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.

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.

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.

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+.

Data for all: Tactile graphics that light up with picture-perfect resolution.

People who are blind do not have access to graphical data and imagery produced by science. This exclusion complicates learning and data sharing between sighted and blind persons. Because blind people use tactile senses to visualize data (and sighted people use eyesight), a single data format that can be easily visualized by both is needed. Here, we report that graphical data can be three-dimensionally printed into tactile graphics that glow with video-like resolution via the lithophane effect. Lithophane forms of gel electropherograms, micrographs, electronic and mass spectra, and textbook illustrations could be interpreted by touch or eyesight at ≥79% accuracy (n = 360). The lithophane data format enables universal visualization of data by people regardless of their level of eyesight.

Visualizing 3D imagery by mouth using candy-like models.

Handheld models help students visualize three-dimensional (3D) objects, especially students with blindness who use large 3D models to visualize imagery by hand. The mouth has finer tactile sensors than hand, which could improve visualization using microscopic models that are portable, inexpensive, and disposable. The mouth remains unused in tactile learning. Here, we created bite-size 3D models of protein molecules from "gummy bear" gelatin or nontoxic resin. Models were made as small as rice grain and could be coded with flavor and packaged like candy. Mouth, hands, and eyesight were tested at identifying specific structures. Students recognized structures by mouth at 85.59% accuracy, similar to recognition by eyesight using computer animation. Recall accuracy of structures was higher by mouth than hand for 40.91% of students, equal for 31.82%, and lower for 27.27%. The convenient use of entire packs of tiny, cheap, portable models can make 3D imagery more accessible to students.

Measuring how two proteins affect each other's net charge in a crowded environment.

Theory predicts that the net charge (Z) of a protein can be altered by the net charge of a neighboring protein as the two approach one another below the Debye length. This type of charge regulation suggests that a protein's charge and perhaps function might be affected by neighboring proteins without direct binding. Charge regulation during protein crowding has never been directly measured due to analytical challenges. Here, we show that lysine specific protein crosslinkers (NHS ester-Staudinger pairs) can be used to mimic crowding by linking two non-interacting proteins at a maximal distance of ~7.9 Å. The net charge of the regioisomeric dimers and preceding monomers can then be determined with lysine-acyl "protein charge ladders" and capillary electrophoresis. As a proof of concept, we covalently linked myoglobin (Zmonomer  = -0.43 ± 0.01) and α-lactalbumin (Zmonomer  = -4.63 ± 0.05). Amide hydrogen/deuterium exchange and circular dichroism spectroscopy demonstrated that crosslinking did not significantly alter the structure of either protein or result in direct binding (thus mimicking crowding). Ultimately, capillary electrophoretic analysis of the dimeric charge ladder detected a change in charge of ΔZ = -0.04 ± 0.09 upon crowding by this pair (Zdimer  = -5.10 ± 0.07). These small values of ΔZ are not necessarily general to protein crowding (qualitatively or quantitatively) but will vary per protein size, charge, and solvent conditions.

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.

Kinetic Variability in Seeded Formation of ALS-Linked SOD1 Fibrils Across Multiple Generations.

The unseeded aggregation of superoxide dismutase-1 (SOD1) into amyloid-like fibrils occurs stochastically in vitro and in vivo, that is, isolated populations of SOD1 proteins (within microplate wells or living cells) self-assemble into amyloid at rates that span a probability distribution. This stochasticity has been attributed to variable degrees of monomer depletion by competing pathways of amorphous and fibrillar aggregation (inter alia). Here, microplate-based thioflavin-T (ThT) fluorescence assays were performed at high iteration (∼300) to establish whether this observed stochasticity persists when progenitor ("parent") SOD1 fibrils are used to seed the formation of multiple generations of progeny fibrils (daughter, granddaughter, and great-granddaughter fibrils). Populations of progenitor fibrils formed stochastically at different rates and fluorescence intensity, however, progeny fibrils formed at more similar rates regardless of the formation rate of the progenitor fibril. For example, populations of progenitor fibrils that formed with a lag time of ∼30 h or ∼15 h both produced progeny fibrils with lag times of ∼8 h. Likewise, populations of progenitor fibrils with high or low maximum fluorescence (e.g., ∼450 or ∼75 A.U.) both produced progeny fibrils with more similar maximum fluorescence (∼125 A.U.). The rate of propagation was found to be more dependent on monomer concentration than seed concentration. These results can be rationalized by classical rate laws for primary nucleation and monomer-dependent secondary nucleation. We also find that the seeding propensity of some "families" of in vitro grown fibrils exhibit a finite lifetime (similar to that observed in the seeding of small molecule crystals and colloids). The single biological takeaway of this study is that the concentration of native SOD1 in a cell can have a stronger effect on rates of seeded aggregation than the concentration of prion-like seed that infected the cell.

Autonomous early detection of eye disease in childhood photographs.

The "red reflex test" is used to screen children for leukocoria ("white eye") in a standard pediatric examination, but is ineffective at detecting many eye disorders. Leukocoria also presents in casual photographs. The clinical utility of screening photographs for leukocoria is unreported. Here, a free smartphone application (CRADLE: ComputeR-Assisted Detector of LEukocoria) was engineered to detect photographic leukocoria and is available for download under the name "White Eye Detector." This study determined the sensitivity, specificity, and accuracy of CRADLE by retrospectively analyzing 52,982 longitudinal photographs of children, collected by parents before enrollment in this study. The cohort included 20 children with retinoblastoma, Coats' disease, cataract, amblyopia, or hyperopia and 20 control children. For 80% of children with eye disorders, the application detected leukocoria in photographs taken before diagnosis by 1.3 years (95% confidence interval, 0.4 to 2.3 years). The CRADLE application allows parents to augment clinical leukocoria screening with photography.

