Metal-free superoxide dismutase-1 and three different amyotrophic lateral sclerosis variants share a similar partially unfolded beta-barrel at physiological temperature.

The structure and unfolding of metal-free (apo) human wild-type SOD1 and three pathogenic variants of SOD1 (A4V, G93R, and H48Q) that cause familial amyotrophic lateral sclerosis have been studied with amide hydrogen/deuterium exchange and mass spectrometry. The results indicate that a significant proportion of each of these proteins exists in solution in a conformation in which some strands of the beta-barrel (i.e. beta2) are well protected from exchange at physiological temperature (37 degrees C), whereas other strands (i.e. beta3 and beta4) appear to be unprotected from hydrogen/deuterium exchange. Moreover, the thermal unfolding of these proteins does not result in the uniform incorporation of deuterium throughout the polypeptide but involves the local unfolding of different residues at different temperatures. Some regions of the proteins (i.e. the "Greek key" loop, residues 104-116) unfold at a significantly higher temperature than other regions (i.e. beta3 and beta4, residues 21-53). Together, these results show that human wild-type apo-SOD1 and variants have a partially unfolded beta-barrel at physiological temperature and unfold non-cooperatively.

Detergent-insoluble aggregates associated with amyotrophic lateral sclerosis in transgenic mice contain primarily full-length, unmodified superoxide dismutase-1.

Determining the composition of aggregated superoxide dismutase 1 (SOD1) species associated with amyotrophic lateral sclerosis (ALS), especially with respect to co-aggregated proteins and post-translational modifications, could identify cellular or biochemical factors involved in the formation of these aggregates and explain their apparent neurotoxicity. The results of mass spectrometric and shotgun-proteomic analyses of SOD1-containing aggregates isolated from spinal cords of symptomatic transgenic ALS mice using two different isolation strategies are presented, including 1) resistance to detergent extraction and 2) size exclusion-coupled anti-SOD1 immunoaffinity chromatography. Forty-eight spinal cords from three different ALS-SOD1 mutant mice were analyzed, namely G93A, G37R, and the unnatural double mutant H46R/H48Q. The analysis consistently revealed that the most abundant proteins recovered from aggregate species were full-length unmodified SOD1 polypeptides. Although aggregates from some spinal cord samples contained trace levels of highly abundant proteins, such as vimentin and neurofilament-3, no proteins were consistently found to co-purify with mutant SOD1 in stoichiometric quantities. The results demonstrate that the principal protein in the high molecular mass aggregates whose appearance correlates with symptoms of the disease is the unmodified, full-length SOD1 polypeptide.

Binding of a single zinc ion to one subunit of copper-zinc superoxide dismutase apoprotein substantially influences the structure and stability of the entire homodimeric protein.

The thermodynamics of zinc binding to metal-free (apo) human and bovine copper-zinc superoxide dismutases (SOD1) were measured using isothermal titration calorimetry. The apparent thermodynamics of zinc binding to the apoproteins were favorable (Ka > 108 M-1), with an observed stoichiometry of one zinc per homodimer. The change in heat capacity for the one-zinc binding event was large and negative (approximately -650 cal mol-1 K-1), suggestive of significant structural changes to the protein upon zinc binding. We further characterized the one-zinc derivative by circular dichroism and determined that this derivative had nearly the same secondary structure as the two-zinc derivative and that both are structurally distinct from the metal-free protein. In addition, we monitored the effect of zinc binding on hydrogen-deuterium exchange and accessibility of histidyl residues to modification by diethyl pyrocarbonate and observed that more than 50% protection was afforded by the binding of one zinc in both assays. Differential scanning calorimetry on the human SOD1 zinc derivatives also showed increased thermostability of the protein due to zinc binding. Further, the melting transitions observed for the one-zinc derivative closely resembled those of the two-zinc derivative. Finally, we observed that the quaternary structure of the protein is stabilized upon binding of one and two zinc ions in analytical ultracentrifugation experiments. Combined, these results suggest communication between the two monomers of SOD1 such that the binding of one zinc ion per homodimer has a more profound effect on the homodimeric protein structure than the binding of subsequent metal ions. The relevance of these findings to amyotrophic lateral sclerosis is discussed.

