Exposure of Solvent-Inaccessible Regions in the Amyloidogenic Protein Human SOD1 Determined by Hydroxyl Radical Footprinting.

Solvent-accessibility change plays a critical role in protein misfolding and aggregation, the culprit for several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Mass spectrometry-based hydroxyl radical (·OH) protein footprinting has evolved as a powerful and fast tool in elucidating protein solvent accessibility. In this work, we used fast photochemical oxidation of protein (FPOP) hydroxyl radical (·OH) footprinting to investigate solvent accessibility in human copper-zinc superoxide dismutase (SOD1), misfolded or aggregated forms of which underlie a portion of ALS cases. ·OH-mediated modifications to 56 residues were detected with locations largely as predicted based on X-ray crystallography data, while the interior of SOD1 ß-barrel is hydrophobic and solvent-inaccessible and thus protected from modification. There were, however, two notable exceptions-two closely located residues inside the ß-barrel, predicted to have minimal or no solvent accessibility, that were found modified by FPOP (Phe20 and Ile112). Molecular dynamics (MD) simulations were consistent with differential access of peroxide versus quencher to SOD1's interior complicating surface accessibility considerations. Modification of these two residues could potentially be explained either by local motions of the ß-barrel that increased peroxide/solvent accessibility to the interior or by oxidative events within the interior that might include long-distance radical transfer to buried sites. Overall, comparison of modification patterns for the metal-free apoprotein versus zinc-bound forms demonstrated that binding of zinc protected the electrostatic loop and organized the copper-binding site. Our study highlights SOD1 hydrophobic groups that may contribute to early events in aggregation and discusses caveats to surface accessibility conclusions. Graphical Abstract.

Structural Characterization of Native Proteins and Protein Complexes by Electron Ionization Dissociation-Mass Spectrometry.

Mass spectrometry (MS) has played an increasingly important role in the identification and structural and functional characterization of proteins. In particular, the use of tandem mass spectrometry has afforded one of the most versatile methods to acquire structural information for proteins and protein complexes. The unique nature of electron capture dissociation (ECD) for cleaving protein backbone bonds while preserving noncovalent interactions has made it especially suitable for the study of native protein structures. However, the intra- and intermolecular interactions stabilized by hydrogen bonds and salt bridges can hinder the separation of fragments even with preactivation, which has become particularly problematic for the study of large macromolecular proteins and protein complexes. Here, we describe the capabilities of another activation method, 30 eV electron ionization dissociation (EID), for the top-down MS characterization of native protein-ligand and protein-protein complexes. Rich structural information that cannot be delivered by ECD can be generated by EID. EID allowed for the comparison of the gas-phase and the solution-phase structural stability and unfolding process of human carbonic anhydrase I (HCA-I). In addition, the EID fragmentation patterns reflect the structural similarities and differences among apo-, Zn-, and Cu,Zn-superoxide dismutase (SOD1) dimers. In particular, the structural changes due to Cu-binding and a point mutation (G41D) were revealed by EID-MS. The performance of EID was also compared to that of 193 nm ultraviolet photodissociation (UVPD), which allowed us to explore their qualitative similarities and differences as potential valuable tools for the MS study of native proteins and protein complexes.

Complete Molecular Weight Profiling of Low-Molecular Weight Heparins Using Size Exclusion Chromatography-Ion Suppressor-High-Resolution Mass Spectrometry.

Low-molecular weight heparins (LMWH) prepared by partial depolymerization of unfractionated heparin are used globally to treat coagulation disorders on an outpatient basis. Patent protection for several LMWH has expired and abbreviated new drug applications have been approved by the Food and Drug Administration. As a result, reverse engineering of LMWH for biosimilar LMWH has become an active global endeavor. Traditionally, the molecular weight distributions of LMWH preparations have been determined using size exclusion chromatography (SEC) with optical detection. Recent advances in liquid chromatography-mass spectrometry methods have enabled exact mass measurements of heparin saccharides roughly up to degree-of-polymerization 20, leaving the high molecular weight half of the LMWH preparation unassigned. We demonstrate a new LC-MS system capable of determining the exact masses of complete LMWH preparations, up to dp30. This system employed an ion suppressor cell to desalt the chromatographic effluent online prior to the electrospray mass spectrometry source. We expect this new capability will impact the ability to define LMWH mixtures favorably.

Insights into the role of the unusual disulfide bond in copper-zinc superoxide dismutase.

