Publications by authors named "Bryan F Shaw"

37 Publications

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

Protein Sci 2021 Apr 29. Epub 2021 Apr 29.

Department of Chemistry and Biochemistry, Baylor University, Waco, Texas, USA.

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  = -0.43 ± 0.01) and α-lactalbumin (Z  = -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  = -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.
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http://dx.doi.org/10.1002/pro.4092DOI Listing
April 2021

Supercharging prions via amyloid-selective lysine acetylation.

Angew Chem Int Ed Engl 2021 Apr 19. Epub 2021 Apr 19.

Baylor University, Department of Chemistry and Biochemistry, 1301 S University Parks Dr, 76706, Waco, UNITED STATES.

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  = -8,100 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).
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http://dx.doi.org/10.1002/anie.202103548DOI Listing
April 2021

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

Angew Chem Int Ed Engl 2020 06 28;59(27):10989-10995. Epub 2020 Apr 28.

Department of Chemistry and Biochemistry, Baylor University, Waco, TX, USA.

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., ΔZ =0.05±0.08 per electron, compared to ΔZ =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 pK '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.
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http://dx.doi.org/10.1002/anie.202001452DOI Listing
June 2020

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

ACS Chem Neurosci 2020 02 15;11(3):304-313. Epub 2020 Jan 15.

Department of Chemistry and Biochemistry , Baylor University , Waco , Texas 76706 , United States.

The unseeded aggregation of superoxide dismutase-1 (SOD1) into amyloid-like fibrils occurs stochastically and , 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 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.
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http://dx.doi.org/10.1021/acschemneuro.9b00464DOI Listing
February 2020

Autonomous early detection of eye disease in childhood photographs.

Sci Adv 2019 10 2;5(10):eaax6363. Epub 2019 Oct 2.

Department of Chemistry and Biochemistry, Baylor University, Waco, TX, USA.

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.
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http://dx.doi.org/10.1126/sciadv.aax6363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6774731PMC
October 2019

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

Chemistry 2019 Jun 10;25(32):7581-7590. Epub 2019 Apr 10.

Department of Chemistry and Biochemistry, Baylor University, Waco, TX, 76706, USA.

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 pK . 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 pK 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 (ΔΔG ) account for most of the outer sphere reorganization energy associated with electron transfer? Or would ΔΔG 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.
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http://dx.doi.org/10.1002/chem.201900178DOI Listing
June 2019

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

ACS Chem Neurosci 2018 07 20;9(7):1743-1756. Epub 2018 Apr 20.

Department of Chemistry and Biochemistry , Baylor University , Waco , Texas 76706 , United States.

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 (RCH(OCH)), but headgroups of different net charge and hydrophobicity (i.e., R(CH)N(CH), RPO(CH)N(CH), and RPO). 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.
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http://dx.doi.org/10.1021/acschemneuro.8b00086DOI Listing
July 2018

Direct Measurement of Charge Regulation in Metalloprotein Electron Transfer.

Angew Chem Int Ed Engl 2018 05 25;57(19):5364-5368. Epub 2018 Mar 25.

Department of Chemistry and Biochemistry, Baylor University, 1301 S University Parks Dr., Waco, TX, 76706, USA.

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  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.
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http://dx.doi.org/10.1002/anie.201712306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6033162PMC
May 2018

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

J Biol Chem 2017 11 3;292(47):19366-19380. Epub 2017 Oct 3.

From the Department of Chemistry and Biochemistry and

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, hydrophobic/steric effects electrostatic effects. Moreover, the degree to which the acylation might alter the prion-like seeding of SOD1 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 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.
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http://dx.doi.org/10.1074/jbc.M117.805283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5702675PMC
November 2017

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

ACS Chem Neurosci 2017 06 23;8(6):1378-1389. Epub 2017 Mar 23.

Department of Chemistry and Biochemistry, Baylor University , Waco, Texas 76706, United States.

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 ∼10 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 (R = 0.67; Pearson's r = 0.82). No correlation exists between Hazard ratios of fibrillization and age of initial onset of ALS (R = 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 (R = 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).
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http://dx.doi.org/10.1021/acschemneuro.7b00029DOI Listing
June 2017

How Do Gyrating Beads Accelerate Amyloid Fibrillization?

Biophys J 2017 Jan;112(2):250-264

Department of Chemistry and Biochemistry, Baylor University, Waco, Texas. Electronic address:

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 = 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 = 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.
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http://dx.doi.org/10.1016/j.bpj.2016.12.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5266089PMC
January 2017

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

J Am Chem Soc 2016 04 18;138(16):5351-62. Epub 2016 Apr 18.

