Thermal stability effects of removing the type-2 copper ligand His306 at the interface of nitrite reductase subunits |
| |
Authors: | Andrea Stirpe Luigi Sportelli Hein Wijma Martin Ph Verbeet Rita Guzzi |
| |
Institution: | (1) Dipartimento di Fisica e Unità CNISM, Università della Calabria, Ponte P. Bucci, Cubo 31C, 87036 Arcavacata di Rende, Cosenza, Italy;(2) Gorleaus Laboratories, Metallo Protein Group, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands |
| |
Abstract: | Nitrite reductase (NiR) is a highly stable trimeric protein, which denatures via an intermediate,
(N—native, U—unfolded and F—final). To understand the role of interfacial residues on protein stability, a type-2 copper
site ligand, His306, has been mutated to an alanine. The characterization of the native state of the mutated protein highlights
that this mutation prevents copper ions from binding to the type-2 site and eliminates catalytic activity. No significant
alteration of the geometry of the type-1 site is observed. Study of the thermal denaturation of this His306Ala NiR variant
by differential scanning calorimetry shows an endothermic irreversible profile, with maximum heat absorption at T
max ≈ 85°C, i.e., 15°C lower than the corresponding value found for wild-type protein. The reduction of the protein thermal stability
induced by the His306Ala replacement was also shown by optical spectroscopy. The denaturation pathway of the variant is compatible
with the kinetic model
where the protein irreversibly passes from the native to the final state. No evidence of subunits’ dissociation has been
found within the unfolding process. The results show that the type-2 copper sites, situated at the interface of two monomers,
significantly contribute to both the stability and the denaturation mechanism of NiR. |
| |
Keywords: | Nitrite reductase mutant Thermal stability Type-2 copper Two-state irreversible model |
本文献已被 PubMed SpringerLink 等数据库收录! |
|