4. The holoazurin Trp 'Lb absorption band red shifts ca. 1 nm compared to apoazurin, while the 'Bb transition red shifts ca. 2 nm. The hydrophobicity of the Trp environment appears little changed between Cu(I) azurin, Cu(II) azurin, and apoazurin as evidenced by the narrowness of the Trp 'Lb 0-0 bands and from the Raman band shape for the Trp 1354-cm'1 band. The Trp 0-0 'Li, absorption spectral shifts most likely derive from subtle environmental changes, possibly alterations at sites somewhat distant from the Trp ring, such as alterations in electrostatic interaction from the Cu site.5. The absorption difference spectra (oxidized minus reduced as well as holoazurin minus apoazurin) show large changes in the 200-300-nm spectral region which presumably derive from alterations in sulfur and imidazole -* Cu(II) charge-transfer transitions. Numerous charge-transfer transitions can occur since the Cu is ligated to two histidines, one cysteine, and one methionine; simple Cu(II)-(imidazole)4 complexes show increased absorbances throughout the 200-300-nm spectral region (Figure 6).We thus conclude that facile energy transfer between the Trp and the Cu-ligand complex is responsible for the fluorescence quenching in Trp. Presumably this quenching occurs between the Trp 'Laib states and a Cu-ligand charge-transfer state. In addition, the Trp absorption spectral shifts between the Cu(I) azurin, Cu(II) azurin, and apoazurin suggest intimate coupling between the electronic transitions of the Cu-ligand complex and those of Trp. It is possible that strong excitonic interactions are present.

1D and 2D NMR spectroscopy is used to determine the helical stability of two Aib-rich peptides, iBoc-(Aib)3-DkNap-Leu-Aib-Ala-(Aib)2-NH(CH2)2OCH3 (Dk4[7/9]) and Ac-(Aib)2-beta-(1'-naphthyl)Ala-(Aib)2-Phe-(Aib)2-NHMe (Nap3Phe6[6/8]), where the bracket notation indicates the number of Aib-class residues/total number of residues. 2D ROESY experiments, carried out previously on Nap3Phe6[6/8] in DMSO (Basu & Kuki, 1993), showed that this compound adopts the 3(10)-helical conformation at 20 degrees C. The first step in the present work is to apply this technique to the peptide Dk4[7/9], demonstrating that it likewise adopts the 3(10)-helical conformation in chloroform at 20 degrees C. The amide proton shifts of Nap3-Phe6[6/8] in DMSO and Dk4[7/9] in C2D2Cl4 were then monitored by means of 1D NMR over a large temperature range, up to 150 and 120 degrees C, respectively. The nonamer Dk4[7/9] exhibits no evidence of any conformational or unfolding transition as the temperature is raised. The nearly temperature independent amide proton chemical shifts of this nonamer are an indication of retention of the intrahelical hydrogen bonding, which was then verified directly by solvent perturbation with DMSO at 120 degrees C. The resulting hydrogen-bonding pattern confirms that Dk4[7/9] retains its 3(10)-helical conformation in C2D2Cl4 over the entire temperature range. This conformational quietness is exploited to examine the intrinsic temperature dependence of free versus intrahelically hydrogen bonded amide proton shifts within the same peptide structure. It is also shown that Nap3Phe6[6/8] retains its 3(10)-helical conformation over the entire temperature range in the stronger hydrogen-bonding solvent DMSO. The extreme thermal stability of these octameric and nonameric Aib-rich peptides in both solvents is contrasted with that of much longer alanine-rich peptides in water.

Model calculations have been carried out to characterize the detailed nature of the benzenewater intermolecular interaction potential energy surface. At equilibrium, the calculations show a binding energy of 1303 an-'. The optimum water molecule's position is above the plane of the benzene ring and shifted from benzene's symmetry axis by 0.82 A. Of course, the equilibrium position alone does not satisfactorily characterize the benzene-water interactions because of the extreme floppiness of the complex. The calculations indicate that even at the zero-point level, the water molecule is capable of two very-low-energy routes interconverting between indistinguishable structures which involve water motions over large fractions of the benzene ring. One is the nearly free internal rotation of the benzene ring. The other is a combined translation and torsion of the water molecule where the hydrogens of the water molecule are exchanged, a process that is really a swapping or interchange of hydrogen bonds. The reason for its remarkably-low-energy barrier is discussed along with the connection between the surface features and experimental observations.

No abstract

No abstract