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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).

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 (Z (monomer) = −0.43 ± 0.01) and α‐lactalbumin (Z (monomer) = −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 (Z (dimer) = −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.

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.

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 (ΔG

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.

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.

Edible and nonedible models of complex 3D imagery can be identified by mouth at accuracies equivalent to eyesight., 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.

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 ...

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 pK a 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.

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.

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 ...

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.

A smartphone app searches baby pictures for common and rare eye disorders., 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.

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 ( R 2 = 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 ( R 2 = 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.

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.

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.

This article utilized “protein charge ladders”—chemical derivatives of proteins with similar structure, but systematically altered net charge—to quantify how missense mutations that cause amyotrophic lateral sclerosis (ALS) affect the net negative charge (Z) of superoxide dismutase-1 (SOD1) as a function of subcellular pH and Zn2+ stoichiometry. Capillary electrophoresis revealed that the net charge of ALS-variant SOD1 can be different in sign and in magnitude—by up to 7.4 units per dimer at lysosomal pH—than values predicted from standard pKa values of amino acids and formal oxidation states of metal ions. At pH 7.4, the G85R, D90A, and G93R substitutions diminished the net negative charge of dimeric SOD1 by up to +2.29 units more than predicted; E100K lowered net charge by less than predicted. The binding of a single Zn2+ to mutant SOD1 lowered its net charge by an additional +2.33 ± 0.01 to +3.18 ± 0.02 units, however, each protein regulated net charge when binding a second, third, or fourth Zn2+ (ΔZ < 0.44 ± 0.07 per additional Zn2+). Both metalated and apo-SOD1 regulated net charge across subcellular pH, without inverting from negative to positive at the theoretical pI. Differential scanning calorimetry, hydrogen-deuterium exchange, and inductively coupled plasma mass spectrometry confirmed that the structure, stability, and metal content of mutant proteins were not significantly affected by lysine acetylation. Measured values of net charge should be used when correlating the biophysical properties of a specific ALS-variant SOD1 protein with its observed aggregation propensity or clinical phenotype.

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 by1 kJ mol(-1). Medicinal targeting of heterodimer thermodynamics represents a plausible strategy for prolonging life in SOD1-linked ALS.

This paper describes the interaction between ubiquitin (UBI) and three sodium n-alkyl sulfates (SCnS) that have the same charge (Z = −1), but different hydrophobicity (n = 10, 12 and 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 ΔGbinding of surfactant for UBI), and (iii) an increase in the number of surfactant molecules (p) 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.

The amino acid substitution or post-translational modification of a cytosolic protein can cause unpredictable changes to its electrophoretic mobility during SDS-PAGE. This type of “gel shifting” has perplexed biochemists and biologists for decades. We identify a mechanism for “gel shifting” that predominates among a set of ALS (amyotrophic lateral sclerosis) mutant hSOD1 (superoxide dismutase) proteins, post-translationally modified hSOD1 proteins, and homologous SOD1 proteins from different organisms. By first comparing how 39 amino acid substitutions throughout hSOD1 affected SDS-PAGE migration, we found that substitutions that caused gel shifting occurred within a single polyacidic domain (residues ∼80–101), and were nonisoelectric. Substitutions that decreased the net negative charge of domain 80–101 increased migration; only one substitution increased net negative charge and slowed migration. Capillary electrophoresis, circular dichroism, and size exclusion chromatography demonstrated that amino acid substitutions increase migration during SDS-PAGE by promoting the binding of three to four additional SDS molecules, without significantly altering the secondary structure or Stokes radius of hSOD1-SDS complexes. The high negative charge of domain 80–101 is required for SOD1 gel shifting: neutralizing the polyacidic domain (via chimeric mouse-human SOD1 fusion proteins) inhibited amino acid substitutions from causing gel shifting. These results demonstrate that the pattern of gel shifting for mutant cytosolic proteins can be used to: (i) identify domains in the primary structure that control interactions between denatured cytosolic proteins and SDS and (ii) identify a predominant chemical mechanism for the interaction (e.g., hydrophobic vs. electrostatic).

