Lynda M. McDowell

Enzymatic redesigning of biologically active heparan sulfate

Heparan sulfate carries a wide range of biological activities, regulating blood coagulation, cell differentiation, and inflammatory responses. The sulfation patterns of the polysaccharide are essential for the biological activities. In this study, we report an enzymatic method for the sulfation of multimilligram amounts of heparan sulfate with specific functions using immobilized sulfotransferases combined with a 3′-phosphoadenosine 5′-phosphosulfate regeneration system. By selecting appropriate enzymatic modification steps, an inactive precursor has been converted to the heparan sulfate having three distinct biological activities, associated with binding to antithrombin, fibroblast growth factor-2, and herpes simplex virus envelope glycoprotein D. Because the recombinant sulfotransferases are expressed in bacteria, and the method uses a low cost sulfo donor, it can be readily utilized to synthesize large quantities of anticoagulant heparin drug or other biologically active heparan sulfates.

High-resolution NMR of biological solids.

Solid-state NMR experiments have recently provided a number of biochemical insights: motionally averaged 2H lineshapes have shown that the motion of a backbone loop protecting a protein binding site is not ligand gated; isotropic 13C chemical shifts of freeze-quenched enzyme-ligand intermediates have revealed mechanistic details of reaction pathways; multiple heteronuclear distance determinations have characterized the binding-site geometry of a 46 kDa noncrystalline enzyme complex; and homonuclear recoupling experiments have established that insoluble amyloid fibrils form a pleated beta sheet.

INHIBITION OR ACTIVATION OF APERT SYNDROME FGFR2 (S252W) SIGNALING BY SPECIFIC GLYCOSAMINOGLYCANS

Most Apert syndrome patients harbor a single amino acid mutation (S252W) in fibroblast growth factor (FGF) receptor 2 (FGFR2), which leads to abnormal FGF/FGFR2 signaling. Here we show that specific combinations of FGFs and glycosaminoglycans activate both alternative splice forms of the mutant but not of the wild-type FGF receptors. More importantly, 2-O- and N-sulfated heparan sulfate, prepared by a combined chemical and enzymatic synthesis, antagonized the over-activated FGFR2b (S252W) to basal levels at nanomolar concentrations. These studies demonstrated that specific glycosaminoglycans could be useful in treating ligand-dependent FGFR signaling-related diseases, such as Apert syndrome and cancer.

Charge transfer and charge resonance states of the primary electron donor in wild-type and mutant bacterial reaction centers

Low-temperature subpicosecond measurements on heterodimer-containing reaction centers from the HisM200 → Leu mutant of Rhodobacter capsulatus are reported. As at room temperature, primary electron transfer is initiated from a state of the bacteriochlorophyll (BChl)/bacteriopheophytin (BPh) heterodimer with substantial [BChl+BPh−] intradimer charge transfer character. The electronic composition of this transient state is discussed in terms of mixing of the exciton and charge resonance states of the BChl/BPh dimer and compared with the nature of the excited states of the BChl/BChl dimer in wild-type reaction centers.

Glycosaminoglycans in Human and Bovine serum: Detection of Twenty-Four Heparan sulfate and chondroitin sulfate Motifs Including a Novel Sialic Acid-modified Chondroitin Sulfate Linkage Hexasaccharide

Heterogeneous heparan sulfate and chondroitin sulfate glycosaminoglycan (GAG) polysaccharides are important components of blood circulation. Changes in GAG quantity and structure in blood have been indicated in cancers and other human diseases. However, GAG quantities and structures have not been fully characterized due to lack of robust and sensitive analytical tools. To develop such tools, we isolated GAGs from serum and plasma. We employed liquid chromatography (LC) for GAG quantification and LC/mass spectrometry (MS) for GAG structural analysis. Twenty-four heparan and chondroitin sulfate motifs were identified, including linkage hexasaccharides, repeating disaccharide compositions, reducing, and non-reducing end mono-, di-, tri-, and tetrasaccharide structures. Disaccharides were detectable at picomolar level without radiolabeling or derivitization, so only a few ml of human and fetal bovine serum was required for this study. The detection of different reducing end structures distinct from GAG linkage hexasaccharides revealed that free GAG chains generated by GAG degradation enzymes co-existed with proteoglycans in serum. In addition, a novel sialic acid-modified linkage hexasaccharide was found conjugated to bikunin, the most abundant serum proteoglycan.