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.

Glycerolipid Headgroups Control Rate and Mechanism of Superoxide Dismutase-1 Aggregation and Accelerate Fibrillization of Slowly Aggregating Amyotrophic Lateral Sclerosis Mutants.

Interactions between superoxide dismutase-1 (SOD1) and lipid membranes might be directly involved in the toxicity and intercellular propagation of aggregated SOD1 in amyotrophic lateral sclerosis (ALS), but the chemical details of lipid-SOD1 interactions and their effects on SOD1 aggregation remain unclear. This paper determined the rate and mechanism of nucleation of fibrillar apo-SOD1 catalyzed by liposomal surfaces with identical hydrophobic chains (RCH2(O2C18H33)2), but headgroups of different net charge and hydrophobicity (i.e., R(CH2)N+(CH3)3, RPO4-(CH2)2N+(CH3)3, and RPO4-). Under semiquiescent conditions (within a 96 well microplate, without a gyrating bead), the aggregation of apo-SOD1 into thioflavin-T-positive (ThT(+)) amyloid fibrils did not occur over 120 h in the absence of liposomal surfaces. Anionic liposomes triggered aggregation of apo-SOD1 into ThT(+) amyloid fibrils; cationic liposomes catalyzed fibrillization but at slower rates and across a narrower lipid concentration; zwitterionic liposomes produced nonfibrillar (amorphous) aggregates. The inability of zwitterionic liposomes to catalyze fibrillization and the dependence of fibrillization rate on anionic lipid concentration suggests that membranes catalyze SOD1 fibrillization by a primary nucleation mechanism. Membrane-catalyzed fibrillization was also examined for eight ALS variants of apo-SOD1, including G37R, G93R, D90A, and E100G apo-SOD1 that nucleate slower than or equal to WT SOD1 in lipid-free, nonquiescent amyloid assays. All ALS variants (with one exception) nucleated faster than WT SOD1 in the presence of anionic liposomes, wherein the greatest acceleratory effects were observed among variants with lower net negative surface charge (G37R, G93R, D90A, E100G). The exception was H46R apo-SOD1, which did not form ThT(+) species.

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.

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.

Kaplan-Meier Meets Chemical Kinetics: Intrinsic Rate of SOD1 Amyloidogenesis Decreased by Subset of ALS Mutations and Cannot Fully Explain Age of Disease Onset.

Over 150 mutations in SOD1 (superoxide dismutase-1) cause amyotrophic lateral sclerosis (ALS), presumably by accelerating SOD1 amyloidogenesis. Like many nucleation processes, SOD1 fibrillization is stochastic (in vitro), which inhibits the determination of aggregation rates (and obscures whether rates correlate with patient phenotypes). Here, we diverged from classical chemical kinetics and used Kaplan-Meier estimators to quantify the probability of apo-SOD1 fibrillization (in vitro) from ∼103 replicate amyloid assays of wild-type (WT) SOD1 and nine ALS variants. The probability of apo-SOD1 fibrillization (expressed as a Hazard ratio) is increased by certain ALS-linked SOD1 mutations but is decreased or remains unchanged by other mutations. Despite this diversity, Hazard ratios of fibrillization correlated linearly with (and for three mutants, approximately equaled) Hazard ratios of patient survival (R2 = 0.67; Pearson's r = 0.82). No correlation exists between Hazard ratios of fibrillization and age of initial onset of ALS (R2 = 0.09). Thus, Hazard ratios of fibrillization might explain rates of disease progression but not onset. Classical kinetic metrics of fibrillization, i.e., mean lag time and propagation rate, did not correlate as strongly with phenotype (and ALS mutations did not uniformly accelerate mean rate of nucleation or propagation). A strong correlation was found, however, between mean ThT fluorescence at lag time and patient survival (R2 = 0.93); oligomers of SOD1 with weaker fluorescence correlated with shorter survival. This study suggests that SOD1 mutations trigger ALS by altering a property of SOD1 or its oligomers other than the intrinsic rate of amyloid nucleation (e.g., oligomer stability; rates of intercellular propagation; affinity for membrane surfaces; and maturation rate).

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.

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.

Stochastic Formation of Fibrillar and Amorphous Superoxide Dismutase Oligomers Linked to Amyotrophic Lateral Sclerosis.

Recent reports suggest that the nucleation and propagation of oligomeric superoxide dismutase-1 (SOD1) is effectively stochastic in vivo and in vitro. This perplexing kinetic variability-observed for other proteins and frequently attributed to experimental error-plagues attempts to discern how SOD1 mutations and post-translational modifications linked to amyotrophic lateral sclerosis (ALS) affect SOD1 aggregation. This study used microplate fluorescence spectroscopy and dynamic light scattering to measure rates of fibrillar and amorphous SOD1 aggregation at high iteration (ntotal = 1.2 × 10(3)). Rates of oligomerization were intrinsically irreproducible and populated continuous probability distributions. Modifying reaction conditions to mimic random and systematic experimental error could not account for kinetic outliers in standard assays, suggesting that stochasticity is not an experimental artifact, rather an intrinsic property of SOD1 oligomerization (presumably caused by competing pathways of oligomerization). Moreover, mean rates of fibrillar and amorphous nucleation were not uniformly increased by mutations that cause ALS; however, mutations did increase kinetic noise (variation) associated with nucleation and propagation. The stochastic aggregation of SOD1 provides a plausible statistical framework to rationalize how a pathogenic mutation can increase the probability of oligomer nucleation within a single cell, without increasing the mean rate of nucleation across an entire population of cells.