The functional and structural significance of the intrasubunit disulfide bond in copper-zinc superoxide dismutase (SOD1) was studied by characterizing mutant forms of human SOD1 (hSOD) and yeast SOD1 lacking the disulfide bond. We determined x-ray crystal structures of metal-bound and metal-deficient hC57S SOD1. C57S hSOD1 isolated from yeast contained four zinc ions per protein dimer and was structurally very similar to wild type. The addition of copper to this four-zinc protein gave properly reconstituted 2Cu,2Zn C57S hSOD, and its spectroscopic properties indicated that the coordination geometry of the copper was remarkably similar to that of holo wild type hSOD1. In contrast, the addition of copper and zinc ions to apo C57S human SOD1 failed to give proper reconstitution. Using pulse radiolysis, we determined SOD activities of yeast and human SOD1s lacking disulfide bonds and found that they were enzymatically active at ∼10% of the wild type rate. These results are contrary to earlier reports that the intrasubunit disulfide bonds in SOD1 are essential for SOD activity. Kinetic studies revealed further that the yeast mutant SOD1 had less ionic attraction for superoxide, possibly explaining the lower rates. Saccharomyces cerevisiae cells lacking the sod1 gene do not grow aerobically in the absence of lysine, but expression of C57S SOD1 increased growth to 30-50% of the growth of cells expressing wild type SOD1, supporting that C57S SOD1 retained a significant amount of activity.

Yeast copper-zinc superoxide dismutase can be activated in the absence of its copper chaperone.

Copper-zinc superoxide dismutase (Sod1) is an abundant intracellular enzyme that catalyzes the disproportionation of superoxide to give hydrogen peroxide and dioxygen. In most organisms, Sod1 acquires copper by a combination of two pathways, one dependent on the copper chaperone for Sod1 (CCS), and the other independent of CCS. Examples have been reported of two exceptions: Saccharomyces cerevisiae, in which Sod1 appeared to be fully dependent on CCS, and Caenorhabditis elegans, in which Sod1 was completely independent of CCS. Here, however, using overexpressed Sod1, we show there is also a significant amount of CCS-independent activation of S. cerevisiae Sod1, even in low-copper medium. In addition, we show CCS-independent oxidation of the disulfide bond in S. cerevisiae Sod1. There appears to be a continuum between CCS-dependent and CCS-independent activation of Sod1, with yeast falling near but not at the CCS-dependent end.

Tetramerization reinforces the dimer interface of MnSOD.

Two yeast manganese superoxide dismutases (MnSOD), one from Saccharomyces cerevisiae mitochondria (ScMnSOD) and the other from Candida albicans cytosol (CaMnSODc), have most biochemical and biophysical properties in common, yet ScMnSOD is a tetramer and CaMnSODc is a dimer or "loose tetramer" in solution. Although CaMnSODc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of CaMnSODc in vivo depends upon the formation of the tetrameric structure. To elucidate further the functional significance of MnSOD quaternary structure, wild-type and mutant forms of ScMnSOD (K182R, A183P mutant) and CaMnSODc (K184R, L185P mutant) with the substitutions at dimer interfaces were analyzed with respect to their oligomeric states and resistance to pH, heat, and denaturant. Dimeric CaMnSODc was found to be significantly more subject to thermal or denaturant-induced unfolding than tetrameric ScMnSOD. The residue substitutions at dimer interfaces caused dimeric CaMnSODc but not tetrameric ScMnSOD to dissociate into monomers. We conclude that the tetrameric assembly strongly reinforces the dimer interface, which is critical for MnSOD activity.

Six-coordinate manganese(3+) in catalysis by yeast manganese superoxide dismutase.

Reduction of superoxide (O2-) by manganese-containing superoxide dismutase occurs through either a "prompt protonation" pathway, or an "inner-sphere" pathway, with the latter leading to formation of an observable Mn-peroxo complex. We recently reported that wild-type (WT) manganese superoxide dismutases (MnSODs) from Saccharomyces cerevisiae and Candida albicans are more gated toward the "prompt protonation" pathway than human and bacterial MnSODs and suggested that this could result from small structural changes in the second coordination sphere of manganese. We report here that substitution of a second-sphere residue, Tyr34, by phenylalanine (Y34F) causes the MnSOD from S. cerevisiae to react exclusively through the "inner-sphere" pathway. At neutral pH, we have a surprising observation that protonation of the Mn-peroxo complex in the mutant yeast enzyme occurs through a fast pathway, leading to a putative six-coordinate Mn(3+) species, which actively oxidizes O2- in the catalytic cycle. Upon increasing pH, the fast pathway is gradually replaced by a slow proton-transfer pathway, leading to the well-characterized five-coordinate Mn(3+). We here propose and compare two hypothetical mechanisms for the mutant yeast enzyme, differing in the structure of the Mn-peroxo complex yet both involving formation of the active six-coordinate Mn(3+) and proton transfer from a second-sphere water molecule, which has substituted for the -OH of Tyr34, to the Mn-peroxo complex. Because WT and the mutant yeast MnSOD both rest in the 2+ state and become six-coordinate when oxidized up from Mn(2+), six-coordinate Mn(3+) species could also actively function in the mechanism of WT yeast MnSODs.

