Proton-coupled electron transfer at a mis-metalated zinc site detected with protein charge ladders.

Distinguishing proton-coupled electron transfer (PCET) from uncoupled electron transfer (ET) in proteins can be challenging. A recent investigation [J. C. Koone, M. Simmang, D. L. Saenger, M. L. Hunsicker-Wang and B. F. Shaw, J. Am. Chem. Soc., 145, 16488-16497] reported that protein charge ladders and capillary electrophoresis can distinguish between single ET, PCET, and two-proton coupled ET (2PCET) by directly measuring the change in protein net charge upon reduction/oxidation (ΔZET). The current study used similar methods to assess PCET in zinc-free, "double copper" superoxide dismutase-1 (4Cu-SOD1), where one copper is bound at the copper site of each monomer and one copper is bound at the bridging zinc site, resulting in a quasi-type III Cu center. At pH 7.4, the net charge (Z) of the 4Cu-SOD1 dimer was unaffected by reduction of all four Cu2+ ions, i.e., ΔZ4ET = -0.09 ± 0.05 per dimer (-0.02 ± 0.01 per copper atom). These values suggest that PCET is taking place at all four Cu atoms of the homodimer. Molecular dynamics and Poisson-Boltzmann calculations suggest that a metal-coordinating histidine at the zinc site (His71) is the proton acceptor. These data show how ligands of a naturally occurring zinc site can help facilitate PCET when the right redox metal is bound.

Metal migration and subunit swapping in ALS-linked SOD1: Zn2+ transfer between mutant and wild-type occurs faster than the rate of heterodimerization.

The heterodimerization of WT Cu, Zn superoxide dismutase-1 (SOD1), and mutant SOD1 might be a critical step in the pathogenesis of SOD1-linked amyotrophic lateral sclerosis (ALS). Rates and free energies of heterodimerization (ΔGHet) between WT and ALS-mutant SOD1 in mismatched metalation states-where one subunit is metalated and the other is not-have been difficult to obtain. Consequently, the hypothesis that under-metalated SOD1 might trigger misfolding of metalated SOD1 by "stealing" metal ions remains untested. This study used capillary zone electrophoresis and mass spectrometry to track heterodimerization and metal transfer between WT SOD1, ALS-variant SOD1 (E100K, E100G, D90A), and triply deamidated SOD1 (modeled with N26D/N131D/N139D substitutions). We determined that rates of subunit exchange between apo dimers and metalated dimers-expressed as time to reach 30% heterodimer-ranged from t30% = 67.75 ± 9.08 to 338.53 ± 26.95 min; free energies of heterodimerization ranged from ΔGHet = -1.21 ± 0.31 to -3.06 ± 0.12 kJ/mol. Rates and ΔGHet values of partially metalated heterodimers were more similar to those of fully metalated heterodimers than apo heterodimers, and largely independent of which subunit (mutant or WT) was metal-replete or metal-free. Mass spectrometry and capillary electrophoresis demonstrated that mutant or WT 4Zn-SOD1 could transfer up to two equivalents of Zn2+ to mutant or WT apo-SOD1 (at rates faster than the rate of heterodimerization). This result suggests that zinc-replete SOD1 can function as a chaperone to deliver Zn2+ to apo-SOD1, and that WT apo-SOD1 might increase the toxicity of mutant SOD1 by stealing its Zn2+.

Charge Regulation in a Rieske Proton Pump Pinpoints Zero, One, and Two Proton-Coupled Electron Transfer.

The degree to which redox-driven proton pumps regulate net charge during electron transfer (ΔZET) remains undetermined due to difficulties in measuring the net charge of solvated proteins. Values of ΔZET can reflect reorganization energies or redox potentials associated with ET and can be used to distinguish ET from proton(s)-coupled electron transfer (PCET). Here, we synthesized protein "charge ladders" of a Rieske [2Fe-2S] subunit from Thermus thermophilus (truncTtRp) and made 120 electrostatic measurements of ΔZET across pH. Across pH 5-10, truncTtRp is suspected of transitioning from ET to PCET, and then to two proton-coupled ET (2PCET). Upon reduction, we found that truncTtRp became more negative at pH 6.0 by one unit (ΔZET = -1.01 ± 0.14), consistent with single ET; was isoelectric at pH 8.8 (ΔZET = -0.01 ± 0.45), consistent with PCET; and became more positive at pH 10.6 (ΔZET = +1.37 ± 0.60), consistent with 2PCET. These ΔZET values are attributed to protonation of H154 and H134. Across pH, redox potentials of TtRp (measured previously) correlated with protonation energies of H154 and H134 and ΔZET for truncTtRp, supporting a discrete proton pumping mechanism for Rieske proteins at the Fe-coordinating histidines.

