Probing the unfolding of myoglobin and domain C of PARP-1 with covalent labeling and top-down ultraviolet photodissociation mass spectrometry.

Ultraviolet photodissocation (UVPD) mass spectrometry was used for high mass accuracy top-down characterization of two proteins labeled by the chemical probe, S-ethylacetimidate (SETA), in order to evaluate conformational changes as a function of denaturation. The SETA labeling/UVPD-MS methodology was used to monitor the mild denaturation of horse heart myoglobin by acetonitrile, and the results showed good agreement with known acetonitrile and acid unfolding pathways of myoglobin. UVPD outperformed electron transfer dissociation (ETD) in terms of sequence coverage, allowing the SETA reactivity of greater number of lysine amines to be monitored and thus providing a more detailed map of myoglobin. This strategy was applied to the third zinc-finger binding domain, domain C, of PARP-1 (PARP-C), to evaluate the discrepancies between the NMR and crystal structures which reported monomer and dimer forms of the protein, respectively. The trends reflected from the reactivity of each lysine as a function of acetonitrile denaturation in the present study support that PARP-C exists as a monomer in solution with a close-packed C-terminal α helix. Additionally, those lysines for which the SETA reactivity increased under denaturing conditions were found to engage in tertiary polar contacts such as salt bridging and hydrogen bonding, providing evidence that the SETA/UVPD-MS approach offers a versatile means to probe the interactions responsible for conformational changes in proteins.

Chromogenic chemical probe for protein structural characterization via ultraviolet photodissociation mass spectrometry.

A chemical probe/ultraviolet photodissociation (UVPD) mass spectrometry strategy for evaluating structures of proteins and protein complexes is reported, as demonstrated for lysozyme and beta-lactoglobulin with and without bound ligands. The chemical probe, NN, incorporates a UV chromophore that endows peptides with high cross sections at 351 nm, a wavelength not absorbed by unmodified peptides. Thus, NN-modified peptides can readily be differentiated from nonmodified peptides in complex tryptic digests created upon proteolysis of proteins after their exposure to the NN chemical probe. The NN chemical probe also affords two diagnostic reporter ions detected upon UVPD of the NN-modified peptide that provides a facile method for the identification of NN peptides within complex mixtures. Quantitation of the modified and unmodified peptides allows estimation of the surface accessibilities of lysine residues based on their relative reactivities with the NN chemical probe.

Peptide-conjugated pterins as inhibitors of ricin toxin A.

Several 7-peptide-substituted pterins were synthesized and tested as competitive active-site inhibitors of ricin toxin A (RTA). Focus began on dipeptide conjugates, and these results further guided the construction of several tripeptide conjugates. The binding of these compounds to RTA was studied via a luminescence-based kinetic assay, as well as through X-ray crystallography. Despite the relatively polar, solvent exposed active site, several hydrophobic interactions, most commonly π-interactions not predicted by modeling programs, were identified in all of the best-performing inhibitors. Nearly all of these compounds provide IC50 values in the low micromolar range.

Sulfur incorporation generally improves Ricin inhibition in pterin-appended glycine-phenylalanine dipeptide mimics.

Several 7-aminoamido-pterins were synthesized to evaluate the electronic and biochemical subtleties observed in the 'linker space' when N-{N-(pterin-7-yl)carbonylglycyl}-l-phenylalanine 1 was bound to the active site of RTA. The gylcine-phenylalanine dipeptide analogs included both amides and thioamides. Decarboxy gly-phe analog 2 showed a 6.4-fold decrease in potency (IC50 = 128 µM), yet the analogous thioamide 7 recovered the lost activity and performed similarly to the parent inhibitor (IC50 = 29 µM). Thiourea 12 exhibited an IC50 nearly six times lower than the oxo analog 13. All inhibitors showed the pterin head-group firmly bound in their X-ray structures yet the pendants were not fully resolved suggesting that all pendants are not firmly bound in the RTA linker space. Calculated log P values do not correlate to the increase in bioactivity suggesting other factors dominate.

Optimized 5-membered heterocycle-linked pterins for the inhibition of Ricin Toxin A.

The optimization of a series of pterin amides for use as Ricin Toxin A (RTA) inhibitors is reported. Based upon crystallographic data of a previous furan-linked pterin, various expanded furans were synthesized, linked to the pterin and tested for inhibition. Concurrently, hetero-analogs of furan were explored, leading to the discovery of more potent triazol-linked pterins. Additionally, we discuss a dramatic improvement in the synthesis of these pterin amides via a dual role by diazabicycloundecene (DBU). This synthetic enhancement facilitates rapid diversification of the previously challenging pterin heterocycle, potentially aiding future medicinal research involving this structure.

7-Substituted pterins provide a new direction for ricin A chain inhibitors.

Ricin is a potent toxin found in castor seeds. The A chain, RTA, enzymaticlly depurinates a specific adenosine in ribosomal RNA, inhibiting protein synthesis. Ricin is a known chemical weapons threat having no effective antidote. This makes the discovery of new inhibitors of great importance. We have previously used 6-substituted pterins, such as pteroic acid, as an inhibitor platform with moderate success. We now report the success of 7-carboxy pterin (7CP) as an RTA inhibitor; its binding has been monitored using both kinetic and temperature shift assays and by X-ray crystallography. We also discuss the synthesis of various derivatives of 7CP, and their binding affinity and inhibitory effects, as part of a program to make effective RTA inhibitors.

Acyl radical insertion for the direct formation of new 7-substituted pterin analogs.

A variety of pterin molecules were synthesized via an under-utilized acyl radical insertion, using aldehydes and alpha-keto esters as the acyl source. These reactions gave complete regiospecificity for the 7-isomer, with reaction times ranging in minutes, often with instantaneous product precipitation. This approach led to the construction of new pterin analogs unaccessable via traditional Friedel-Crafts acylation. The compounds were characterized by NMR spectroscopy and high-resolution mass spectroscopy.

Lanthanide complexes as fluorescent indicators for neutral sugars and cancer biomarkers.

Simple water-soluble lanthanum and europium complexes are effective at detecting neutral sugars as well as glycolipids and phospholipids. In solutions at physiologically relevant pH the fluorescent lanthanum complex binds neutral sugars with apparent binding constants comparable to those of arylboronic acids. Interference from commonly occurring anions is minimal. The europium complex detects sialic acid-containing gangliosides at pH 7.0 over an asialoganglioside. This selectivity is attributed, in large part, to the cooperative complexation of the oligosaccharide and sialic acid residues to the metal center, based on analogous prior studies. In MeOH, lysophosphatidic acid (LPA), a biomarker for several pathological conditions including ovarian cancer, is selectively detected by the europium complex. LPA is also detected via a fluorescence increase in human plasma samples. The 2-sn-OH moiety of LPA plays a key role in promoting binding to the metal center. Other molecules found in common brain ganglioside and phospholipid extracts do not interfere in the ganglioside or LPA fluorescence assays.