Local unfolding in a destabilized, pathogenic variant of superoxide dismutase 1 observed with H/D exchange and mass spectrometry.

Hydrogen exchange monitored by mass spectrometry has been used to study the structural behavior of the pathogenic A4V variant of superoxide dismutase 1 (SOD1) in the metal-free (apo) form. Mass spectrometric data revealed that in the disulfide-intact (S-S) form, the A4V variant is destabilized at residues 50-53, in the disulfide subloop of the dimer interface, but many other regions of the A4V protein exhibited hydrogen exchange properties identical to that of the wild type protein. Additionally, mass spectrometry revealed that A4V apoSOD1(S-S) undergoes slow localized unfolding in a large segment of the beta-barrel that included beta3, beta4, and loops II and III. In the disulfide-reduced form, A4V apoSOD1 exchanged like a "random coil" polypeptide at 20 degrees C and began to populate folded states at 4 degrees C. These local and global unfolding events could facilitate intermolecular protein-protein interactions that cause the aggregation or neurotoxicity of A4V SOD1.

Destabilization of apoprotein is insufficient to explain Cu,Zn-superoxide dismutase-linked ALS pathogenesis.

The relative stabilities and structural properties of a representative set of 20 ALS-mutant Cu,Zn-superoxide dismutase apoproteins were examined by using differential scanning calorimetry and hydrogen-deuterium (H/D) exchange followed by MS. Contrary to recent reports from other laboratories, we found that ALS-mutant apoproteins are not universally destabilized by the disease-causing mutations. For example, several of the apoproteins with substitutions at or near the metal binding region (MBR) (MBR mutants) exhibited melting temperatures (Tm) in the range 51.6 degrees C to 56.2 degrees C, i.e., similar to or higher than that of the WT apoprotein (Tm = 52.5 degrees C). The apoproteins with substitutions remote from the MBR (WT-like mutants) showed a wide range of Tms, 40.0 degrees C to 52.4 degrees C. The H/D exchange properties of the mutants were also wide-ranging: the MBR mutant apoproteins exhibited H/D exchange kinetics similar to the WT apoprotein, as did some of the more stable WT-like mutant apoproteins, whereas the less stable apoproteins exhibited significantly less protection from H/D exchange than the WT apoprotein. Most striking were the three mutant apoproteins, D101N, E100K, and N139K, which have apparently normal metallation properties, and differ little from the WT apoprotein in either thermal stability or H/D exchange kinetics. Thus, the ALS mutant Cu,Zn-superoxide dismutase apoproteins do not all share reduced global stability, and additional properties must be identified and understood to explain the toxicity of all of the mutant proteins.

Spectroscopic investigation of stellacyanin mutants: axial ligand interactions at the blue copper site.

Detailed electronic and geometric structural descriptions of the blue copper sites in wild-type (WT) stellacyanin and its Q99M and Q99L axial mutants have been obtained using a combination of XAS, resonance Raman, MCD, EPR, and DFT calculations. The results show that the origin of the short Cu-S(Cys) bond in blue copper proteins is the weakened axial interaction, which leads to a shorter (based on EXAFS results) and more covalent (based on S K-edge XAS) Cu-S bond. XAS pre-edge energies show that the effective nuclear charge on the copper increases going from O(Gln) to S(Met) to no axial (Leu) ligand, indicating that the weakened axial ligand is not fully compensated for by the increased donation from the thiolate. This is further supported by EPR results. MCD data show that the decreased axial interaction leads to an increase in the equatorial ligand field, indicating that the site acquires a more trigonally distorted tetrahedral structure. These geometric and electronic structural changes, which result from weakening the bonding interaction of the axial ligand, allow the site to maintain efficient electron transfer (high H(DA) and low reorganization energy), while modulating the redox potential of the site to the biologically relevant range. These spectroscopic studies are complemented by DFT calculations to obtain insight into the factors that allow stellacyanin to maintain a trigonally distorted tetrahedral structure with a relatively strong axial Cu(II)-oxygen bond.