Investigations on the conformational stability and metal substitution of the zinc ion-dependent anthrax lethal factor protease and new strategies for the quantification of divalent metal ions in metalloproteins

Anthrax lethal factor (LF) has been shown to be a critical virulence factor in the pathogenesis of anthrax, a disease caused by the Gram-positive bacterium Bacillus anthracis. LF is a 90 kDa Zn2+ -dependent metalloprotease which has been implicated in impairing important intracellular signaling pathways by cleaving mitogen-activated protein kinase kinases. In order for LF to breach the host cell membrane and to reach its intracellular targets, the protein must bind to protective antigen (PA), a membrane-spanning, pore-forming protein secreted by B. anthracis that is believed to function as a passage way for LF to access the cytosol. Since PA's 14-stranded beta-barrel pore measures only 12 A at its constriction site, LF is likely translocated in a partially unfolded state. However, it is not yet known whether such unfolding induces the dissociation of the Zn2+ ion from LF as a consequence of structural changes in the enzyme's active site.;This study addresses this issue by probing changes in LF's global protein structure, activity and metal status induced by the exposure to chemical denaturants. Accordingly, the conformational stability of LF in its holo-, apo- and Co 2+-substituted forms was assessed by intrinsic tryptophan fluorescence spectroscopy using guanidine hydrochloride (GdnHCI) as the denaturant. It was determined that the presence of a metal (Zn2+ or Co 2+) in LF's active site does confer some measure of conformational stability to the protein as compared to that noted for the apo-protein. An analysis of the influence of GdnHCI on the activity of LF revealed the enzyme to be inactivated at denaturant concentrations much lower than those required to unfold the protein. To establish whether this phenomenon is due to subtle structural changes in LF's active site (not traceable by fluorescence spectroscopy), the effect of a variety of denaturing and non-denaturing agents (urea, guanidine isothiocyanate, guanidine sulfate, and alkali/alkaline earth metals ions) on the enzyme's activity was investigated. These studies revealed the loss of LF's function to be a consequence of an ionic strength effect rather than a reflection of structural changes in the protein's active site. The Zn 2+ ion status during GdnHCI-induced unfolding of LF was probed by monitoring the susceptibility of the metal ion to chelation by the chromophoric chelator 4-(2-pyridylazo)resorcinol (PAR). Through these investigations, it was determined that Zn2+ tends to resist dissociation from the protein upon unfolding. Such finding raises the possibility of Zn 2+ remaining LF-bound during PA-mediated translocation.;As mentioned above, the susceptibility of Co2+-substituted LF (CoLF) towards denaturation was investigated. The conditions for the preparation of CoLF were optimized in this study. CoLF was obtained by a direct exchange method, in which the zinc protein was first exposed to an excess of Co 2+, followed by the removal of any protein-unbound metal ions by extensive dialysis. CoLF was found to be approximately twice as active as the native zinc protein. The dissociation constants for both LF in its native and Co2+-substituted forms were determined and found to be 1 pM and 80 pM, respectively.;As a secondary focus of this work, two novel methods for metal content determination in substituted metalloprotein preparations, employing the use of the colorimetric metal chelators PAR and 2-carboxy-2'hydroxy-5'sulfoformazylbenzene (Zincon) were developed. In the first method, PAR was utilized to directly and simultaneously determine low micromolar concentrations (1 to 10 muM) of both Zn2+ and Co2+ ions. The method, which is based on the fitting of absorption spectra to a linear addition of Beer-Lambert law, circumvents the need for separating or masking one of the metal ions before their quantification. The developed method is useful for determining the metal content of Co2+-substituted zinc proteins including CoLF. In the second method, the spectral features of Zincon (in its free form and complexed to a variety of biologically relevant transition metals) in the presence of guanidine hydrochloride and urea were studied with a view to apply the chelator to the determination of metal ions in metalloproteins. As a consequence, a method for the quantification of Zn2+, Cu2+ and Co2+ with Zincon (with detection limits in the high nanomolar range) was developed with borate-containing urea (8 M, pH 9.0) serving as the optimal buffer medium. Finally, a simple two-step method for the determination of both Zn2+ and Cu2+ in the same sample with Zincon was developed, and verified using the prototypical Cu2+/Zn2+-protein superoxide dismutase. The outlined methods may be useful for the quantification of metal ions in Zn 2+-, Cu2+-, Co2+- and Zn2+/Cu 2+-containing proteins.