Biophysical characterization of the recombinant importin-α from Neurospora crassa.
Neurospora crassa has been widely used as a model organism and contributed to the development of biochemistry and molecular biology by allowing the identification of many metabolic pathways and mechanisms responsible for gene regulation. Nuclear proteins are synthesized in the cytoplasm and need to be translocated to the nucleus to exert their functions which the importin-α receptor has a key role for the classical nuclear import pathway. In an attempt to get structural information of the nuclear transport process in N. crassa, we present herein the cloning, expression, purification and structural studies with N-terminally truncated IMPα from N. crassa (IMPα-Nc). Circular dichroism analysis revealed that the IMPα-Nc obtained is correctly folded and presents a high structural conservation compared to other importins-α. Dynamic light scattering, analytical size-exclusion chromatography experiments and molecular dynamics simulations indicated that the IMPα-Nc unbound to any ligand may present low stability in solution. The IMPα-Nc theoretical model displayed high similarity of its inner concave surface, which binds the cargo proteins containing the nuclear localization sequences, among IMPα from different species. However, the presence of non-conserved amino acids relatively close to the NLS binding region may influence the binding specificity of IMPα-Nc to cargo proteins.
Departamento de Bioquimica e Tecnologia Quimica, Instituto de Biociências, Universidade Estadual Paulista, Botucatu, SP, Brazil.
Agnes A S
AA
Takeda
Fernanda Z
FZ
Freitas
Angelo J
AJ
Magro
Natalia E
NE
Bernardes
Carlos A H
CA
Fernandes
Rodrigo D
RD
Gonçalves
Maria Célia
MC
Bertolini
Marcos R M
MR
Fontes
D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D008024:Ligands; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D009492:Neurospora crassa / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000378:metabolism*; D055550:Protein Stability; D016415:Sequence Alignment; D028901:alpha Karyopherins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D028961:beta Karyopherins / Q000378:metabolism
D008024:Ligands; D019913:Nuclear Localization Signals; D028901:alpha Karyopherins; D028961:beta Karyopherins