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- Amino acid (1)
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We report surface plasmon resonance (spr) and confocal fluorescence results concerning the interaction of a gene-5-protein (g5p) with single-stranded DNA oligonucleotides (dT25) tethered to a gold surface. The spr data show that a highly stable g5pssDNA complex is readily formed on a gold surface with koff = 1.7 × 10-3 s-1. The extent of the complexion indicated involvement of the cooperative proteinprotein interactions within the binding to DNA. In the experiments where dT25 coexist with g5p in the solution, the fluorescence data show that g5p also mediates the binding between the non-complementary oligonucleotides in the solution and those immobilised on the surface.
Microorganisms accumulate molar concentrations of compatible solutes like ectoine to prevent proteins from denaturation. Direct structural or spectroscopic information on the mechanism and about the hydration shell around ectoine are scarce. We combined surface plasmon resonance (SPR), confocal Raman spectroscopy, molecular dynamics simulations, and density functional theory (DFT) calculations to study the local hydration shell around ectoine and its influence on the binding of a gene-S-protein (G5P) to a single-stranded DNA (dT(25)). Due to the very high hygroscopicity of ectoine, it was possible to analyze the highly stable hydration shell by confocal Raman spectroscopy. Corresponding molecular dynamics simulation results revealed a significant change of the water dielectric constant in the presence of a high molar ectoine concentration as compared to pure water. The SPR data showed that the amount of protein bound to DNA decreases in the presence of ectoine, and hence, the protein-DNA dissociation constant increases in a concentration-dependent manner. Concomitantly, the Raman spectra in terms of the amide I region revealed large changes in the protein secondary structure. Our results indicate that ectoine strongly affects the molecular recognition between the protein and the oligonudeotide, which has important consequences for osmotic regulation mechanisms.