Evidence for the formation of disulfide radicals in protein crystals upon X-ray irradiation.

Irradiation of proteins with intense X-ray radiation produced by third-generation synchrotron sources generates specific structural and chemical alterations, including breakage of disulfide bonds and decarboxylation. In this paper, disulfide bond lengths in irradiated crystals of the enzyme Torpedo californica acetylcholinesterase are examined based on quantum simulations and on experimental data published previously. The experimental data suggest that one disulfide bond elongates by approximately 0.

Joint refinement as a tool for thorough comparison between NMR and X-ray data and structures of HU protein.

Joint refinement, i.e., the simultaneous refinement of a structure against both nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic data, was performed on the HU protein from Bacillus stearothermophilus (HUBst).

Specific chemical and structural damage to proteins produced by synchrotron radiation.

Radiation damage is an inherent problem in x-ray crystallography. It usually is presumed to be nonspecific and manifested as a gradual decay in the overall quality of data obtained for a given crystal as data collection proceeds. Based on third-generation synchrotron x-ray data, collected at cryogenic temperatures, we show for the enzymes Torpedo californica acetylcholinesterase and hen egg white lysozyme that synchrotron radiation also can cause highly specific damage.

Completeness of NOEs in protein structure: a statistical analysis of NMR.

The completeness of experimentally observed NOE restraints of a set of 97 NMR protein structures deposited in the PDB has been assessed. Completeness is defined as the ratio of the number of experimentally observed NOEs and the number of 'expected NOEs'. A practical definition of 'expected NOEs' based on inter-proton distances in the structures up to a given cut-off distance is proposed.

Validation of nuclear magnetic resonance structures of proteins and nucleic acids: hydrogen geometry and nomenclature.

A statistical analysis is reported of 1,200 of the 1,404 nuclear magnetic resonance (NMR)-derived protein and nucleic acid structures deposited in the Protein Data Bank (PDB) before 1999. Excluded from this analysis were the entries not yet fully validated by the PDB and the more than 100 entries that contained < 95% of the expected hydrogens. The aim was to assess the geometry of the hydrogens in the remaining structures and to provide a check on their nomenclature.

Crystal structures of aged phosphonylated acetylcholinesterase: nerve agent reaction products at the atomic level.

Organophosphorus acid anhydride (OP) nerve agents are potent inhibitors which rapidly phosphonylate acetylcholinesterase (AChE) and then may undergo an internal dealkylation reaction (called "aging") to produce an OP-enzyme conjugate that cannot be reactivated. To understand the basis for irreversible inhibition, we solved the structures of aged conjugates obtained by reaction of Torpedo californica AChE (TcAChE) with diisopropylphosphorofluoridate (DFP), O-isopropylmethylphosponofluoridate (sarin), or O-pinacolylmethylphosphonofluoridate (soman) by X-ray crystallography to 2.3, 2.

Static Laue diffraction studies on acetylcholinesterase.

Acetylcholinesterase (AChE) is one of nature's fastest enzymes, despite the fact that its three-dimensional structure reveals its active site to be deeply sequestered within the molecule. This raises questions with respect to traffic of substrate to, and products from, the active site, which may be investigated by time-resolved crystallography. In order to address one aspect of the feasibility of performing time-resolved studies on AChE, a data set has been collected using the Laue technique on a trigonal crystal of Torpedo californica AChE soaked with the reversible inhibitor edrophonium, using a total X-ray exposure time of 24 ms.

Structure of acetylcholinesterase complexed with the nootropic alkaloid, (-)-huperzine A.

(-)-Huperzine A (HupA) is found in an extract from a club moss that has been used for centuries in Chinese folk medicine. Its action has been attributed to its ability to strongly inhibit acetylcholinesterase (AChE). The crystal structure of the complex of AChE with optically pure HupA at 2.

Soluble monomeric acetylcholinesterase from mouse: expression, purification, and crystallization in complex with fasciculin.

A soluble, monomeric form of acetylcholinesterase from mouse (mAChE), truncated at its carboxyl-terminal end, was generated from a cDNA encoding the glycophospholipid-linked form of the mouse enzyme by insertion of an early stop codon at position 549. Insertion of the cDNA behind a cytomegalovirus promoter and selection by aminoglycoside resistance in transfected HEK cells yielded clones secreting large quantities of mAChE into the medium. The enzyme sediments as a soluble monomer at 4.

A metastable state of Torpedo californica acetylcholinesterase generated by modification with organomercurials.

Chemical modification of Torpedo californica acetylcholinesterase by various sulfhydryl reagents results in its conversion to one of two principal states. One of these states, viz., that produced by disulfides and alkylating agents, is stable.