Supercharging Prions via Amyloid-Selective Lysine Acetylation.

Repulsive electrostatic forces between prion-like proteins are a barrier against aggregation. In neuropharmacology, however, a prion's net charge (Z) is not a targeted parameter. Compounds that selectively boost prion Z remain unreported. Here, we synthesized compounds that amplified the negative charge of misfolded superoxide dismutase-1 (SOD1) by acetylating lysine-NH3+ in amyloid-SOD1, without acetylating native-SOD1. Compounds resembled a "ball and chain" mace: a rigid amyloid-binding "handle" (benzothiazole, stilbene, or styrylpyridine); an aryl ester "ball"; and a triethylene glycol chain connecting ball to handle. At stoichiometric excess, compounds acetylated up to 9 of 11 lysine per misfolded subunit (ΔZfibril =-8100 per 103 subunits). Acetylated amyloid-SOD1 seeded aggregation more slowly than unacetylated amyloid-SOD1 in vitro and organotypic spinal cord (these effects were partially due to compound binding). Compounds exhibited reactivity with other amyloid and non-amyloid proteins (e.g., fibrillar α-synuclein was peracetylated; serum albumin was partially acetylated; carbonic anhydrase was largely unacetylated).

Complete Charge Regulation by a Redox Enzyme Upon Single Electron Transfer.

The degree by which metalloproteins partially regulate net charge (Z) upon electron transfer (ET) was recently measured for the first time using "protein charge ladders" of azurin, cytochrome c, and myoglobin [Angew. Chem. Int. Ed. 2018, 57(19), 5364-5368; Angew. Chem. 2018, 130, 5462-5466]. Here, we show that Cu, Zn superoxide dismutase (SOD1) is unique among proteins in its ability to resist changes in net charge upon single ET (e.g., ΔZET(SOD1) =0.05±0.08 per electron, compared to ΔZET(Cyt-c) =1.19±0.02). This total regulation of net charge by SOD1 is attributed to the protonation of the bridging histidine upon copper reduction, yielding redox centers that are isoelectric at both copper oxidation states. Charge regulation by SOD1 would prevent long range coulombic perturbations to residue pKa 's upon ET at copper, allowing SOD1's "electrostatic loop" to attract superoxide with equal affinity (at both redox states of copper) during diffusion-limited reduction and oxidation of superoxide.

What Are We Missing by Not Measuring the Net Charge of Proteins?

The net electrostatic charge (Z) of a folded protein in solution represents a bird's eye view of its surface potentials-including contributions from tightly bound metal, solvent, buffer, and cosolvent ions-and remains one of its most enigmatic properties. Few tools are available to the average biochemist to rapidly and accurately measure Z at pH≠pI. Tools that have been developed more recently seem to go unnoticed. Most scientists are content with this void and estimate the net charge of a protein from its amino acid sequence, using textbook values of pKa . Thus, Z remains unmeasured for nearly all folded proteins at pH≠pI. When marveling at all that has been learned from accurately measuring the other fundamental property of a protein-its mass-one wonders: what are we missing by not measuring the net charge of folded, solvated proteins? A few big questions immediately emerge in bioinorganic chemistry. When a single electron is transferred to a metalloprotein, does the net charge of the protein change by approximately one elementary unit of charge or does charge regulation dominate, that is, do the pKa values of most ionizable residues (or just a few residues) adjust in response to (or in concert with) electron transfer? Would the free energy of charge regulation (ΔΔGz ) account for most of the outer sphere reorganization energy associated with electron transfer? Or would ΔΔGz contribute more to the redox potential? And what about metal binding itself? When an apo-metalloprotein, bearing minimal net negative charge (e.g., Z=-2.0) binds one or more metal cations, is the net charge abolished or inverted to positive? Or do metalloproteins regulate net charge when coordinating metal ions? The author's group has recently dusted off a relatively obscure tool-the "protein charge ladder"-and used it to begin to answer these basic questions.

