surface. NMR study revealed conformational heterogeneity of SLURP-1 in aqueous solution, which was associated with cis-trans isomerization of the Tyr39-Pro40 peptide bond. The two structural forms of the protein have almost equal population in aqueous solution, and exchange process between them takes place with characteristic time of about 4 ms. Almost complete (1)H and (15)N resonance assignment was obtained for both structural forms of SLURP-1. The secondary structure of SLURP-1 involves two antiparallel β-sheets formed from five β-strands and closely resembles those of three-finger snake neurotoxins.
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia. ekaterina-lyukmanova@yandex.ru
authors.forename: M A
authors.initials: MA
authors.lastname: Shulepko
authors.forename: E N
authors.initials: EN
authors.lastname: Lyukmanova
authors.forename: A S
authors.initials: AS
authors.lastname: Paramonov
authors.forename: A A
authors.initials: AA
authors.lastname: Lobas
authors.forename: Z O
authors.initials: ZO
authors.lastname: Shenkarev
authors.forename: I E
authors.initials: IE
authors.lastname: Kasheverov
authors.forename: D A
authors.initials: DA
authors.lastname: Dolgikh
authors.forename: V I
authors.initials: VI
authors.lastname: Tsetlin
authors.forename: A S
authors.initials: AS
authors.lastname: Arseniev
authors.forename: M P
authors.initials: MP
authors.lastname: Kirpichnikov
mesh_terms: D000595:Amino Acid Sequence; D000950:Antigens, Ly / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D018377:Neurotransmitter Agents / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011978:Receptors, Nicotinic / Q000378:metabolism*; D016415:Sequence Alignment; D012996:Solutions / Q000737:chemistry; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000950:Antigens, Ly; D018377:Neurotransmitter Agents; D011978:Receptors, Nicotinic; C048963:SLURP1 protein, human; D012996:Solutions; D014568:Urokinase-Type Plasminogen Activator