SOD1 aggregation and ALS: role of metallation states and disulfide status.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the death of motor neurons. About 10% of ALS cases are inherited (familial), and a large subset of them are caused by mutations in the gene encoding the copper-zinc superoxide dismutase (SOD1). The detection of SOD1-positive inclusions in familial ALS patients suggests the role of SOD1 aggregation underlying the pathology of familial ALS. Although SOD1 mutant proteins are different in structure, stability and activity, they all exhibit a higher aggregation propensity than wild-type SOD1. We here review the recent studies on the role of metallation states and disulfide status in the unfolding, misfolding, and aggregation of SOD1. Investigations of the mechanism of SOD1 aggregation enhance our understanding of onset and progression of ALS and have implications for therapeutic approaches for treating ALS.

Comparison of two yeast MnSODs: mitochondrial Saccharomyces cerevisiae versus cytosolic Candida albicans.

Human MnSOD is significantly more product-inhibited than bacterial MnSODs at high concentrations of superoxide (O(2)(-)). This behavior limits the amount of H(2)O(2) produced at high [O(2)(-)]; its desirability can be explained by the multiple roles of H(2)O(2) in mammalian cells, particularly its role in signaling. To investigate the mechanism of product inhibition in MnSOD, two yeast MnSODs, one from Saccharomyces cerevisiae mitochondria (ScMnSOD) and the other from Candida albicans cytosol (CaMnSODc), were isolated and characterized. ScMnSOD and CaMnSODc are similar in catalytic kinetics, spectroscopy, and redox chemistry, and they both rest predominantly in the reduced state (unlike most other MnSODs). At high [O(2)(-)], the dismutation efficiencies of the yeast MnSODs surpass those of human and bacterial MnSODs, due to very low level of product inhibition. Optical and parallel-mode electron paramagnetic resonance (EPR) spectra suggest the presence of two Mn(3+) species in yeast Mn(3+)SODs, including the well-characterized 5-coordinate Mn(3+) species and a 6-coordinate L-Mn(3+) species with hydroxide as the putative sixth ligand (L). The first and second coordination spheres of ScMnSOD are more similar to bacterial than to human MnSOD. Gln154, an H-bond donor to the Mn-coordinated solvent molecule, is slightly further away from Mn in yeast MnSODs, which may result in their unusual resting state. Mechanistically, the high efficiency of yeast MnSODs could be ascribed to putative translocation of an outer-sphere solvent molecule, which could destabilize the inhibited complex and enhance proton transfer from protein to peroxide. Our studies on yeast MnSODs indicate the unique nature of human MnSOD in that it predominantly undergoes the inhibited pathway at high [O(2)(-)].

Comparative characterization of fungal anthracenone and naphthacenedione biosynthetic pathways reveals an α-hydroxylation-dependent Claisen-like cyclization catalyzed by a dimanganese thioesterase.

The linear tetracyclic TAN-1612 (1) and BMS-192548 (2) were isolated from different filamentous fungal strains and have been examined as potential neuropeptide Y and neurokinin-1 receptor antagonists, respectively. Although the biosynthesis of fungal aromatic polyketides has attracted much interest in recent years, the biosynthetic mechanism for such naphthacenedione-containing products has not been established. Using a targeted genome mining approach, we first located the ada gene cluster responsible for the biosynthesis of 1 in Aspergillus niger ATCC 1015. The connection between 1 and the ada pathway was verified through overexpression of the Zn(2)Cys(6)-type pathway-specific transcriptional regulator AdaR and subsequent gene expression analysis. The enzymes encoded in the ada gene cluster share high sequence similarities to the known apt pathway linked to the biosynthesis of anthraquinone asperthecin 3. Subsequent comparative investigation of these two highly homologous gene clusters by heterologous pathway reconstitution in Saccharomyces cerevisiae revealed a novel α-hydroxylation-dependent Claisen cyclization cascade, which involves a flavin-dependent monooxygenase that hydroxylates the α-carbon of an acyl carrier protein-bound polyketide and a bifunctional metallo-ß-lactamase-type thioesterase (MßL-TE). The bifunctional MßL-TE catalyzes the fourth ring cyclization to afford the naphthacenedione scaffold upon α-hydroxylation, whereas it performs hydrolytic release of an anthracenone product in the absence of α-hydroxylation. Through in vitro biochemical characterizations and metal analyses, we verified that the apt MßL-TE is a dimanganese enzyme and requires both Mn(2+) cations for the observed activities. The MßL-TE is the first example of a thioesterase in polyketide biosynthesis that catalyzes the Claisen-like condensation without an α/ß hydrolase fold and forms no covalent bond with the substrate. These mechanistic features should be general to the biosynthesis of tetracyclic naphthacenedione compounds in fungi.