Lysine acylation in superoxide dismutase-1 electrostatically inhibits formation of fibrils with prion-like seeding.

The acylation of lysine residues in superoxide dismutase-1 (SOD1) has been previously shown to decrease its rate of nucleation and elongation into amyloid-like fibrils linked to amyotrophic lateral sclerosis. The chemical mechanism underlying this effect is unclear, i.e. hydrophobic/steric effects versus electrostatic effects. Moreover, the degree to which the acylation might alter the prion-like seeding of SOD1 in vivo has not been addressed. Here, we acylated a fraction of lysine residues in SOD1 with groups of variable hydrophobicity, charge, and conformational entropy. The effect of each acyl group on the rate of SOD1 fibril nucleation and elongation were quantified in vitro with thioflavin-T (ThT) fluorescence, and we performed 594 iterate aggregation assays to obtain statistically significant rates. The effect of the lysine acylation on the prion-like seeding of SOD1 was assayed in spinal cord extracts of transgenic mice expressing a G85R SOD1-yellow fluorescent protein construct. Acyl groups with >2 carboxylic acids diminished self-assembly into ThT-positive fibrils and instead promoted the self-assembly of ThT-negative fibrils and amorphous complexes. The addition of ThT-negative, acylated SOD1 fibrils to organotypic spinal cord failed to produce the SOD1 inclusion pathology that typically results from the addition of ThT-positive SOD1 fibrils. These results suggest that chemically increasing the net negative surface charge of SOD1 via acylation can block the prion-like propagation of oligomeric SOD1 in spinal cord.

Direct Measurement of Charge Regulation in Metalloprotein Electron Transfer.

Determining whether a protein regulates its net electrostatic charge during electron transfer (ET) will deepen our mechanistic understanding of how polypeptides tune rates and free energies of ET (e.g., by affecting reorganization energy, and/or redox potential). Charge regulation during ET has never been measured for proteins because few tools exist to measure the net charge of a folded protein in solution at different oxidation states. Herein, we used a niche analytical tool (protein charge ladders analyzed with capillary electrophoresis) to determine that the net charges of myoglobin, cytochrome c, and azurin change by 0.62±0.06, 1.19±0.02, and 0.51±0.04 units upon single ET. Computational analysis predicts that these fluctuations in charge arise from changes in the pKa  values of multiple non-coordinating residues (predominantly histidine) that involve between 0.42-0.90 eV. These results suggest that ionizable residues can tune the reactivity of redox centers by regulating the net charge of the entire protein-cofactor-solvent complex.

How Do Gyrating Beads Accelerate Amyloid Fibrillization?

The chemical and physical mechanisms by which gyrating beads accelerate amyloid fibrillization in microtiter plate assays are unclear. Identifying these mechanisms will help optimize high-throughput screening assays for molecules and mutations that modulate aggregation and might explain why different research groups report different rates of aggregation for identical proteins. This article investigates how the rate of superoxide dismutase-1 (SOD1) fibrillization is affected by 12 different beads with a wide range of hydrophobicity, mass, stiffness, and topology but identical diameter. All assays were performed on D90A apo-SOD1, which is a stable and wild-type-like variant of SOD1. The most significant and uniform correlation between any material property of each bead and that bead's effect on SOD1 fibrillization rate was with regard to bead mass. A linear correlation existed between bead mass and rate of fibril elongation (R2 = 0.7): heavier beads produced faster rates and shorter fibrils. Nucleation rates (lag time) also correlated with bead mass, but only for non-polymeric beads (i.e., glass, ceramic, metallic). The effect of bead mass on fibrillization correlated (R2 = 0.96) with variations in buoyant forces and contact forces (between bead and microplate well), and was not an artifact of residual momentum during intermittent gyration. Hydrophobic effects were observed, but only for polymeric beads: lag times correlated negatively with contact angle of water and degree of protein adhesion (surface adhesion and hydrophobic effects were negligible for non-polymeric beads). These results demonstrate that contact forces (alone) explain kinetic variation among non-polymeric beads, whereas surface hydrophobicity and contact forces explain kinetic variation among polymeric beads. This study also establishes conditions for high-throughput amyloid assays of SOD1 that enable the control over fibril morphologies and produce eightfold faster lag times and fourfold less stochasticity than in previous studies.

