Expression, purification and characterisation of the recombinant possum lipocalin vulpeculin.
Brushtail possum
Chemosignalling
Circular dichroism
Fluorescence-binding assay
Vulpeculin
Journal
Biochimica et biophysica acta. General subjects
ISSN: 1872-8006
Titre abrégé: Biochim Biophys Acta Gen Subj
Pays: Netherlands
ID NLM: 101731726
Informations de publication
Date de publication:
11 2022
11 2022
Historique:
received:
30
11
2021
revised:
17
07
2022
accepted:
18
07
2022
pubmed:
1
8
2022
medline:
31
8
2022
entrez:
31
7
2022
Statut:
ppublish
Résumé
Lipocalins are a large family of proteins, which possess a highly conserved eight-stranded antiparallel beta-barrel structure as distinctive trait. This family includes Major Urinary Proteins (MUPs) from rats and mouse, studied for their role in urinary protein-mediated chemosignalling. Vulpeculin has been identified as the most abundant protein in the urine of the common brushtail possum, Trichosurus vulpecula. On the basis of high similarity with other MUPS, we hypothesised that vulpeculin might have a role in possum chemosignalling and investigated its stability and binding ability. We expressed and purified vulpeculin using an E.coli-based system and confirmed correct folding by circular dichroism (CD) spectroscopy. Thermal stability was studied by CD and binding properties were investigated using two optical probes N-phenyl-naphthylamine (NPN) and 8-anilino-1-naphthalene sulphonic acid (ANS). CD revealed a secondary structure typical of a predominantly β-sheet protein, consistent with the beta barrel structure of the lipocalin family. Vulpeculin showed a high level of thermostability, as assessed by CD, exhibiting a small shift in the secondary structure even at 95 °C. Binding assays indicated that vulpeculin cannot accommodate the NPN ligand but can bind ANS. The urinary secretion, high degree of sequence similarity with other lipocalins, its beta sheet structure assessed by CD and potential to bind hydrophobic ligands in the hydrophobic cavity or an external hydrophobic pocket, suggest vulpeculin may be involved in possum chemosignalling. This work represents a first step towards the further investigation of the newly discovered lipocalin and its role in possum chemosignalling.
Sections du résumé
BACKGROUND
Lipocalins are a large family of proteins, which possess a highly conserved eight-stranded antiparallel beta-barrel structure as distinctive trait. This family includes Major Urinary Proteins (MUPs) from rats and mouse, studied for their role in urinary protein-mediated chemosignalling. Vulpeculin has been identified as the most abundant protein in the urine of the common brushtail possum, Trichosurus vulpecula. On the basis of high similarity with other MUPS, we hypothesised that vulpeculin might have a role in possum chemosignalling and investigated its stability and binding ability.
METHODS
We expressed and purified vulpeculin using an E.coli-based system and confirmed correct folding by circular dichroism (CD) spectroscopy. Thermal stability was studied by CD and binding properties were investigated using two optical probes N-phenyl-naphthylamine (NPN) and 8-anilino-1-naphthalene sulphonic acid (ANS).
RESULTS
CD revealed a secondary structure typical of a predominantly β-sheet protein, consistent with the beta barrel structure of the lipocalin family. Vulpeculin showed a high level of thermostability, as assessed by CD, exhibiting a small shift in the secondary structure even at 95 °C. Binding assays indicated that vulpeculin cannot accommodate the NPN ligand but can bind ANS.
CONCLUSION
The urinary secretion, high degree of sequence similarity with other lipocalins, its beta sheet structure assessed by CD and potential to bind hydrophobic ligands in the hydrophobic cavity or an external hydrophobic pocket, suggest vulpeculin may be involved in possum chemosignalling.
GENERAL SIGNIFICANCE
This work represents a first step towards the further investigation of the newly discovered lipocalin and its role in possum chemosignalling.
Identifiants
pubmed: 35908580
pii: S0304-4165(22)00123-4
doi: 10.1016/j.bbagen.2022.130205
pii:
doi:
Substances chimiques
Ligands
0
Lipocalins
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
130205Informations de copyright
Copyright © 2022 Elsevier B.V. All rights reserved.