The influence of ion composition, pH, and peptide concentration on the conformation and activity of the 37-residue human antibacterial peptide LL-37 has been studied. At micromolar concentration in water, LL-37 exhibits a circular dichroism spectrum consistent with a disordered structure. The addition of 15 mM HCO 3 ؊ , SO 4 2؊ , or CF 3 CO 2 ؊ causes the peptide to adopt a helical structure, with approximately equal efficiency, while 160 mM Cl ؊ is less efficient. A cooperative transition from disordered to helical structure is observed as the peptide concentration is increased, consistent with formation of an oligomer. The extent of ␣-helicity correlates with the antibacterial activity of LL-37 against both Gram-positive and Gram-negative bacteria. Two homologous peptides, FF-33 and SK-29, containing 4 and 8 residue deletions at the N terminus, respectively, require higher concentrations of anions for helix formation and are less active than LL-37 against Escherichia coli D21. Below pH 5, the helical content of LL-37 gradually decreases, and at pH 2 it is entirely disordered. In contrast, the helical structure is retained at pH over 13. The minimal inhibitory concentration of LL-37 against E. coli is 5 M, and at 13-25 M the peptide is cytotoxic against several eukaryotic cells. In solutions containing the ion compositions of plasma, intracellular fluid, or interstitial fluid, LL-37 is helical, and hence it could pose a danger to human cells upon release. However, in the presence of human serum, the antibacterial and the cytotoxic activities of LL-37 are inhibited.

Short-chain dehydrogenases/reductases form a large, evolutionarily old family of NAD(P)(H)-dependent enzymes with over 60 genes found in the human genome. Despite low levels of sequence identity (often 10 -30%), the three-dimensional structures display a highly similar ␣/␤ folding pattern. We have analyzed the role of several conserved residues regarding folding, stability, steady-state kinetics, and coenzyme binding using bacterial 3␤/17␤-hydroxysteroid dehydrogenase and selected mutants. Structure determination of the wildtype enzyme at 1.2-Å resolution by x-ray crystallography and docking analysis was used to interpret the biochemical data. Enzyme kinetic data from mutagenetic replacements emphasize the critical role of residues Thr-12, Asp-60, Asn-86, Asn-87, and Ala-88 in coenzyme binding and catalysis. The data also demonstrate essential interactions of Asn-111 with active site residues. A general role of its side chain interactions for maintenance of the active site configuration to build up a proton relay system is proposed. This extends the previously recognized catalytic triad of Ser-Tyr-Lys residues to form a tetrad of Asn-Ser-Tyr-Lys in the majority of characterized short-chain dehydrogenases/reductase enzymes.

Glutaredoxins belong to the thioredoxin superfamily of structurally similar thiol-disulfide oxidoreductases catalyzing thiol-disulfide exchange reactions via reversible oxidation of two active-site cysteine residues separated by two amino acids (CX 1 X 2 C). Standard state redox potential (E°) values for glutaredoxins are presently unknown, and use of glutathione/glutathione disulfide (GSH/GSSG) redox buffers for determining E° resulted in variable levels of GSH-mixed disulfides. To overcome this complication, we have used reversephase high performance liquid chromatography to separate and quantify the oxidized and reduced forms present in the thiol-disulfide exchange reaction at equilibrium after mixing one oxidized and one reduced protein. This allowed for direct and quantitative pairwise comparisons of the reducing capacities of the proteins and mutant forms. Equilibrium constants from pair-wise reaction with thioredoxin or its P34H mutant, which have accurately determined E° values from their redox equilibrium with NADPH catalyzed by thioredoxin reductase, allowed for transformation into standard state values. Using this new procedure, the standard state redox potentials for the Escherichia coli glutaredoxins 1 and 3, which contain identical active site sequences CPYC, were found to be E° ‫؍‬ ؊233 and ؊198 mV, respectively. These values were confirmed independently by using the thermodynamic linkage between the stability of the disulfide bond and the stability of the protein to denaturation. Comparison of calculated E° values from a number of proteins ranging from ؊270 mV for E. coli Trx to ؊124 mV for DsbA obtained using this method with those determined using glutathione redox buffers provides independent confirmation of the standard state redox potential of glutathione as ؊240 mV. Determining redox potentials through direct proteinprotein equilibria is of general interest as it overcomes errors in determining redox potentials calculated from large equilibrium constants with the strongly reducing NADPH or by accumulating mixed disulfides with GSH.Glutaredoxin (Grx1) 1 was discovered as a GSH-dependent hydrogen donor for ribonucleotide reductase in Escherichia coli mutants lacking the first identified electron donor, thioredoxin (1). The presence of an additional hydrogen donor system for ribonucleotide reductase was postulated since a double mutant lacking both Grx1 and Trx was viable (2). The search for this third hydrogen donor system resulted in the isolation of two additional glutaredoxins in E. coli, Grx2 and Grx3 (3). The recent structural characterization of Grx3 showed that in addition to the 33% amino acid sequence identity with Grx1, the two proteins have highly conserved secondary structure elements and overall fold (4). However, despite Grx1 and Grx3 being closely related 9-kDa redox proteins with identical active-site sequences (CPYC), Grx3 exhibits only a fraction of the activity of Grx1 as a reductant of ribonucleotide reductase (NrdAB and NrdEF; Refs. 3 and 5) or of insulin disulfides (4). ...

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