The role of electrostatic interactions between the ionizable Asp158 and the active site thiolate-imidazolium ion pair of some cysteine proteinases has been the subject of controversy for some time. This study reports the expression of wild type procaricain and Asp158Glu, Asp158Asn and Asp158Ala mutants from Escherichia coli. Purification of autocatalytically matured enzymes yielded sufficient fully active material for pH (kcat/Km) profiles to be obtained. Use of both uncharged and charged substrates allowed the effects of different reactive enzyme species to be separated from the complications of electrostatic effects between enzyme and substrate. At least three ionizations are detectable in the acid limb of wild type caricain and the Glu and Asn mutants. Only two pKa values, however, are detectable in the acid limb using the Ala mutant. Comparison of pH activity profiles shows that whilst an ionizable residue at position 158 is not essential for the formation of the thiolate-imidazolium ion pair, it does form a substantial part of the electrostatic field responsible for increased catalytic competence. Changing the position of this ionizable group in any way reduces activity. Complete removal of the charged group reduces catalytic competence even further. This work indicates that hydronations distant to the active site are contributing to the electrostatic effects leading to multiple active ionization states of the enzyme.

Schroeder et al. demonstrate that when peripheral tolerance is relaxed, tier 2 HIV-1–neutralizing antibodies can be elicited and identify new autoreactive antibody specificities against histone H2A capable of neutralizing tier 2 HIV-1.

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The performance of dye-sensitized solar and photoelectrochemical cells is strongly dependent on the electron transfer events at the electrode-sensitizer interface. Surface-bound peptides derivatized with chromophores have not been used in dye-sensitized solar and photoelectrochemical cells, but they have properties for these applications that could be advantageous by exploiting secondary structure and the attachment of multiple chromophores. In this manuscript, we have investigated structure-property relationships for three metallopeptide-based assemblies to solution and chemically bound to nanocrystalline MO(2) (M = Ti, Zr) films. A particular interest was exploring the influence of increasing separation distance between a common chromophore, [Ru(bpy)(2) (4-Me-4'-(NHCO)bpy)](2+) , and the underlying oxide substrate on excited and ground state electron transfer. Rates of Ru(II) oxidation to Ru(III) at the interface were measured by cyclic voltammetry on fluorine-doped tin oxide and cross-surface electron transfer on TiO(2) . Excited state injection by [Ru(III) (bpy)(2) (bpy(-) )](2+) was monitored by transient absorption and time-resolved emission. There are discernible trends in the electron transfer rate data with approximated, fully extended distances between the [Ru(bpy)(2) (4-Me-4'-(NHCO)bpy)](2+) sites and the interface. However, the distance dependences that are observed are smaller than anticipated, a result consistent with a lack of ordered secondary structure in the surface-bound peptide chains and a distribution of local orientations. For the surface-bound excited states, only a small fraction undergo quenching by electron transfer to TiO(2) , presumably from those oriented near the surface.