Insights into the factors controlling the rates of the deactivation processes that compete with charge separation in photosynthetic reaction centers

Abstract This article addresses the factors that underlie the unity quantum yield of charge separation in the bacterial photosynthetic reaction center (RC). Of particular interest are the factors that suppress charge recombination and other deactivation processes which could compete effectively with charge separation. Studies on mutant RCs with enhanced charge recombination show that rates of decay processes can be increased tenfold or more upon a relatively small fractional increase in the free energy gap to the ground state. This behavior is opposite to that expected on the basis of Franck-Condon effects, where an increase in Δ G should decrease the deactivation rate. In the mutants, it is proposed that the enhanced deactivation rates of the intermediate charge-separated states result from (i) increased quantum mechanical mixing with electronic states at slightly higher energy that have inherently strong deactivation properties, and (ii) increased thermal repopulation of such strongly deactivated states. The thermal repopulation mechanism is proposed to play an important role in the heterodimer mutants studied here. This mechanism requires that the free energy gaps between the charge-separated and strongly deactivated states must decrease with temperature to be consistent with the weakly temperature-dependent deactivation behavior. Potential mediating states include the excited dimeric primary electron donor and a state involving the oxidized dimer and the reduced accessory bacteriochlorophyll molecule. It is concluded that increasing the free energy of charge-separated intermediates in order to minimize the Franck-Condon factors can enhance the deactivation rates and thus reduce the quantum yield of charge separation.

Rotational-echo double-resonance NMR-restrained model of the ternary complex of 5-enolpyruvylshikimate-3-phosphate synthase.

The 46-kD enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate-3-phosphate (S3P) and phosphoenolpyruvate to form EPSP. The reaction is inhibited by N-(phosphonomethyl)-glycine (Glp), which, in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. We have used solid-state NMR and molecular modeling to characterize the EPSP synthase–S3P–Glp ternary complex. Modeling began with the crystal coordinates of the unliganded protein, published distance restraints, and information from the chemical modification and mutagenesis literature on EPSP synthase. New inter-ligand and ligand-protein distances were obtained. These measurements utilized the native 31P in S3P and Glp, biosynthetically 13C-labeled S3P, specifically 13C and 15N labeled Glp, and a variety of protein-15N labels. Several models were investigated and tested for accuracy using the results of both new and previously published rotational-echo double resonance (REDOR) NMR experiments. The REDOR model is compared with the recently published X-ray crystal structure of the ternary complex, PDB code 1G6S. There is general agreement between the REDOR model and the crystal structure with respect to the global folding of the two domains of EPSP synthase and the relative positioning of S3P and Glp in the binding pocket. However, some of the REDOR data are in disagreement with predictions based on the coordinates of 1G6S, particularly those of the five arginines lining the binding site. We attribute these discrepancies to substantive differences in sample preparation for REDOR and X-ray crystallography. We applied the REDOR restraints to the 1G6S coordinates and created a REDOR-refined xray structure that agrees with the NMR results.

High affinity glycosaminoglycan and autoantigen interaction explains joint specificity in a mouse model of rheumatoid arthritis

In the K/BxN mouse model of rheumatoid arthritis, autoantibodies specific for glucose-6-phosphate isomerase (GPI) can transfer joint-specific inflammation to most strains of normal mice. Binding of GPI and autoantibody to the joint surface is a prerequisite for joint-specific inflammation. However, how GPI localizes to the joint remains unclear. We show that glycosaminoglycans (GAGs) are the high affinity (83 nm) joint receptors for GPI. The binding affinity and structural differences between mouse paw/ankle GAGs and elbows/knee GAGs correlated with the distal to proximal disease severity in these joints. We found that cartilage surface GPI binding was greatly reduced by either chondroitinase ABC or β-glucuronidase treatment. We also identified several inhibitors that inhibit both GPI/GAG interaction and GPI enzymatic activities, which suggests that the GPI GAG-binding domain overlaps with the active site of GPI enzyme. Our studies raise the possibility that GAGs are the receptors for other autoantigens involved in joint-specific inflammatory responses.

Location of Fluorotryptophan Sequestered in an Amphiphilic Nanoparticle by Rotational-Echo Double-Resonance NMR

Rotational-echo double-resonance (REDOR) 13C NMR spectra (with 19F dephasing) have been obtained of 6-fluorotryptophan complexed by a polymeric amphiphilic nanosphere consisting of a polystyrene core covalently attached to a poly(acrylic acid)-polyacrylamide shell. The REDOR spectra show that aromatic carbons from the polystyrene core and oxygenated carbons in the poly(acrylic acid)-polyacrylamide shell are both proximate to the 19F of 6-fluorotryptophan. Molecular modeling restrained by distances inferred from the REDOR spectra suggests that all of the 6-fluorotryptophans are in the shell but within 10 A of the core-shell interface.

Elucidation of the role of metal-to-ring charge-transfer excited states in the deactivation of photoexcited ruthenium porphyrin carbonyl complexes

Abstract Deactivation of the lowest excited triplet state, 3 (π, π*), of the Ru(II) porphyrins RuP(CO)(L) is more strongly dependent on temperature than decay of 3 (π, π*) in Pt(II)P and H 2 P (metal-free) complexes containing the same macrocycle P. This and other observations support the proposal that 3 (π, π*) in the RuP(CO)(L) complexes decays in part via a metal-to-ring (d, π*) charge-transfer excited state at higher energy.