Department of Chemistry and Biochemistry, Baylor University , Waco, Texas 76798-7348, United States.

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.
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http://dx.doi.org/10.1021/jacs.6b01742DOI Listing
April 2016

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

ACS Chem Neurosci 2016 06 31;7(6):799-810. Epub 2016 Mar 31.

Department of Chemistry and Biochemistry, and ‡Institute of Biomedical Studies, Baylor University , Waco, Texas 76798-7348, United States.

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.
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http://dx.doi.org/10.1021/acschemneuro.6b00048DOI Listing
June 2016

Voltage-Induced Misfolding of Zinc-Replete ALS Mutant Superoxide Dismutase-1.

ACS Chem Neurosci 2015 Oct 5;6(10):1696-707. Epub 2015 Aug 5.

Department of Chemistry and Biochemistry, Baylor University , Waco, Texas 76706, United States.

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.
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http://dx.doi.org/10.1021/acschemneuro.5b00146DOI Listing
October 2015

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

Biophys J 2015 Mar;108(5):1199-212

Department of Chemistry and Biochemistry, Baylor University, Waco, Texas. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bpj.2015.01.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4375441PMC
March 2015

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

J Biol Chem 2015 Jan 28;290(4):2405-18. Epub 2014 Nov 28.

From the Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, the Department of Bioinspired Science, Ewha Womans University, Seoul 120-750, Korea

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.
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http://dx.doi.org/10.1074/jbc.M114.588798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4303690PMC
January 2015

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

Anal Chem 2014 Oct 29;86(20):10303-10. Epub 2014 Sep 29.

Department of Chemistry and Biochemistry, Baylor University , Waco, Texas 76706, United States.

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.
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http://dx.doi.org/10.1021/ac502714vDOI Listing
October 2014

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

BMC Ophthalmol 2014 Sep 9;14:110. Epub 2014 Sep 9.

Department of Computer Science, Baylor University, One Bear Place #97356, Waco, TX 76798-7356, USA.

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.
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http://dx.doi.org/10.1186/1471-2415-14-110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4167153PMC
September 2014

Protein charge ladders reveal that the net charge of ALS-linked superoxide dismutase can be different in sign and magnitude from predicted values.

Protein Sci 2014 Oct 7;23(10):1417-33. Epub 2014 Aug 7.

Department of Chemistry and Biochemistry, Baylor University, Waco, Texas, 76798-7348.

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.
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http://dx.doi.org/10.1002/pro.2526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4287002PMC
October 2014

Colorimetric and longitudinal analysis of leukocoria in recreational photographs of children with retinoblastoma.

PLoS One 2013 30;8(10):e76677. Epub 2013 Oct 30.

Department of Chemistry and Biochemistry, Baylor University, Waco, Texas, United States of America.

Retinoblastoma is the most common primary intraocular tumor in children. The first sign that is often reported by parents is the appearance of recurrent leukocoria (i.e., "white eye") in recreational photographs. A quantitative definition or scale of leukocoria--as it appears during recreational photography--has not been established, and the amount of clinical information contained in a leukocoric image (collected by a parent) remains unknown. Moreover, the hypothesis that photographic leukocoria can be a sign of early stage retinoblastoma has not been tested for even a single patient. This study used commercially available software (Adobe Photoshop®) and standard color space conversion algorithms (operable in Microsoft Excel®) to quantify leukocoria in actual "baby pictures" of 9 children with retinoblastoma (that were collected by parents during recreational activities i.e., in nonclinical settings). One particular patient with bilateral retinoblastoma ("Patient Zero") was photographed >7, 000 times by his parents (who are authors of this study) over three years: from birth, through diagnosis, treatment, and remission. This large set of photographs allowed us to determine the longitudinal and lateral frequency of leukocoria throughout the patient's life. This study establishes: (i) that leukocoria can emerge at a low frequency in early-stage retinoblastoma and increase in frequency during disease progression, but decrease upon disease regression, (ii) that Hue, Saturation and Value (i.e., HSV color space) are suitable metrics for quantifying the intensity of retinoblastoma-linked leukocoria; (iii) that different sets of intraocular retinoblastoma tumors can produce distinct leukocoric reflections; and (iv) the Saturation-Value plane of HSV color space represents a convenient scale for quantifying and classifying pupillary reflections as they appear during recreational photography.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076677PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3813630PMC
August 2014

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

J Am Chem Soc 2013 Oct 10;135(42):15897-908. Epub 2013 Oct 10.