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.

Gibbs Energy of Superoxide Dismutase Heterodimerization Accounts for Variable Survival in Amyotrophic Lateral Sclerosis.

The exchange of subunits between homodimeric mutant Cu, Zn superoxide dismutase (SOD1) and wild-type (WT) SOD1 is suspected to be a crucial step in the onset and progression of amyotrophic lateral sclerosis (ALS). The rate, mechanism, and ΔG of heterodimerization (ΔGHet) all remain undetermined, due to analytical challenges in measuring heterodimerization. This study used capillary zone electrophoresis to measure rates of heterodimerization and ΔGHet for seven ALS-variant apo-SOD1 proteins that are clinically diverse, producing mean survival times between 2 and 12 years (postdiagnosis). The ΔGHet of each ALS variant SOD1 correlated with patient survival time after diagnosis (R(2) = 0.98), with more favorable ΔGHet correlating with shorter survival by 4.8 years per kJ. Rates of heterodimerization did not correlate with survival time or age of disease onset. Metalation diminished the rate of subunit exchange by up to ∼38-fold but only altered ΔGHet by <1 kJ mol(-1). Medicinal targeting of heterodimer thermodynamics represents a plausible strategy for prolonging life in SOD1-linked ALS.

Arresting amyloid with coulomb's law: acetylation of ALS-linked SOD1 by aspirin impedes aggregation.

Although the magnitude of a protein's net charge (Z) can control its rate of self-assembly into amyloid, and its interactions with cellular membranes, the net charge of a protein is not viewed as a druggable parameter. This article demonstrates that aspirin (the quintessential acylating pharmacon) can inhibit the amyloidogenesis of superoxide dismutase (SOD1) by increasing the intrinsic net negative charge of the polypeptide, i.e., by acetylation (neutralization) of multiple lysines. The protective effects of acetylation were diminished (but not abolished) in 100 mM NaCl and were statistically significant: a total of 432 thioflavin-T amyloid assays were performed for all studied proteins. The acetylation of as few as three lysines by aspirin in A4V apo-SOD1-a variant that causes familial amyotrophic lateral sclerosis (ALS)-delayed amyloid nucleation by 38% and slowed amyloid propagation by twofold. Lysines in wild-type- and ALS-variant apo-SOD1 could also be peracetylated with aspirin after fibrillization, resulting in supercharged fibrils, with increases in formal net charge of ∼2 million units. Peracetylated SOD1 amyloid defibrillized at temperatures below unacetylated fibrils, and below the melting temperature of native Cu2,Zn2-SOD1 (e.g., fibril Tm = 84.49°C for acetylated D90A apo-SOD1 fibrils). Targeting the net charge of native or misfolded proteins with small molecules-analogous to how an enzyme's Km or Vmax are medicinally targeted-holds promise as a strategy in the design of therapies for diseases linked to protein self-assembly.

Metal-ion-specific screening of charge effects in protein amide H/D exchange and the Hofmeister series.

In this study, protein charge ladders and mass spectrometry were used to quantify how metal cations in the Hofmeister series (Na(+), K(+), Li(+), Mg(2+), and Ca(2+)) permute the effects of lysine acetylation on the rate of amide H/D exchange in a representative protein (myoglobin, Mb). The successive acetylation of up to 18 Lys-ε-NH3(+) groups in Mb caused a linear decrease in its global rate of amide H/D exchange (as measured by mass spectrometry), despite also decreasing the thermostability of Mb by >10 °C. The ability of a metal cation to screen kinetic electrostatic effects during H/D exchange-and to abolish the protective effect of acetylation against H/D exchange-was found to depend on the position of the cation in the Hofmeister series. Na(+) and K(+) cations did not fully equalize the rates of H/D exchange among each "rung" of the charge ladder, whereas Mg(2+) and Ca(2+) did equalize rates without eliminating the hydrophobic core of the protein (i.e., without unfolding Mb); Li(+) exhibited intermediate effects. The ability of Mg(2+) and Ca(2+) to completely screen electrostatic effects associated with the H/D exchange of charge isomers of Mb suggests that Mg(2+) or Ca(2+) (but not Na(+) or K(+)) can be used to quantify the magnitude by which electrostatic charge contributes to the observed rates of amide H/D exchange in proteins.

