Richard A. Mowery

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

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).

Compositional Analysis of Water-Soluble Materials in Switchgrass

Any valuation of a potential feedstock for bioprocessing is inherently dependent upon detailed knowledge of its chemical composition. Accepted analytical procedures for compositional analysis of biomass water-soluble extracts currently enable near-quantitative mass closure on a dry weight basis. Techniques developed in conjunction with a previous analytical assessment of corn stover have been applied to assess the composition of water-soluble materials in four representative switchgrass samples. To date, analytical characterization of water-soluble material in switchgrass has resulted in >78% mass closures for all four switchgrass samples, three of which have a mass closure of >85%. Over 30 previously unknown constituents in aqueous extracts of switchgrass were identified and quantified using a variety of chromatographic techniques. Carbohydrates (primarily sucrose, glucose, and fructose) were found to be the predominant water-soluble components of switchgrass, accounting for 18-27% of the dry weight of extractives. Total glycans (monomeric and oligomeric sugars) contributed 25-32% to the dry weight of extractives. Additional constituents contributing to the mass balance for extractives included various alditols (2-3%), organic acids (10-13%), inorganic ions (11-13%), and a distribution of oligomers presumed to represent a diverse mixture of lignin-carbohydrate complexes (30-35%). Switchgrass results are compared with previous analyses of corn stover extracts and presented in the context of their potential impact on biomass processing, feedstock storage, and future analyses of feedstock composition.

Rapid analysis of carbohydrates in aqueous extracts and hydrolysates of biomass using a carbonate-modified anion-exchange column.

Quantitative liquid-chromatography techniques used to characterize carbohydrates present in biomass samples can suffer from long analysis times, limited analyte resolution, poor stability, or a combination of these factors. The current manuscript details a novel procedure enabling resolution of glucose, xylose, arabinose, galactose, mannose, fructose, and sucrose via isocratic elution in less than 5 min. Equivalent conditions also enable analysis of cellobiose and maltose with a minimal increase in chromatographic run time (ca. 3 and 6 min, respectively). Noted chromatographic performance requires that a commercially available anion-exchange column be modified with carbonate prior to analysis. Analytical performance of a modified column was assessed over a 5-day period via repeated analyses of 4 samples, resulting from aqueous extraction or quantitative saccharification of a potential biofuel feedstock (i.e., corn stover or switchgrass). A simple solid phase extraction procedure was utilized to clean up each sample prior to analysis. Analytical accuracy of the extraction protocol was assessed by evaluation of matrix spike recoveries which typically ranged from 84% to 98%. The instrumental variability of measured concentrations in real samples over the 5-day period was generally less than 5% RSD for all detected analytes, independent of sample type. Finally, it is important to note that the modified column exhibited exceptional stability over approximately 800 injections of biofeedstock-based samples. These data demonstrate that a carbonate-modified anion-exchange column can be employed for rapid determination of carbohydrates in biomass samples of lignocellulosic origin.

Photochemical tissue bonding using monomeric 4-amino-1,8-naphthalimides.

Certain substituted naphthalimides have been shown to produce, on photochemical activation, mechanically viable bonds between a variety of tissue surfaces. It is believed that these compounds act as photochemically activated oxidants, catalyzing the formation of reactive intermediates in the extracellular matrices of approximated tissue surfaces. The condensation of these intermediates results in the formation of crosslinks between the intimate surfaces. Of particular interest is the application of this technique to the repair of tears in the typically unrepairable avascular zone of menisci. The menisci are collagen-rich fibrocartilaginous tissues that support up to 90% of the load across the knee joint and participate in important functions including shock absorption, joint stabilization, hyperextension prevention, and lubrication of the knee. Preliminary ex vivo and in vivo work in our laboratories has demonstrated that photochemically activated naphthalimides have significant potential for the repair of meniscal lesions. We describe preliminary ex vivo studies assessing the relative abilities of a variety of water-soluble monomeric 4-amino-1,8-naphthalimides to bond bovine knee meniscal tissue on visible light irradiation.

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

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 15N-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.

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

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