Department of Chemistry and Biochemistry, Baylor University , Waco, Texas 76706, United States.

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.
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http://dx.doi.org/10.1021/ja407801xDOI Listing
October 2013

Effect of metal loading and subcellular pH on net charge of superoxide dismutase-1.

J Mol Biol 2013 Nov 17;425(22):4388-404. Epub 2013 Jul 17.

Department of Chemistry and Biochemistry, Baylor University, Waco, TX 76798-7348, USA.

The net charge of a folded protein is hypothesized to influence myriad biochemical processes (e.g., protein misfolding, electron transfer, molecular recognition); however, few tools exist for measuring net charge and this elusive property remains undetermined--at any pH--for nearly all proteins. This study used lysine-acetyl "protein charge ladders" and capillary electrophoresis to measure the net charge of superoxide dismutase-1 (SOD1)--whose aggregation causes amyotrophic lateral sclerosis (ALS)--as a function of coordinated metal ions and pH. The net negative charge of apo-SOD1 was similar to predicted values; however, the binding of a single Zn(2+) or Cu(2+) ion reduced the net negative charge by a greater magnitude than predicted (i.e., ~4 units, instead of 2), whereas the SOD1 protein underwent charge regulation upon binding 2-4 metal ions. From pH5 to pH8 (i.e., a range consistent with the multiple subcellular loci of SOD1), the holo-SOD1 protein underwent smaller fluctuations in net negative charge than predicted (i.e., ~3 units, instead of ~14) and did not undergo charge inversion at its isoelectric point (pI=5.3) but remained anionic. The regulation of SOD1 net charge along its pathways of metal binding, and across solvent pH, provides insight into its metal-induced maturation and enzymatic activity (which remains diffusion-limited across pH5-8). The anionic nature of holo-SOD1 across subcellular pH suggests that ~45 different ALS-linked mutations to SOD1 will reduce its net negative charge regardless of subcellular localization.
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http://dx.doi.org/10.1016/j.jmb.2013.07.018DOI Listing
November 2013

Selective photocrosslinking of functional ligands to antibodies via the conserved nucleotide binding site.

Biomaterials 2013 Jul 16;34(22):5700-10. Epub 2013 Apr 16.

Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, IN 46556, USA.

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.
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http://dx.doi.org/10.1016/j.biomaterials.2013.03.082DOI Listing
July 2013

Ligand-induced protein mobility in complexes of carbonic anhydrase II and benzenesulfonamides with oligoglycine chains.

PLoS One 2013 5;8(3):e57629. Epub 2013 Mar 5.

Department of Chemistry, Tufts University, Medford, Massachusetts, USA.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0057629PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3589393PMC
December 2013

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

J Am Chem Soc 2012 Nov 31;134(45):18739-45. Epub 2012 Oct 31.

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States.

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.
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http://dx.doi.org/10.1021/ja3079863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3518387PMC
November 2012

Abnormal SDS-PAGE migration of cytosolic proteins can identify domains and mechanisms that control surfactant binding.

Protein Sci 2012 Aug;21(8):1197-209

Department of Chemistry and Biochemistry, Baylor University, Waco, Texas 76706, USA.

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).
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http://dx.doi.org/10.1002/pro.2107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3537240PMC
August 2012

Complexes of native ubiquitin and dodecyl sulfate illustrate the nature of hydrophobic and electrostatic interactions in the binding of proteins and surfactants.

J Am Chem Soc 2011 Nov 13;133(44):17681-95. Epub 2011 Oct 13.

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States.

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.
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http://dx.doi.org/10.1021/ja205735qDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3209763PMC
November 2011

Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.

J Am Chem Soc 2010 Dec 19;132(49):17411-25. Epub 2010 Nov 19.

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.

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).
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http://dx.doi.org/10.1021/ja9067035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3135700PMC
December 2010

Taking charge of proteins from neurodegeneration to industrial biotechnology.

Adv Protein Chem Struct Biol 2010 ;79:127-64

Department of Chemistry and Biochemistry, Baylor University, Waco, TX, USA.

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.
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http://dx.doi.org/10.1016/S1876-1623(10)79004-0DOI Listing
May 2011

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

J Biol Chem 2009 Dec 5;284(49):34382-9. Epub 2009 Oct 5.

Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095, USA.

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.
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http://dx.doi.org/10.1074/jbc.M109.052076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2797206PMC
December 2009