Analysis of the stability and function of nucleoplasmin through cysteine mutants.
Xenopus laevis nucleoplasmin is a pentameric nuclear chaperone. The relation between the structure and the multifunctional aspects of the molecule has not yet been clearly established. In the course of analysing a C-terminally His-tagged recombinant version of the region equivalent to the trypsin resistant core (r-NP142) of the molecule, we found that this domain exhibited a substantially decreased oligomerization potential. To better understand the role of the three cysteines of nucleoplasmin on its pentameric functional structure, we have selectively mutated these residues to serine and generated three mutants (C15S, C35S, and C45S) both for the complete recombinant nucleoplasmin (r-NP) and the truncated r-NP142 non-tagged forms. We demonstrate that there are no disulphide bridges stabilizing either the monomer or the pentamer. Neither C15S nor C35S has any structural effects, while the mutation C45S abolishes the ability of r-NP142 to pentamerize. This structural impairment suggests that hydrophobic interactions of Cys 45 are critical for the stability of the protein. Our studies allow to analyse for the first time the structural and functional properties of nucleoplasmin in its monomeric form.
Arnan
Carme
C
Departament d'Enginyeria Química, ETSEIB, Universitat Politècnica de Catalunya, Diagonal, 647, Barcelona E-08028, Spain.
Prieto
Cèlia
C
Chiva
Manel
M
Salvany
Lara
L
Ausió
Juan
J
Subirana
Juan A
JA
Saperas
Núria
N
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003545:Cysteine / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D056489:Nucleoplasmins; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D014357:Trypsin / Q000378:metabolism; D014982:Xenopus laevis
D009687:Nuclear Proteins; D056489:Nucleoplasmins; D010750:Phosphoproteins; D014357:Trypsin; D003545:Cysteine