[en] Energy-dependent protein degradation machines, such as the Escherichia coli protease ClpAP, require regulated interactions between the ATPase component (ClpA) and the protease component (ClpP) for function. Recent studies indicate that the ClpP N-terminus is essential in these interactions, yet the dynamics of this region remain unclear. Here, we use synchrotron hydroxyl radical footprinting and kinetic studies to characterize functionally important conformational changes of the ClpP N-terminus. Footprinting experiments show that the ClpP N-terminus becomes more solvent-exposed upon interaction with ClpA. In the absence of ClpA, deletion of the ClpP N-terminus increases the initial degradation rate of large peptide substrates 5-15-fold. Unlike ClpAP, ClpP?N exhibits a distinct slow phase of product formation that is eliminated by the addition of hydroxylamine, suggesting that truncation of the N-terminus leads to stabilization of the acyl-enzyme intermediate. These results indicate that (1) the ClpP N-terminus acts as a 'gate' controlling substrate access to the active sites, (2) binding of ClpA opens this 'gate', allowing substrate entry and formation of the acyl-enzyme intermediate, and (3) closing of the N-terminal 'gate' stimulates acyl-enzyme hydrolysis.

[en] Synchrotron X-ray protein footprinting is used to study structural changes upon formation of the ClpA hexamer. Comparative solvent accessibilities between ClpA monomer and ClpA hexamer samples are in agreement throughout most of the sequence, with calculations based on two previously proposed hexameric models. The data differ substantially from the proposed models in two parts of the structure: the D1 sensor 1 domain and the D2 loop region. The results suggest that these two regions can access alternate conformations in which their solvent protection is greater than that in the structural models based on crystallographic data. In combination with previously reported structural data, the footprinting data provide support for a revised model in which the D2 loop contacts the D1 sensor 1 domain in the ATP-bound form of the complex. These data provide the first direct experimental support for the nucleotide-dependent D2 loop conformational change previously proposed to mediate substrate translocation.

[en] The chemistry of the Fe(VI) compound, K₂FeO₄, was investigated in aqueous potassium hydroxide electrolyte for its potential use in secondary storage systems. High charge storage K₂FeO₄ material was synthesized using alkaline hypochlorite oxidation of ferric nitrate and characterized by in situ X-ray diffraction and Moessbauer spectroscopy. The discharge-charge profile of the cathode, measured versus a zinc anode, was semiquantitatively correlated with changes in Fe⁶⁺+/Fe³⁺ ratio and the nature of structural transformation at each stage by in situ Moessbauer and in situ synchrotron X-ray diffraction spectroscopy. The data in conjunction with the charge-discharge profile clearly indicates the rechargeability of K₂FeO₄ with a charge efficiency of about 36% of the total current passed under the prevailing experimental conditions. These results are potentially important to the design of Fe⁶⁺ compound analogs for use in secondary energy storage systems