Lilly Wong

Phosphorylation Driven Motions in the COOH-terminal Src Kinase, Csk, Revealed Through Enhanced Hydrogen–Deuterium Exchange and Mass Spectrometry (DXMS)

Previous kinetic studies demonstrated that nucleotide-derived conformational changes regulate function in the COOH-terminal Src kinase. We have employed enhanced methods of hydrogen-deuterium exchange-mass spectrometry (DXMS) to probe conformational changes on CSK in the absence and presence of nucleotides and thereby provide a structural framework for understanding phosphorylation-driven conformational changes. High quality peptic fragments covering approximately 63% of the entire CSK polypeptide were isolated using DXMS. Time-dependent deuterium incorporation into these probes was monitored to identify short peptide segments that exchange differentially with solvent. Regions expected to lie in loops exchange rapidly, whereas other regions expected to lie in stable secondary structure exchange slowly with solvent implying that CSK adopts a modular structure. The ATP analog, AMPPNP, protects probes in the active site and distal regions in the large and small lobes of the kinase domain, the SH2 domain, and the linker connecting the SH2 and kinase domains. The product ADP protects similar regions of the protein but the extent of protection varies markedly in several crucial areas. These areas correspond to the activation loop and helix G in the kinase domain and several inter-domain regions. These results imply that delivery of the gamma phosphate group of ATP induces unique local and long-range conformational changes in CSK that may influence regulatory motions in the catalytic pathway.

Probing the Active Center Gorge of Acetylcholinesterase by Fluorophores Linked to Substituted Cysteines

To examine the influence of individual side chains in governing rates of ligand entry into the active center gorge of acetylcholinesterase and to characterize the dynamics and immediate environment of these residues, we have conjugated reactive groups with selected charge and fluorescence characteristics to cysteines substituted by mutagenesis at specific positions on the enzyme. Insertion of side chains larger than in the native tyrosine at position 124 near the constriction point of the active site gorge confers steric hindrance to affect maximum catalytic throughput (k cat/K m ) and rates of diffusional entry of trifluoroketones to the active center. Smaller groups appear not to present steric constraints to entry; however, cationic side chains selectively and markedly reduce cation ligand entry through electrostatic repulsion in the gorge. The influence of side chain modification on ligand kinetics has been correlated with spectroscopic characteristics of fluorescent side chains and their capacity to influence the binding of a peptide, fasciculin, which inhibits catalysis peripherally by sealing the mouth of the gorge. Acrylodan conjugated to cysteine was substituted for tyrosine at position 124 within the gorge, for histidine 287 on the surface adjacent to the gorge and for alanine 262 on a mobile loop distal to the gorge. The 124 position reveals the most hydrophobic environment and the largest hypsochromic shift of the emission maximum with fasciculin binding. This finding likely reflects a sandwiching of the acrylodan in the complex with the tip of fasciculin loop II. An intermediate spectral shift is found for the 287 position, consistent with partial occlusion by loops II and III of fasciculin in the complex. Spectroscopic properties of the acrylodan at the 262 position are unaltered by fasciculin addition. Hence, combined spectroscopic and kinetic analyses reveal distinguishing characteristics in various regions of acetylcholinesterase that influence ligand association.

Analysis of cholinesterase inactivation and reactivation by systematic structural modification and enantiomeric selectivity.

We show here with a congeneric series of Rp- and Sp-alkoxymethyl phosphonothiolates of known absolute stereochemistry that chiral selectivity in their reaction with acetylcholinesterase can be described in terms of discrete orientational and steric requirements. Stereoselectivity depends on acyl pocket dimensions, which govern leaving group orientation and a productive association of the phosphonyl oxygen in the oxyanion hole. Overall geometry is consistent with a pentavalent intermediate where the attacking serine and leaving group are at apical positions. Oxime reactivation of the phosphonylated enzyme occurs through a similar associative intermediate presumably forming an oxime phosphonate. The oximes of differing structure show distinct angles of attacking the phosphate where the attack angles and access to the phosphorus are constrained in the sterically impacted gorge. Hence, efficacy of oxime reactivation is dependent on both oxime and conjugated phosphonate structures.

