7 research outputs found
Contribution of inter-subunit interactions to the thermostability of Pyrococcus furiosus citrate synthase
Using citrate synthase from the hyperthermophile Pyrococcus furiosus (PfCS) as our test molecule, we show through guanidine hydrochloride-induced unfolding that the dimer separates into folded, but inactive, monomers before individual subunit unfolding takes place. Given that forces across the dimer interface are vital for thermostability, a robust computational method was derived that uses the University of Houston Brownian Dynamics (UHBD) program to calculate both the hydrophobic and electrostatic contribution to the dimerisation energy at 100°C. The results from computational and experimental determination of the lowered stability of interface mutants were correlated, being both of the same order of magnitude and placing the mutant proteins in the same order of stability. This computational method, optimised for hyperthermophilic molecules and tested in the laboratory, after further testing on other examples, could be of widespread use in the prediction of thermostabilising mutations in other oligomeric proteins for which dissociation is the first step in unfolding
In-cell NMR reveals potential precursor of toxic species from SOD1 fALS mutants
Mutations in the superoxide dismutase 1 (SOD1) gene are related to familial cases of
amyotrophic lateral sclerosis (fALS). Here we exploit in-cell NMR to characterize the protein
folding and maturation of a series of fALS-linked SOD1 mutants in human cells and to obtain
insight into their behaviour in the cellular context, at the molecular level. The effect of various
mutations on SOD1 maturation are investigated by changing the availability of metal ions in
the cells, and by coexpressing the copper chaperone for SOD1, hCCS. We observe for most of
the mutants the occurrence of an unstructured SOD1 species, unable to bind zinc.
This species may be a common precursor of potentially toxic oligomeric species, that are
associated with fALS. Coexpression of hCCS in the presence of copper restores the correct
maturation of the SOD1 mutants and prevents the formation of the unstructured species,
confirming that hCCS also acts as a molecular chaperone
Insights into SOD1-linked amyotrophic lateral sclerosis from NMR studies of Ni2+- and other metal-ion-substituted wild-type copper–zinc superoxide dismutases
The dimeric copper-zinc superoxide dismutase Cu(2)Zn(2)SOD1 is a particularly interesting system for biological inorganic chemical studies because substitutions of the native Cu and/or Zn ions by a non-native metal ion cause minimal structural changes and result in high enzymatic activity for those derivatives with Cu remained in the Cu site. The pioneering NMR studies by Ivano Bertini and coworkers of the magnetically coupled derivative Cu(2)Co(2)SOD1 are of particular importance in this regard. In addition to Co(2+), Ni(2+) is also a versatile metal ion for substitution into Cu(2)Zn(2)SOD1, showing very little disturbance of the structure in Cu(2)Ni(2)SOD1 and a very good mimic of the native Cu ion in Ni(2)Zn(2)SOD1. The NMR studies presented here were inspired by and indebted to Professor Bertini's paramagnetic NMR pursuits of metalloproteins. In the current study, we report Ni(2+) binding to apo-wild type SOD1 and a time-dependent Ni(2+) ion migration from the Zn site to the Cu site and preparation and characterization of Ni(2)Ni(2)SOD1, which shows similar coordination properties to those of Cu(2)Cu(2)SOD1, namely a different anion binding property from the wild type and a possibly broken bridging His. Mutations in the human SOD1 gene can cause familial amyotrophic lateral sclerosis (ALS), and mutant SOD1 proteins with significantly altered metal binding behaviors are implicated in causing the disease. We therefore conclude by discussing the effects of the ALS mutations on the remarkable stabilities and metal-binding properties of wild type SOD1 proteins and the implications concerning the causes of SOD1-linked ALS