Human neuromodulator SLURP-1: Bacterial expression, binding to muscle-type nicotinic acetylcholine receptor, secondary structure, and conformational heterogeneity in solution
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...
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Published in | Biochemistry (Moscow) Vol. 78; no. 2; pp. 204 - 211 |
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Main Authors | , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
Dordrecht
SP MAIK Nauka/Interperiodica
01.02.2013
Springer Nature B.V |
Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0006-2979 1608-3040 |
DOI: | 10.1134/S0006297913020090 |