In contrast to the WTL class, fALS-associated MBR mutations in SOD1 are located at the metal binding sites themselves or in the lengthy zinc and electrostatic loop elements. Metal analyses of a number of these MBR mutants purified from insect and yeast cells using inductively coupled plasma mass spectrome-try demonstrate that these proteins have a significantly decreased metal content relative to the wild-type enzyme [47,122]. MBR mutants display decreased thermal stabilities of their metal bound forms when compared to the metal bound forms of the wild type and WTL mutant proteins [47,116,122]. Lowered stability of these mutants is likely to be directly due to the decrease in metal binding. In contrast, the metal free forms of these mutants have equivalent, and in some cases, increased thermal stabilities in comparison to metal free wild-type protein .
As shown in Figure 1A and Figure 4, loss of metal ions is likely to affect the conformations of the zinc and electrostatic loop elements while leaving the P-barrel and dimer interface unaltered relative to the wild-type protein. Interestingly, the metal free form of MBR mutant H46R has the highest thermal stability of all of the mutants tested and patients harboring this mutation have one of the longest time courses for the disease, over 20 years . While it is tempting to correlate disease progression with relative stabilities of the various SOD1 mutants, most of the fALS SOD1 patient pools are quite small in number, and it is currently difficult to generate reliable statistics or to quantify the effects of other factors (such as diet and genetic background) on disease progression.
Crystallographic studies of two MBR mutants, H46R and S134N, were the first metal-deficient SOD1 structures to be determined . As discussed below, it is precisely the metal deficiency of these proteins that led to the discovery of their propensity to form higher-order assemblies.
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