Human neuromodulator SLURP-1: Bacterial expression, binding to muscle-type nicotinic acetylcholine receptor, secondary structure, and conformational heterogeneity in solution

• Published in: Biochemistry (Moscow) Vol. 78; no. 2; pp. 204 - 211
• Main Authors: Shulepko, M. A., Lyukmanova, E. N., Paramonov, A. S., Lobas, A. A., Shenkarev, Z. O., Kasheverov, I. E., Dolgikh, D. A., Tsetlin, V. I., Arseniev, A. S., Kirpichnikov, M. P.
• Format: Journal Article
• Language: English
• Published: Dordrecht SP MAIK Nauka/Interperiodica 01.02.2013; Springer Nature B.V
• Subjects: Amino Acid Sequence; Antigens, Ly - chemistry; Antigens, Ly - genetics; Antigens, Ly - metabolism; Bacteria; Binding, Competitive; Biochemistry; Biomedical and Life Sciences; Biomedicine; Bioorganic Chemistry; Cloning, Molecular; Escherichia coli; Escherichia coli - genetics; Gene expression; Humans; Life Sciences; Magnetic Resonance Spectroscopy; Microbiology; Molecular Conformation; Molecular Sequence Data; Neurotransmitter Agents - metabolism; Protein Binding; Protein Folding; Protein Structure, Secondary; Proteins; Receptors, Nicotinic - metabolism; Sequence Alignment; Solutions - chemistry; Torpedo californica; Toxins; Urokinase-Type Plasminogen Activator - chemistry; Urokinase-Type Plasminogen Activator - genetics; Urokinase-Type Plasminogen Activator - metabolism; three-finger snake neurotoxin; bacterial expression; Lynx; conformational exchange; NMR spectroscopy; nicotinic acetylcholine receptor
• ISSN: 0006-2979; 1608-3040
• DOI: 10.1134/S0006297913020090

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.