No abstract

The effect of biotin binding on streptavidin (STV) structure and stability was studied using differential scanning calorimetry, Fourier transform infrared spectroscopy (FT-IR), and fluorescence spectroscopy. Biotin increases the midpoint temperature T m , of thermally induced denaturation of STV from 75°C in unliganded protein to 112°C at full ligand saturation. The cooperativity of thermally induced unfolding of STV changes substantially in presence of biotin. Unliganded STV monomer has at least one domain that unfolds independently. The dimer bound to biotin undergoes a single coupled denaturation process. Simulations of thermograms of STV denaturation that take into account only the thermodynamic effects of the ligand with a K a ϳ10 15 reproduce the behavior observed, but the estimated values of T m are 15-20°C lower than those experimentally determined. This increased stability is attributed to an enhanced cooperativity of the thermal unfolding of STV. The increment in the cooperativity is as consequence of a stronger intersubunit association and an increased structural order upon binding. FT-IR and fluorescence spectroscopy data reveal that unordered structure found in unliganded STV disappears under fully saturating conditions. The data provide a rationale for previous suggestions that biotin binding induces an increase in protein tightness (structural cooperativity) leading, in turn, to a higher thermostability.The biological function of many proteins is triggered and modulated by the binding of ligands. For this reason, an understanding of the mechanism of protein-ligand interactions is essential for a detailed knowledge of protein function at the molecular level. Ligand binding, in most cases, involves the formation of noncovalent bonds at specific interacting surfaces between the protein and the ligand. The binding of a ligand can be accompanied by conformational changes at the protein site that sometimes are propagated throughout the entire protein.It is desirable to have a way to monitor these structural changes to understand any new properties acquired by the complex. The high affinity of the biotin-streptavidin binding not only offers useful bioanalytical advantages (1), but it also makes this system an attractive model for studying proteinligand interactions (2-5). The biotin⅐STV 1 association constant of about 10 15 is the highest known in biochemistry. In the present work we explore protein thermostability by heating STV in the absence of ligand or under conditions of partial or full ligand saturation. A dramatic increase in the T m of protein denaturation, from 75°C in absence of biotin to 112°C at full ligand saturation, was revealed using differential scanning calorimetry (DSC). An analysis of the cooperativity of the denaturation was made on the basis of a reversible nontwo-state model of protein unfolding to gain understanding of the system that unfolds differently if biotin is present.Conformational changes were characterized by FT-IR and fluorescence spectroscopy. The large changes in the...

Survival of Pseudomonas aeruginosa in cystic fibrosis (CF) chronic infections is based on a genetic adaptation process consisting of mutations in specific genes, which can produce advantageous phenotypic switches and ensure its persistence in the lung. Among these, mutations inactivating the regulators MucA (alginate biosynthesis), LasR (quorum sensing) and MexZ (multidrug-efflux pump MexXY) are the most frequently observed, with those inactivating the DNA mismatch repair system (MRS) being also highly prevalent in P. aeruginosa CF isolates, leading to hypermutator phenotypes that could contribute to this adaptive mutagenesis by virtue of an increased mutation rate. Here, we characterized the mutations found in the mucA, lasR, mexZ and MRS genes in P. aeruginosa isolates obtained from Argentinean CF patients, and analyzed the potential association of mucA, lasR and mexZ mutagenesis with MRS-deficiency and antibiotic resistance. Thus, 38 isolates from 26 chronically infected CF patients were characterized for their phenotypic traits, PFGE genotypic patterns, mutations in the mucA, lasR, mexZ, mutS and mutL gene coding sequences and antibiotic resistance profiles. The most frequently mutated gene was mexZ (79%), followed by mucA (63%) and lasR (39%) as well as a high prevalence (42%) of hypermutators being observed due to loss-of-function mutations in mutL (60%) followed by mutS (40%). Interestingly, mutational spectra were particular to each gene, suggesting that several mechanisms are responsible for mutations during chronic infection. However, no link could be established between hypermutability and mutagenesis in mucA, lasR and mexZ, indicating that MRS-deficiency was not involved in the acquisition of these mutations. Finally, although inactivation of mucA, lasR and mexZ has been previously shown to confer resistance/tolerance to antibiotics, only mutations in MRS genes could be related to an antibiotic resistance increase. These results help to unravel the mutational dynamics that lead to the adaptation of P. aeruginosa to the CF lung.