A previous study, using capillary electrophoresis (CE) [J. Am. Chem. Soc. 2008, 130, 17384-17393], reported that six discrete complexes of ubiquitin (UBI) and sodium dodecyl sulfate (SDS) form at different concentrations of SDS along the pathway to unfolding of UBI in solutions of SDS. One complex (which formed between 0.8 and 1.8 mM SDS) consisted of native UBI associated with approximately 11 molecules of SDS. The current study used CE and (15)N/(13)C-(1)H heteronuclear single quantum coherence (HSQC) NMR spectroscopy to identify residues in folded UBI that associate specifically with SDS at 0.8-1.8 mM SDS, and to correlate these associations with established biophysical and structural properties of this well-characterized protein. The ability of the surface charge and hydrophobicity of folded UBI to affect the association with SDS (at concentrations below the CMC) was studied, using CE, by converting lys-ε-NH(3)(+) to lys-ε-NHCOCH(3) groups. According to CE, the acetylation of lysine residues inhibited the binding of 11 SDS ([SDS]2 mM) and decreased the number of complexes of composition UBI-(NHAc)(8)·SDS(n) that formed on the pathway of unfolding of UBI-(NHAc)(8) in SDS. A comparison of (15)N-(1)H HSQC spectra at 0 mM and 1 mM SDS with calculated electrostatic surface potentials of folded UBI (e.g., solutions to the nonlinear Poisson-Boltzmann (PB) equation) suggested, however, that SDS binds preferentially to native UBI at hydrophobic residues that are formally neutral (i.e., Leu and Ile), but that have positive electrostatic surface potential (as predicted from solutions to nonlinear PB equations); SDS did not uniformly interact with residues that have formal positive charge (e.g., Lys or Arg). Cationic functional groups, therefore, promote the binding of SDS to folded UBI because these groups exert long-range effects on the positive electrostatic surface potential (which extend beyond their own van der Waals radii, as predicted from PB theory), and not because cationic groups are necessarily the site of ionic interactions with sulfate groups. Moreover, SDS associated with residues in native UBI without regard to their location in α-helix or β-sheet structure (although residues in hydrogen-bonded loops did not bind SDS). No correlation was observed between the association of an amino acid with SDS and the solvent accessibility of the residue or its rate of amide H/D exchange. This study establishes a few (of perhaps several) factors that control the simultaneous molecular recognition of multiple anionic amphiphiles by a folded cytosolic protein.

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.

This paper describes a method for the purification of monoclonal antibodies (rat anti-2,4-dinitrophenyl IgG: IgG(DNP); and mouse antidigoxin IgG: IgG(Dgn)) from ascites fluid. This procedure (for IgG(DNP)) has three steps: (i) precipitation of proteins heavier than immunoglobulins with ammonium sulfate; (ii) formation of cyclic complexes of IgG(DNP) by causing it to bind to synthetic multivalent haptens containing multiple DNP groups; (iii) selective precipitation of these dimers, trimers, and higher oligomers of the target antibody, followed by regeneration of the free antibody. This procedure separates the targeted antibody from a mixture of antibodies, as well as from other proteins and globulins in a biological fluid. This method is applicable to antibodies with a wide range of monovalent binding constants (0.1 microM to 0.1 nM). The multivalent ligands we used (derivatives of DNP and digoxin) isolated IgG(DNP) and IgG(Dgn) from ascites fluid in yields of >80% and with >95% purity. This technique has two advantages over conventional chromatographic methods for purifying antibodies: (i) it is selective for antibodies with two active Fab binding sites (both sites are required to form the cyclic complexes) over antibodies with one or zero active Fab binding sites; (ii) it does not require chromatographic separation. It has the disadvantage that the structure of the hapten must be compatible with the synthesis of bi- and/or trivalent analogues.

This paper reports that the acetylation of lysine epsilon-NH3(+) groups of alpha-amylase--one of the most important hydrolytic enzymes used in industry--produces highly negatively charged variants that are enzymatically active, thermostable, and more resistant than the wild-type enzyme to irreversible inactivation on exposure to denaturing conditions (e.g., 1 h at 90 degrees C in solutions containing 100-mM sodium dodecyl sulfate). Acetylation also protected the enzyme against irreversible inactivation by the neutral surfactant TRITON X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)phenyl ether), but not by the cationic surfactant, dodecyltrimethylammonium bromide (DTAB). The increased resistance of acetylated alpha-amylase toward inactivation is attributed to the increased net negative charge of alpha-amylase that resulted from the acetylation of lysine ammonium groups (lysine epsilon-NH3(+) --epsilon-NHCOCH3). Increases in the net negative charge of proteins can decrease the rate of unfolding by anionic surfactants, and can also decrease the rate of protein aggregation. The acetylation of lysine represents a simple, inexpensive method for stabilizing bacterial alpha-amylase against irreversible inactivation in the presence of the anionic and neutral surfactants that are commonly used in industrial applications.

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.