Detection of leukocoria using a soft fusion of expert classifiers under non-clinical settings.

BACKGROUND: Leukocoria is defined as a white reflection and its manifestation is symptomatic of several ocular pathologies, including retinoblastoma (Rb). Early detection of recurrent leukocoria is critical for improved patient outcomes and can be accomplished via the examination of recreational photography. To date, there exists a paucity of methods to automate leukocoria detection within such a dataset. METHODS: This research explores a novel classification scheme that uses fuzzy logic theory to combine a number of classifiers that are experts in performing multichannel detection of leukocoria from recreational photography. The proposed scheme extracts features aided by the discrete cosine transform and the Karhunen-Loeve transformation. RESULTS: The soft fusion of classifiers is significantly better than other methods of combining classifiers with p = 1.12 × 10-5. The proposed methodology performs at a 92% accuracy rate, with an 89% true positive rate, and an 11% false positive rate. Furthermore, the results produced by our methodology exhibit the lowest average variance. CONCLUSIONS: The proposed methodology overcomes non-ideal conditions of image acquisition, presenting a competent approach for the detection of leukocoria. Results suggest that recreational photography can be used in combination with the fusion of individual experts in multichannel classification and preprocessing tools such as the discrete cosine transform and the Karhunen-Loeve transformation.

Heat-induced structural and chemical changes to a computationally designed miniprotein.

The de novo design of miniprotein inhibitors has recently emerged as a new technology to create proteins that bind with high affinity to specific therapeutic targets. Their size, ease of expression, and apparent high stability makes them excellent candidates for a new class of protein drugs. However, beyond circular dichroism melts and hydrogen/deuterium exchange experiments, little is known about their dynamics, especially at the elevated temperatures they seemingly tolerate quite well. To address that and gain insight for future designs, we have focused on identifying unintended and previously overlooked heat-induced structural and chemical changes in a particularly stable model miniprotein, EHEE_rd2_0005. Nuclear magnetic resonance (NMR) studies suggest the presence of dynamics on multiple time and temperature scales. Transiently elevating the temperature results in spontaneous chemical deamidation visible in the NMR spectra, which we validate using both capillary electrophoresis and mass spectrometry (MS) experiments. High temperatures also result in greatly accelerated intrinsic rates of hydrogen exchange and signal loss in NMR heteronuclear single quantum coherence spectra from local unfolding. These losses are in excellent agreement with both room temperature hydrogen exchange experiments and hydrogen bond disruption in replica exchange molecular dynamics simulations. Our analysis reveals important principles for future miniprotein designs and the potential for high stability to result in long-lived alternate conformational states.

Universal pictures: A lithophane codex helps teenagers with blindness visualize nanoscopic systems.

People with blindness have limited access to the high-resolution graphical data and imagery of science. Here, a lithophane codex is reported. Its pages display tactile and optical readouts for universal visualization of data by persons with or without eyesight. Prototype codices illustrated microscopy of butterfly chitin-from N-acetylglucosamine monomer to fibril, scale, and whole insect-and were given to high schoolers from the Texas School for the Blind and Visually Impaired. Lithophane graphics of Fischer-Spier esterification reactions and electron micrographs of biological cells were also 3D-printed, along with x-ray structures of proteins (as millimeter-scale 3D models). Students with blindness could visualize (describe, recall, distinguish) these systems-for the first time-at the same resolution as sighted peers (average accuracy = 88%). Tactile visualization occurred alongside laboratory training, synthesis, and mentoring by chemists with blindness, resulting in increased student interest and sense of belonging in science.

Deamidation of asparagine to aspartate destabilizes Cu, Zn superoxide dismutase, accelerates fibrillization, and mirrors ALS-linked mutations.

