An extended-X-ray-absorption-fine-structure study of freeze-dried and solution ovotransferrin. Evidence for water co-ordination at the metal-binding sites.

Our previous extended-X-ray-absorption-fine-structure (e.x.a.f.s.) study has shown that the probable iron environment in chicken ovotransferrin involves two low-Z ligands (consistent with phenolate linkages) at 0.185(1) nm and four low-Z ligands at 0.204(1) nm [Garratt, Evans, Hasnain & Lindley (1986) Biochem. J. 233, 479-484]. Herein we provide additional information from the e.x.a.f.s. and near-edge structure suggestive of a decrease in the co-ordination number of ovotransferrin-bound iron upon freeze-drying. These effects are reversible, and exposure of the freeze-dried material to a humid atmosphere results in reversion to the solution spectra. Progressive rehydration was monitored by using e.p.r. spectroscopy and was confirmed by recording the high-resolution X-ray-absorption near-edge structure (x.a.n.e.s.). The results suggest the presence of a labile water molecule at the iron-binding sites of ovotransferrin in solution.     

Serine Protease(s) Secreted by the Nematode Trichuris muris Degrade the Mucus Barrier

The polymeric mucin component of the intestinal mucus barrier changes during nematode infection to provide not only physical protection but also to directly affect pathogenic nematodes and aid expulsion. Despite this, the direct interaction of the nematodes with the mucins and the mucus barrier has not previously been addressed. We used the well-established Trichuris muris nematode model to investigate the effect on mucins of the complex mixture of immunogenic proteins secreted by the nematode called excretory/secretory products (ESPs). Different regimes of T. muris infection were used to simulate chronic (low dose) or acute (high dose) infection. Mucus/mucins isolated from mice and from the human intestinal cell line, LS174T, were treated with ESPs. We demonstrate that serine protease(s) secreted by the nematode have the ability to change the properties of the mucus barrier, making it more porous by degrading the mucin component of the mucus gel. Specifically, the serine protease(s) acted on the N-terminal polymerising domain of the major intestinal mucin Muc2, resulting in depolymerisation of Muc2 polymers. Importantly, the respiratory/gastric mucin Muc5ac, which is induced in the intestine and is critical for worm expulsion, was protected from the depolymerising effect exerted by ESPs. Furthermore, serine protease inhibitors (Serpins) which may protect the mucins, in particular Muc2, from depolymerisation, were highly expressed in mice resistant to chronic infection. Thus, we demonstrate that nematodes secrete serine protease(s) to degrade mucins within the mucus barrier, which may modify the niche of the parasite to prevent clearance from the host or facilitate efficient mating and egg laying from the posterior end of the parasite that is in intimate contact with the mucus barrier. However, during a T_H2-mediated worm expulsion response, serpins, Muc5ac and increased levels of Muc2 protect the barrier from degradation by the nematode secreted protease(s).     

Variable metallation of human superoxide dismutase: atomic resolution crystal structures of Cu-Zn, Zn-Zn and as-isolated wild-type enzymes

Human Cu-Zn superoxide dismutase (SOD1) protects cells from the effects of oxidative stress. Mutations in SOD1 are linked to the familial form of amyotrophic lateral sclerosis. Several hypotheses for their toxicity involve the mis-metallation of the enzyme. We present atomic-resolution crystal structures and biophysical data for human SOD1 in three metallation states: Zn-Zn, Cu-Zn and as-isolated. These data represent the first atomic-resolution structures for human SOD1, the first structure of a reduced SOD1, and the first structure of a fully Zn-substituted SOD1 enzyme. Recombinantly expressed as-isolated SOD1 contains a mixture of Zn and Cu at the Cu-binding site. The Zn-Zn structure appears to be at least as stable as the correctly (Cu-Zn) metallated enzyme. These data raise the possibility that in a cellular environment with low availability of free copper, Zn-Zn may be the preferred metallation state of SOD1 prior to its interaction with the copper chaperone.     

Biophysical characterization and unfolding of LEF4 factor of RNA polymerase from AcNPV

Late expression factor 4 (LEF4) is one of the four subunits of Autographa californica nuclear polyhedrosis virus (AcNPV) RNA polymerase. LEF4 was overexpressed in Escherichia coli and recombinant protein was subjected to structural characterization. Chemical induced unfolding of LEF4 was investigated using intrinsic fluorescence, hydrophobic dye binding, fluorescence quenching, and circular dichroism (CD) techniques. The unfolding of LEF4 was found to be a non‐two state, biphasic transition. Intermediate states of LEF4 at 2M GnHCl and 4M urea shared some common structural features and hence may lie on the same pathway of protein folding. Steady‐state fluorescence and far‐UV CD showed that while there was considerable shift in the wavelength of emission maximum (λ_max), the secondary structure of LEF4 intermediates at 2M GnHCl and 4M urea remained intact. Further, temperature induced denaturation of LEF4 was monitored using far‐UV CD. This study points to the structural stability of LEF4 under the influence of denaturants like urea and temperature. Although LEF4 is an interesting model protein to study protein folding intermediates, in terms of functional significance the robust nature of this protein might reflect one of the several strategies adapted by the virus to survive under very adverse environmental and physiological conditions. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 574–582, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Gating mechanisms for biological electron transfer: Integrating structure with biophysics reveals the nature of redox control in cytochrome P450 reductase and copper-dependent nitrite reductase

Biological electron transfer is a fundamentally important reaction. Despite the apparent simplicity of these reactions (in that no bonds are made or broken), their experimental interrogation is often complicated because of adiabatic control exerted through associated chemical and conformational change. We have studied the nature of this control in several enzyme systems, cytochrome P450 reductase, methionine synthase reductase and copper-dependent nitrite reductase. Specifically, we review the evidence for conformational control in cytochrome P450 reductase and methionine synthase reductase and chemical control i.e. proton coupled electron transfer in nitrite reductase. This evidence has accrued through the use and integration of structural, spectroscopic and advanced kinetic methods. This integrated approach is shown to be powerful in dissecting control mechanisms for biological electron transfer and will likely find widespread application in the study of related biological redox systems.