The monomerization of Cu, Zn superoxide dismutase (SOD1) is an early step along pathways of misfolding linked to amyotrophic lateral sclerosis (ALS). Monomerization requires the reversal of two post-translational modifications that are thermodynamically favorable: (i) dissociation of active-site metal ions and (ii) reduction of intramolecular disulfide bonds. This study found, using amide hydrogen/deuterium (H/D) exchange, capillary electrophoresis, and lysine-acetyl protein charge ladders, that ALS-linked A4V SOD1 rapidly monomerizes and partially unfolds in an external electric field (of physiological strength), without loss of metal ions, exposure to disulfide-reducing agents, or Joule heating. Voltage-induced monomerization was not observed for metal-free A4V SOD1, metal-free WT SOD1, or metal-loaded WT SOD1. Computational modeling suggested a mechanism for this counterintuitive effect: subunit macrodipoles of dimeric SOD1 are antiparallel and amplified 2-fold by metal coordination, which increases torque at the dimer interface as subunits rotate to align with the electric field.

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.

Dysregulation of matrix metalloproteinases (MMPs) and ineffective fibrinolysis are associated with the deposition of extracellular matrix (ECM). We hypothesized that elevated plasminogen activator inhibitor (PAI)-1 promotes ECM deposition in the asthmatic airway by inhibiting MMP-9 activity and fibrinolysis. Degree of airway inflammation was similar in PAI-1(-/-) and wild type (WT) mice after ovalbumin (OVA) challenge. PAI-1 production, deposition of collagen and fibrin, and MMP-9 activity in the lung tissue or airways were greater after OVA challenge compared with saline challenge. However, in PAI-1(-/-) mice, collagen deposition was 2-fold less, fibrin deposition was 4-fold less, and MMP-9 activity was 3-fold higher. This is the first direct evidence that the plasmin system regulates ECM deposition in the airways of a murine asthma model, independently of the effect of PAI-1 on inflammatory cells. The results suggest that the PAI-1-dependent inhibition of MMP-9 activity and fibrinolysis is a major mechanism by which ECM deposition occurs.

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.

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 by10 °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.

This article utilized "protein charge ladders"-chemical derivatives of proteins with similar structure, but systematically altered net charge-to quantify how missense mutations that cause amyotrophic lateral sclerosis (ALS) affect the net negative charge (Z) of superoxide dismutase-1 (SOD1) as a function of subcellular pH and Zn(2+) stoichiometry. Capillary electrophoresis revealed that the net charge of ALS-variant SOD1 can be different in sign and in magnitude-by up to 7.4 units per dimer at lysosomal pH-than values predicted from standard pKa values of amino acids and formal oxidation states of metal ions. At pH 7.4, the G85R, D90A, and G93R substitutions diminished the net negative charge of dimeric SOD1 by up to +2.29 units more than predicted; E100K lowered net charge by less than predicted. The binding of a single Zn(2+) to mutant SOD1 lowered its net charge by an additional +2.33 ± 0.01 to +3.18 ± 0.02 units, however, each protein regulated net charge when binding a second, third, or fourth Zn(2+) (ΔZ 0.44 ± 0.07 per additional Zn(2+) ). Both metalated and apo-SOD1 regulated net charge across subcellular pH, without inverting from negative to positive at the theoretical pI. Differential scanning calorimetry, hydrogen-deuterium exchange, and inductively coupled plasma mass spectrometry confirmed that the structure, stability, and metal content of mutant proteins were not significantly affected by lysine acetylation. Measured values of net charge should be used when correlating the biophysical properties of a specific ALS-variant SOD1 protein with its observed aggregation propensity or clinical phenotype.

We use Convolutional Neural Networks to detect leukocoria, or white-eye reflections, in recreational photography. Leukocoria is the most prominent symptom of retinoblastoma, a solid-tumor cancer of the eye that occurs most often in young children. We trained several networks for the task, using training images downloaded from Flickr. We achieved low error rates (

Retinoblastoma is a pediatric ocular cancer typically indicated by leukocoria (white-eye pupillary reflex). Early detection of leukocoria can improve health outcomes when it indicates disease, and it can be easily seen in recreational photographs. As part of a system for automatic leukocoria detection, we propose an image processing algorithm for detecting the exact location and radius of the smallest circle containing the iris in an eye image. Our algorithms use both median filters and two-dimensional stationary wavelet transforms and achieve low error rates.

