Native and Methylated Cyclodextrins with Positive and Negative Solubility Coefficients in Water Studied by SAXS and SANS

While the solubility of native alpha-, beta-, gamma-cyclodextrins (CDs) in water rises with temperature, the opposite is true for their methylated derivatives (mCDs; per-dimethylated beta-CD and per-trimethylated gamma-CD). The mCDs are well-soluble in cold water and crystallize upon heating, which we associate with the hydrophobic effect. To study the hydrophobic effect and hydration of CDs and mCDs dissolved in water (D 2O), we performed small-angle X-ray and neutron scattering (SAXS and SANS) measurements. The experimental scattering curves were put on absolute scale and compared to scattering curves calculated from crystal structures using the cube method. The results of the comparison indicate that (i) in solution, CDs and mCDs are in monomeric form, (ii) van der Waals and solute excluded volumes can be related by introducing a shell of a thickness that correlates with the solute's structure and solute-water interactions, and (iii) the SAXS curves calculated under the assumption of a uniform distribution of electron density in the solute molecules agree with experimental ones for CDs, but not for mCDs. The temperature and concentration dependence of SAXS curves is significant for mCDs and weak for CDs and is discussed in terms of solute-solute interactions. Specifically, these interactions become more attractive in solutions of mCDs with increasing temperature, concentration, or both, in accord with mCDs' negative temperature coefficient of solubility in water

Resonant X-Ray Scattering and Absorption for the Global and Local Structures of Cu-modified Metallothioneins in Solution

With Cd and Zn metal ions removed from the native rabbit-liver metallothionein upon unfolding, Cu-modified metallothioneins (Cu-MTs) were obtained during refolding in solutions containing CuI or CuII ions. X-ray absorption near-edge spectroscopic results confirm the respectively assigned oxidation states of the copper ions in CuI-MT and CuII-MT. Global and local structures of the Cu-MTs were subsequently characterized by anomalous small-angle x-ray scattering (ASAXS) and extended x-ray absorption fine structure. Energy-dependent ASAXS results indicate that the morphology of CuII-MT resembles that of the native MT, whereas CuI-MT forms oligomers with a higher copper content. Both dummy-residue simulation and model-shape fitting of the ASAXS data reveal consistently rodlike morphology for CuII-MT. Clearly identified Cu-S, Cu-O, and Cu-Cu contributions in the extended x-ray absorption fine structure analysis indicate that both CuI and CuII ions are bonded with O and S atoms of nearby amino acids in a four-coordination environment, forming metal clusters smaller than metal thiolate clusters in the native MT. It is demonstrated that a combination of resonant x-ray scattering and x-ray absorption can be particularly useful in revealing complementary global and local structures of metalloproteins due to the atom specific characteristics of the two techniques

Solubilization of Poly{1,4-phenylene-[9,9-bis(4-phenoxy-butylsulfonate)] fluorene-2,7-diyl} in Water by Nonionic Amphiphiles

In the presence of the nonionic alkyloxyethylene surfactant n-dodecylpentaoxyethylene glycol ether (C12E5), the anionic conjugated polyelectrolyte (CPE) poly{1,4-phenylene-[9,9-bis(4-phenoxy-butylsulfonate)]fluorene-2,7-diyl} (PBS-PFP) dissolves in water, leading to a blue shift in fluorescence and dramatic increases in fluorescence quantum yields above the surfactant critical micelle concentration (cmc). No significant changes were seen with a poly(ethylene oxide) of similar size to the surfactant headgroup, confirming that specific surfactant−polyelectrolyte interactions are important. From UV−visible and fluorescence spectroscopy, dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM), and electrical conductivity, together with our published NMR and small-angle neutron scattering (SANS) results, we provide a coherent model for this behavior in terms of breakup of PBS-PFP clusters through polymer−surfactant association leading to cylindrical aggregates containing isolated polymer chains. This is supported by molecular dynamics simulations, which indicate stable polymer−surfactant structures and also provide indications of the tendency of C12E5 to break up polymer clusters to form these mixed polymer−surfactant aggregates. Radial electron density profiles of the cylindrical cross section obtained from SAXS results reveal the internal structure of such inhomogeneous species. DLS and cryo-TEM results show that at higher surfactant concentrations the micelles start to grow, possibly partially due to formation of long, threadlike species. Other alkyloxyethylene surfactants, together with poly(propylene glycol) and hydrophobically modified poly(ethylene glycol), also solubilize this polymer in water, and it is suggested that this results from a balance between electrostatic (or ion-dipole), hydrophilic, and hydrophobic interactions. There is a small, but significant, dependence of the emission maximum on the local environment

Urea-Induced Denaturation Process on Defatted Human Serum Albumin and in the Presence of Palmitic Acid

We report a study on the unfolding behavior of the most abundant protein contained in plasma, human serumalbumin. The unfolding mechanisms in denaturing conditions induced by urea are studied for the defattedform (HSA) and for the palmitic acid:albumin (HSAPalm) complex. We employed the singular valuedecomposition method to determine the minimum number of structural states present in the unfolding processes.Low-resolution three-dimensional structures are reconstructed from the one-dimensional small-angle X-rayscattering patterns and are correlated with the parameters obtained from static and dynamic light scatteringexperiments. The unfolding process is pointed out by both ab initio and rigid body fitting methods that highlighta stepwise evolution of the protein structure toward open conformations. The superimpositions of the 3Dstructures provided independently by the two methods show very good agreements. The hydrodynamic radiiestimated for the protein best fitting conformations are in satisfactory agreement with the experimental ones.The results show that the HSA unfolding process is consistent with previous spectroscopic studies that suggesta multistep unfolding pathway. In particular, a scheme in which domains I and II are opened in sequence andthe presence of two intermediates are evidenced is presented. The opening sequence is different from thatfound using guanidine hydrochloride as denaturant agent. The stabilizing role of the fatty acids in the ureadenaturation process is evident. The palmitic acid ligand strongly stabilizes the protein, which remains in thenative form up to high denaturant concentrations. In this case, the unfolding process is characterized by asingle-step mechanism