The reactivity of asparagine residues in Cu, Zn superoxide dismutase (SOD1) to deamidate to aspartate remains uncharacterized; its occurrence in SOD1 has not been investigated, and the biophysical effects of deamidation on SOD1 are unknown. Deamidation is, nonetheless, chemically equivalent to Asn-to-Asp missense mutations in SOD1 that cause amyotrophic lateral sclerosis (ALS). This study utilized computational methods to identify three asparagine residues in wild-type (WT) SOD1 (i.e., N26, N131, and N139) that are predicted to undergo significant deamidation (i.e., to >20%) on time scales comparable to the long lifetime (>1 year) of SOD1 in large motor neurons. Site-directed mutagenesis was used to successively substitute these asparagines with aspartate (to mimic deamidation) according to their predicted deamidation rate, yielding: N26D, N26D/N131D, and N26D/N131D/N139D SOD1. Differential scanning calorimetry demonstrated that the thermostability of N26D/N131D/N139D SOD1 is lower than WT SOD1 by ~2-8 °C (depending upon the state of metalation) and <3 °C lower than the ALS mutant N139D SOD1. The triply deamidated analog also aggregated into amyloid fibrils faster than WT SOD1 by ~2-fold (p < 0.008**) and at a rate identical to ALS mutant N139D SOD1 (p > 0.2). A total of 534 separate amyloid assays were performed to generate statistically significant comparisons of aggregation rates among WT and N/D SOD1 proteins. Capillary electrophoresis and mass spectrometry demonstrated that ~23% of N26 is deamidated to aspartate (iso-aspartate was undetectable) in a preparation of WT human SOD1 (isolated from erythrocytes) that has been used for decades by researchers as an analytical standard. The deamidation of asparagine--an analytically elusive, sub-Dalton modification--represents a plausible and overlooked mechanism by which WT SOD1 is converted to a neurotoxic isoform that has a similar structure, instability, and aggregation propensity as ALS mutant N139D SOD1.

Oxidation of Dueling Cysteine Promotes Subunit Exchange in SOD1.

The heterodimerization of wild-type (WT) Cu, Zn superoxide dismutase-1 (SOD1) and mutant SOD1 might be a critical step in the pathogenesis of SOD1-linked amyotrophic lateral sclerosis (ALS). Post-translational modifications that accelerate SOD1 heterodimerization remain unidentified. Here, we used capillary electrophoresis to quantify the effect of cysteine-111 oxidation on the rate and free energy of ALS mutant/WT SOD1 heterodimerization. The oxidation of Cys111-ß-SH to sulfinic and sulfonic acid (by hydrogen peroxide) increased rates of heterodimerization (with unoxidized protein) by ∼3-fold. Cysteine oxidation drove the equilibrium free energy of SOD1 heterodimerization by up to ΔΔG = -5.11 ± 0.36 kJ mol-1. Molecular dynamics simulations suggested that this enhanced heterodimerization, between oxidized homodimers and unoxidized homodimers, was promoted by electrostatic repulsion between the two "dueling" Cys111-SO2-/SO3-, which point toward one another in the homodimeric state. Together, these results suggest that oxidation of Cys-111 promotes subunit exchange between oxidized homodimers and unoxidized homodimers, regardless of whether they are mutant or WT dimers.

Effect of surfactant hydrophobicity on the pathway for unfolding of ubiquitin.

This paper describes the interaction between ubiquitin (UBI) and three sodium n-alkyl sulfates (SC(n)S) that have the same charge (Z = -1) but different hydrophobicity (n = 10, 12, or 14). Increasing the hydrophobicity of the n-alkyl sulfate resulted in (i) an increase in the number of distinct intermediates (that is, complexes of UBI and surfactant) that form along the pathway of unfolding, (ii) a decrease in the minimum concentrations of surfactant at which intermediates begin to form (i.e., a more negative ΔG(binding) of surfactant for UBI), and (iii) an increase in the number of surfactant molecules bound to UBI in each intermediate or complex. These results demonstrate that small changes in the hydrophobicity of a surfactant can significantly alter the binding interactions with a folded or unfolded cytosolic protein.

How do ALS-associated mutations in superoxide dismutase 1 promote aggregation of the protein?

More than 100 different mutations in the gene encoding copper-zinc superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS)--a fatal neurodegenerative disease in which aggregation of the SOD1 protein is considered to be the primary mode of pathogenesis. Recent results show that these mutations have remarkably diverse and unexpected effects on the structure, activity and native state stability of SOD1. Intriguingly, many mutations seem to have no measurable effect on the biophysical and biochemical properties of SOD1, except for decreasing the net charge of the protein. Thus, it seems likely that different ALS-associated mutations promote SOD1 aggregation by fundamentally distinct mechanisms. Understanding this complexity has implications for drug development and treatment of the disease.