This paper describes a biophysical investigation of residual mobility in complexes of bovine carbonic anhydrase II (BCA) and para-substituted benzenesulfonamide ligands with chains of 1-5 glycine subunits, and explains the previously observed increase in entropy of binding with chain length. The reported results represent the first experimental demonstration that BCA is not the rigid, static globulin that has been typically assumed, but experiences structural fluctuations upon binding ligands. NMR studies with (15)N-labeled ligands demonstrated that the first glycine subunit of the chain binds without stabilization or destabilization by the more distal subunits, and suggested that the other glycine subunits of the chain behave similarly. These data suggest that a model based on ligand mobility in the complex cannot explain the thermodynamic data. Hydrogen/deuterium exchange studies provided a global estimate of protein mobility and revealed that the number of exchanged hydrogens of BCA was higher when the protein was bound to a ligand with five glycine subunits than when bound to a ligand with only one subunit, and suggested a trend of increasing number of exchanged hydrogens with increasing chain length of the BCA-bound ligand, across the series. These data support the idea that the glycine chain destabilizes the structure of BCA in a length-dependent manner, causing an increase in BCA mobility. This study highlights the need to consider ligand-induced mobility of even "static" proteins in studies of protein-ligand binding, including rational ligand design approaches.

The conserved nucleotide binding site (NBS), found in the Fab variable domain of all antibody isotypes, remains a not-so-widely known and under-utilized site. Here, we describe a UV photocrosslinking method (UV-NBS) that utilizes the NBS for site-specific covalent functionalization of antibodies, while preserving antibody activity. We identified a small molecule, indole-3-butyric acid (IBA), which has affinity for the NBS (K(d) = 1-8 μM) and can be photocrosslinked to antibodies upon UV energy exposure. By synthesizing their IBA conjugated versions, we have successfully photocrosslinked various types of functional ligands to antibodies at the NBS, including affinity tags (biotin), fluorescent molecules (FITC), peptides (iRGD), and chemotherapeutics (paclitaxel). An optimal UV exposure of 1-2 J/cm(2) yielded the most efficient photocrosslinking and resulted in 1-2 conjugations per antibody, while preserving the antigen binding activity and Fc related functions. Analysis of the photocrosslinked conjugates using western blotting, mass spectrometry, and computational docking simulations demonstrated that the photocrosslinking specifically takes place at the Y/F42 residue in framework region 2 of the antibody light chain. Taken together, the UV-NBS method provides a practical, site-specific, and chemically efficient method to functionalize antibodies with significant implications in diagnostic and therapeutic settings.

This paper combines two techniques--mass spectrometry and protein charge ladders--to examine the relationship between the surface charge and hydrophobicity of a representative globular protein (bovine carbonic anhydrase II; BCA II) and its rate of amide hydrogen-deuterium (H/D) exchange. Mass spectrometric analysis indicated that the sequential acetylation of surface lysine-ε-NH3(+) groups--a type of modification that increases the net negative charge and hydrophobicity of the surface of BCA II without affecting its secondary or tertiary structure--resulted in a linear decrease in the aggregate rate of amide H/D exchange at pD 7.4, 15 °C. According to analysis with MS, the acetylation of each additional lysine generated between 1.4 and 0.9 additional hydrogens that are protected from H/D exchange during the 2 h exchange experiment at 15 °C, pD 7.4. NMR spectroscopy demonstrated that none of the hydrogen atoms which became protected upon acetylation were located on the side chain of the acetylated lysine residues (i.e., lys-ε-NHCOCH3) but were instead located on amide NHCO moieties in the backbone. The decrease in rate of exchange associated with acetylation paralleled a decrease in thermostability: the most slowly exchanging rungs of the charge ladder were the least thermostable (as measured by differential scanning calorimetry). This observation--that faster rates of exchange are associated with slower rates of denaturation--is contrary to the usual assumptions in protein chemistry. The fact that the rates of H/D exchange were similar for perbutyrated BCA II (e.g., [lys-ε-NHCO(CH2)2CH3]18) and peracetylated BCA II (e.g., [lys-ε-NHCOCH3]18) suggests that the electrostatic charge is more important than the hydrophobicity of surface groups in determining the rate of H/D exchange. These electrostatic effects on the kinetics of H/D exchange could complicate (or aid) the interpretation of experiments in which H/D exchange methods are used to probe the structural effects of non-isoelectric perturbations to proteins (i.e., phosphorylation, acetylation, or the binding of the protein to an oligonucleotide or to another charged ligand or protein).