Detection of mercury ion by infrared fluorescent protein and its hydrogel-based paper assay.

Mercury is a highly hazardous and widespread pollutant with bioaccumulative properties. Novel approaches that meet the criteria of desired selectivity, high sensitivity, good biocompatibility, and low background interference in natural settings are continuously being explored. We herein describe a new strategy utilizing the combination of infrared fluorescent protein (IFP) and its chromophore as an infrared fluorescence probe for mercury ion (Hg(II)) detection. Hg(II) has been validated to have specific binding affinity to a cysteine residue (C24) of IFP, thereby inhibiting the conjugation of IFP chromophore biliverdin (BV) to C24 and "turning off" the infrared emission of IFP. The IFP/BV sensor has high selectivity toward Hg(II) among other metal ions over a broad pH range. The in vitro detection limit was determined to be less than 50 nM. As a genetically encoded probe, we demonstrate the IFP/BV sensor can serve as a tool to detect Hg(II) in living organisms or tissues. Moreover, we have exploited a protein-agarose hydrogel-based paper assay to immobilize IFP for detection of Hg(II) in a portable and robust fashion.

Investigation of the highly active manganese superoxide dismutase from Saccharomyces cerevisiae.

Manganese superoxide dismutase (MnSOD) from different species differs in its efficiency in removing high concentrations of superoxide (O(2)(-)), due to different levels of product inhibition. Human MnSOD exhibits a substantially higher level of product inhibition than the MnSODs from bacteria. In order to investigate the mechanism of product inhibition and whether it is a feature common to eukaryotic MnSODs, we purified MnSOD from Saccharomyces cerevisiae (ScMnSOD). It was a tetramer with 0.6 equiv of Mn per monomer. The catalytic activity of ScMnSOD was investigated by pulse radiolysis and compared with human and two bacterial (Escherichia coli and Deinococcus radiodurans) MnSODs. To our surprise, ScMnSOD most efficiently facilitates removal of high concentrations of O(2)(-) among these MnSODs. The gating value k(2)/k(3) that characterizes the level of product inhibition scales as ScMnSOD > D. radiodurans MnSOD > E. coli MnSOD > human MnSOD. While most MnSODs rest as the oxidized form, ScMnSOD was isolated in the Mn(2+) oxidation state as revealed by its optical and electron paramagnetic resonance spectra. This finding poses the possibility of elucidating the origin of product inhibition by comparing human MnSOD with ScMnSOD.

New metal-organic frameworks with large cavities: selective sorption and desorption of solvent molecules.

Five novel transition metal complexes [Cd(II) (3)(tpba-2)(2)(SCN)(6)].6 THF.3 H(2)O (1), [Cu(II) (3)(tpba-2)(2)(SCN)(6)].6 THF.3 H(2)O (2), [Ni(II) (3)(tpba-2)(2)(SCN)(6)].6 THF.3 H(2)O (3), [Cd(II) (2)(tpba-2)(SCN)(3)]ClO(4) (4), [Cu(I) (3)(SCN)(6)(H(3)tpba-2)] (5) [TPBA-2 = N',N'',N'''-tris(pyrid-2-ylmethyl)-1,3,5-benzenetricarboxamide, THF=tetrahydrofuran] were obtained by reactions of the corresponding transition metal salts with TPBA-2 ligand in the presence of NH(4)SCN using layering or solvothermal method, respectively. The results of X-ray crystallographic analysis showed that complexes 1, 2 and 3 are isostructural and have the same 2D honeycomb network structure with Kagomé lattice, in which all the M(II) (M = Cd, Cu, Ni) atoms are six-coordinated, and the TPBA-2 ligands adopt cis,cis,cis conformation while the thiocyanate anions act as terminal ligands. Capsule-like motifs are found in 1, 2 and 3, in which six THF molecules are hosted, and the results of XPRD and solid-state (13)C NMR spectral measurements showed that the compound 1 can selectively desorb and adsorb THF molecules occurring along with the re-establishment of its crystallinity. In contrast to 1, 2 and 3, complex 4 has different 2D network structure, resulting from TPBA-2 ligands with cis,trans,trans conformation, thiocyanate anions serving as end-to-end bridging ligands, and the incomplete replacement of perchlorate anions, which further link the 2D layers into 3D framework by the hydrogen bonds. In complex 5, the Cu(II) atoms are reduced to Cu(I) during the process of solvothermal reaction, and the Cu(I) atoms are connected by thiocyanate anions to form a 3D porous framework, in which the protonated TPBA-2 ligands are hosted in the cavities as templates.