Nanosecond Dynamics of Acetylcholinesterase Near the Active Center Gorge

To delineate the role of peptide backbone flexibility and rapid molecular motion in acetylcholinesterase catalysis and inhibitor association, we investigated the decay of fluorescence anisotropy at three sites of fluorescein conjugation to cysteine-substitution mutants of the enzyme. One cysteine was placed in a loop at the peripheral site near the rim of the active center gorge (H287C); a second was in a helical region outside of the active center gorge (T249C); a third was at the tip of a small, flexible ω loop well separated from the gorge (A262C). Mutation and fluorophore conjugation did not appreciably alter catalytic or inhibitor binding parameters of the enzyme. The results show that each site examined was associated with a high degree of segmental motion; however, the A262C and H287C sites were significantly more flexible than the T249C site. Association of the active center inhibitor, tacrine, and the peripheral site peptide inhibitor, fasciculin, had no effect on the anisotropy decay of fluorophores at positions 249 and 262. Fasciculin, but not tacrine, on the other hand, dramatically altered the decay profile of the fluorophore at the 287 position, in a manner consistent with fasciculin reducing the segmental motion of the peptide chain in this local region. The results suggest that the motions of residues near the active center gorge and across from the Cys69-Cys96 ω loop are uncoupled and that ligand binding at the active center or the peripheral site does not influence acetylcholinesterase conformational dynamics globally, but induces primarily domain localized decreases in flexibility proximal to the bound ligand.

The solvent in CNBr cleavage reactions determines the fragmentation efficiency of ketosteroid isomerase fusion proteins used in the production of recombinant peptides

Abnormal fragmentation during cyanogen bromide polypeptide cleavage rarely occurs, although parallel side reactions are known to typically accompany normal cleavage. We have observed that cyanogen bromide cleavage of highly hydrophobic fusion proteins utilized for production of recombinant peptides results in almost complete abolishment of the expected reaction products when the reaction is carried out in 70% trifluoroacetic acid. On the basis of mass spectrometric analysis of the reaction products, we have identified a number of fragments whose origin can be attributed to incomplete fragmentation of the fusion protein, and to unspecific degradation affecting the carrier protein. Substituting the solvent in the reaction media with 70% formic acid or with a matrix composed of 6M guanidinium hydrochloride in 0.1M HCl, however, was found to alleviate polypeptide cleavage. We have attributed the poor yields of the CNBr cleavage carried out in 70% TFA to the increased hydrophobicity of our particular fusion proteins, and to the poor solubilizing ability of this reaction medium. We propose the utilization of chaotropic agents in the presence of diluted acids as the preferred cyanogen bromide cleavage medium of fusion proteins in order to maximize cleavage efficiency of hydrophobic sequences and to prevent deleterious degradation and structural modifications of the target peptides.

Reactivation of Enantiomeric Organophosphonyl Conjugates of Acetylcholinesterase Mutants, F295L and F297I by Mono- and Bis-Quarternary Oximes

Single site acetylcholinesterase (AChE) mutants, F295L and F297I, were inhibited with enantiomeric Sp- and Rp- cycloheptyl (CHMP), isopropyl (iPrMP), and dimethylbutyl (DMBMP) methylphosphonyl thiocholine. The resulting conjugates were subjected to reactivation with 2-(hydroxyiminomethyl)-1-methylpyridinium methiodide (2-PAM) and 1 -(2′ -hydroxyiminomethyl-1′ -pyridinium)-3 -(4″-carbamoy1-1″-pyridinium)-2 -oxapropane dichloride (HI6). Rates of reactivation obtained for mutants were compared to wildtype AChE. HI6 was able to reactivate all conjugates except for the Rp- CHMP conjugates and Rp- DMBMP-F297I conjugate. The F295L mutant exhibited a 6–8 fold faster reactivation rate compared to wildtype for Sp- CHMP and Sp- DMBMP conjugates, while only a 2–3 fold difference was seen for the Sp- iPrMP conjugate. The Rp- DMBMP-F295L conjugate showed biphasic behavior upon reactivation with HI6. The F297I mutant only showed a 2–3 fold higher reactivation rate for all three Sp- enantiomeric conjugates compared to wildtype. The Rp- conjugates that underwent reactivation were slow and only slightly modified by these mutations. 2-PAM was able to reactivate all conjugates except for Rp- CHMP-AChE and Rp- CHMP-F295L. The F295L mutant did not show an enhancement in reactivation rates compared to wildtype for both Sp- and Rp- conjugates. The F297I mutant showed a 8–10 fold faster reactivation rates for the Sp- conjugates while no enhancement was seen with the Rp- conjugates. HI6 proved to be a more potent reactivator. Its maximum rate of enhancement (kmax) of the deacylation step is anywhere from 10–100 fold faster than 2-PAM. HI6 shows a greater enantiomeric selectivity than 2-PAM in reactivation. Kinetic analysis reveals that acyl pocket dimensions play a major role in controlling the reactivation of organophosphonyl conjugates.