The aggregation and precipitation of a soluble protein-within a motor neuron, or a pharmaceutical vial, or even inside a industrial-scale hydrolysis chamber-is a problem in human health and in biotechnology. A growing body of research is suggesting that the magnitude of the net charge of a protein is a determinant of the rate at which proteins self-assemble in solution into aggregates with amorphous or fibrillar (or uncharacterized) morphologies. This chapter discusses how this apparently simple electrostatic effect might explain-in part or entirely-the pathogenicity of some mutations that cause familial protein aggregation diseases-especially the familial forms of amyotrophic lateral sclerosis that are caused by mutations in the gene encoding superoxide dismutase-1 (SOD1). In parallel, this chapter also discusses how understanding these electrostatic effects can guide the engineering of industrial enzymes (such as alpha-amylase from Bacillus licheniformis) into forms that are more resistant to aggregation and thermal precipitation than the enzymes that are currently used, for example, in the production of ethanol from starch or cellulose.

The aggregation and precipitation of a soluble protein-within a motor neuron, or a pharmaceutical vial, or even inside a industrial-scale hydrolysis chamber-is a problem in human health and in biotechnology. A growing body of research is suggesting that the magnitude of the net charge of a protein is a determinant of the rate at which proteins self-assemble in solution into aggregates with amorphous or fibrillar (or uncharacterized) morphologies. This chapter discusses how this apparently simple electrostatic effect might explain-in part or entirely-the pathogenicity of some mutations that cause familial protein aggregation diseases-especially the familial forms of amyotrophic lateral sclerosis that are caused by mutations in the gene encoding superoxide dismutase-1 (SOD1). In parallel, this chapter also discusses how understanding these electrostatic effects can guide the engineering of industrial enzymes (such as alpha-amylase from Bacillus licheniformis) into forms that are more resistant to aggregation and thermal precipitation than the enzymes that are currently used, for example, in the production of ethanol from starch or cellulose.

This paper describes the phase separation of millimetre-scale spheres based on electrostatic charge. Initially, polymeric (Teflon, T; Nylon-6,6, N) and metallic (gold-coated Nylon-6,6, Au(N)) spheres are uniformly mixed in a two-dimensional (2D) monolayer on a gold-coated plate. Oscillating the plate vertically caused the spheres to charge by contact electrification (tribocharging). Positively charged N and negatively charged T spheres attracted each other more strongly than they attracted the capacitively charged, Au(N) spheres. The T and N spheres formed 2D Coulombic crystals, and these crystals separated from the Au(N) spheres. The extent and rate of separation increased with increasing amplitude of agitation during tribocharging, and with decreasing density of spheres on the surface. At high surface density, the T and N spheres did not separate from the Au(N) spheres. This system models the 2D nucleation of an ionic crystal from a polarizable liquid.

Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1) are one of the causes of familial amyotrophic lateral sclerosis (FALS). Fibrillar inclusions containing SOD1 and SOD1 inclusions that bind the amyloid-specific dye thioflavin S have been found in neurons of transgenic mice expressing mutant SOD1. Therefore, the formation of amyloid fibrils from human SOD1 was investigated. When agitated at acidic pH in the presence of low concentrations of guanidine or acetonitrile, metalated SOD1 formed fibrillar material which bound both thioflavin T and Congo red and had circular dichroism and infrared spectra characteristic of amyloid. While metalated SOD1 did not form amyloid-like aggregates at neutral pH, either removing metals from SOD1 with its intramolecular disulfide bond intact or reducing the intramolecular disulfide bond of metalated SOD1 was sufficient to promote formation of these aggregates. SOD1 formed amyloid-like aggregates both with and without intermolecular disulfide bonds, depending on the incubation conditions, and a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1) formed amyloid-like aggregates at neutral pH under reducing conditions. ALS mutations enhanced the ability of disulfide-reduced SOD1 to form amyloid-like aggregates, and apo-AS-SOD1 formed amyloid-like aggregates at pH 7 only when an ALS mutation was also present. These results indicate that some mutations related to ALS promote formation of amyloid-like aggregates by facilitating the loss of metals and/or by making the intramolecular disulfide bond more susceptible to reduction, thus allowing the conversion of SOD1 to a form that aggregates to form resembling amyloid. Furthermore, the occurrence of amyloid-like aggregates per se does not depend on forming intermolecular disulfide bonds, and multiple forms of such aggregates can be produced from SOD1.

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.

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.

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 ( T m ) in the range 51.6°C to 56.2°C, i.e., similar to or higher than that of the WT apoprotein ( T m = 52.5°C). The apoproteins with substitutions remote from the MBR (WT-like mutants) showed a wide range of T m s, 40.0°C to 52.4°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.