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Biochemistry (Moscow)

, Volume 78, Issue 2, pp 204–211 | Cite as

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

  • M. A. Shulepko
  • E. N. LyukmanovaEmail author
  • A. S. Paramonov
  • A. A. Lobas
  • Z. O. Shenkarev
  • I. E. Kasheverov
  • D. A. Dolgikh
  • V. I. Tsetlin
  • A. S. Arseniev
  • M. P. Kirpichnikov
Article

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 15N-labeled analog were obtained. The recombinant SLURP-1 competed with 125I-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 1H and 15N 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.

Key words

nicotinic acetylcholine receptor bacterial expression Lynx conformational exchange three-finger snake neurotoxin NMR spectroscopy 

Abbreviations

α-Bgtx

α-bungarotoxin

nAChR

nicotinic acetylcholine receptor

STII

signal peptide of E. coli heat-stable enterotoxin II

TRX

thioredoxin

ws-Lynx1

water-soluble domain of human Lynx1

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Copyright information

© Pleiades Publishing, Ltd. 2013

Authors and Affiliations

  • M. A. Shulepko
    • 1
    • 2
  • E. N. Lyukmanova
    • 1
    Email author
  • A. S. Paramonov
    • 1
  • A. A. Lobas
    • 1
  • Z. O. Shenkarev
    • 1
  • I. E. Kasheverov
    • 1
  • D. A. Dolgikh
    • 1
    • 2
  • V. I. Tsetlin
    • 1
  • A. S. Arseniev
    • 1
  • M. P. Kirpichnikov
    • 1
    • 2
  1. 1.Shemyakin and Ovchinnikov Institute of Bioorganic ChemistryRussian Academy of SciencesMoscowRussia
  2. 2.Biological FacultyLomonosov Moscow State UniversityMoscowRussia

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