To clarify the role of metal ion coordination in horseradish peroxidase C (HRPC), the effect of pressure and of an externally applied electric field on spectral holes was compared for both metal-free and Mg-mesoporphyrin-substituted horseradish peroxidase C (MP-HRP and MgMP-HRP), as affected by the binding of 2-naphthohydroxamic acid (NHA). The data are compared to earlier studies performed on the same derivatives. Results obtained for MP-HRP show the presence of a predominant MP tautomer, as well as that of another small population with different pocket field and isothermal compressibility (0.12 vs 0.24 GPa(-1)). Binding NHA induces the formation of two new almost equal populations of MP-HRP tautomer complexes and the protein compressibility in both forms is increased to 0.50 and 0.36 GPa(-1). The protein structure becomes much softer than in the absence of NHA. Binding the same substrate to MgMP-HRP resulted in MgMP adopting a single conformation with no compressibility changes, while without NHA, two forms were possible. Stark effect results show charge rearrangement upon substrate binding in both cases. We propose that it is the presence of the metal that stabilizes the structure during the reorganization of the protein matrix induced by the substrate binding event. With the metal, only one conformation is adopted, without significant structural rearrangement but with charge redistribution. The dissociation constants determined for NHA binding to both derivatives and to native HRPC show that studies using mesoporphyrin and Mg-mesoporphyrin derivatives are relevant to investigating the specificity of the substrate-binding pocket in this enzyme.
The effect of trehalose on the interaction of human serum albumin (HSA) with neutral and negatively charged small unilamellar vesicles (SUVs) composed of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) or of mixtures of DMPC (19:1 w/w) with 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol (DMPG) was studied by time-resolved fluorescence and dynamic light scattering measurements. The results are interpreted with supporting nonbond calculations describing the nonbond domains most likely to be involved in the protein-SUV interaction. In the absence of trehalose, lifetime measurements of the single Trp of HSA are indicative of two different SUV-HSA associative mechanisms depending on the [lipid]/[HSA] concentration ratios. At low ratios, depletion of phospholipid molecules from vesicles by HSA occurs independently of the lipid composition of the vesicles via favorable hydrophobic contacts. At higher ratios, vesicle-HSA assocation is favored by electrostatic interactions for the negatively charged SUVs. For neutral SUVs, hydrophobically driven penetration of HSA is proposed. All association mechanisms are damped in the presence of trehalose, due to its capacity to coat the interacting surfaces. The results of dynamic light scattering experiments clearly show that the aging of the liposomes is dependent on the lipid composition. The aging of DMPC vesicles is faster and not affected by the presence of either HSA or trehalose. The aging of DMPC/DMPG liposomes is more pronounced in the presence of HSA. These SUVs are stabilized by trehalose through different mechanisms depending on whether they are covered by HSA or not.
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