The S-layer of Bacillus sphaericus strain JG-A12, isolated from a uranium-mining site, exhibits a high metal-binding capacity, indicating that it may provide a protective function by preventing the cellular uptake of heavy metals and radionuclides. This property has allowed the use of this and other S-layers as self-assembling organic templates for the synthesis of nanosized heavy metal cluster arrays. However, little is known about the molecular basis of the metal-protein interactions and their impact on secondary structure. We have studied the secondary structure, protein stability, and Pd((II)) coordination in S-layers from the B. sphaericus strains JG-A12 and NCTC 9602 to elucidate the molecular basis of their biological function and of the metal nanocluster growth. Fourier transform infrared spectroscopy reveals similar secondary structures, containing approximately 35% beta-sheets and little helical structure. pH-induced infrared absorption changes of the side-chain carboxylates evidence a remarkably low pK < 3 in both strains and a structural stabilization when Pd((II)) is bound. The COO(-)-stretching absorptions reveal a predominant Pd((II)) coordination by chelation/bridging by Asp and Glu residues. This agrees with XANES and EXAFS data revealing oxygens as coordinating atoms to Pd((II)). The additional participation of nitrogen is assigned to side chains rather than to the peptide backbone. The topology of nitrogen- and carboxyl-bearing side chains appears to mediate heavy metal binding to the large number of Asp and Glu in both S-layers at particularly low pH as an adaptation to the environment from which the strain JG-A12 has been isolated. These side chains are thus prime targets for the design of engineered S-layer-based nanoclusters.

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The conformational substates B(I) and B(II) of the phosphodiester backbone in B-DNA are thought to contribute to DNA flexibility and protein recognition. We have studied by rapid scan FTIR spectroscopy the isothermal B(I)-B(II) transition on its intrinsic time scale. Correlation analysis of IR absorption changes occurring within seconds after a reversible incremental growth of the DNA hydration shell identifies water populations w(1) (PO(2)(-)-bound) and w(2) (non-PO(2)(-)-bound) exhibiting weaker and stronger H-bonds, respectively, than those dominating in bulk water. The B(II) substate is stabilized by w(2). The water H-bond imbalance of 3-4 kJ mol(-1) is equalized at little enthalpic cost upon formation of a contiguous water network (at 12-14 H(2)O molecules per DNA phosphate) of reduced ν(OH) bandwidth. In this state, hydration water cooperatively stabilizes the B(I) conformer via the entropically favored replacement of w(2)-DNA interactions by additional w(2)-water contacts, rather than binding to B(I)-specific hydration sites. Such water rearrangements contribute to the recognition of DNA by indolicidin, an antimicrobial 13-mer peptide from bovine neutrophils which, despite little intrinsic structure, preferentially binds to the B(I) conformer in a water-mediated induced fit. The FTIR spectra resolve sequential steps leading from PO(2)(-)-solvation to substate transition and eventually to base stacking changes in the complex. In combination with CD-spectral titrations, the data indicate that, in the absence of a bulk aqueous phase, as in molecular crowded environments, water relocation within the DNA hydration shell allows for entropic contributions similar to those assigned to water upon DNA ligand recognition in solution.

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