A structural comparison between the Normal and the Expanded isomers of the human serum albumin has been carried out by using small angle X-ray scattering (SAXS) and light scattering (LS) techniques. Geometrical bodies, recovered structures (GA_STRUCT code) and rigid body modeling (CRYSOL and BUNCH software) were used to obtain low-resolution 3D structures from one-dimensional scattering patterns. These restored shapes were also exploited to perform a correlation between SAXS and LS data. By attempting a detailed description of globular and unfolded protein structures in solution, we tried to propose a suitable approach to follow the path of folding/unfolding processes and to isolate and characterize possible partially folded intermediate states.

We report a kinetic study of the supramolecular tubule formation of the bile salt derivative [3 beta, 5 beta, 7 alpha, 12 alpha]-3-(4-t-butylbenzoilamine)-7,12-dihydroxycholan-24-oic acid sodium salt (Na-tbutPhC). At high bicarbonate buffer concentration (pH similar to 10) this salt shows gelator properties. Starting from gels or viscous solutions, the tubule formation is triggered by increasing the temperature beyond the critical value of 34-36 degrees C. For gels, when the process takes place, the transition to sols occurs. The process is easily triggered and can be followed by several techniques. We used static light scattering (SLS), circular dichroism (CD), small angle X-ray scattering (SAXS) along with transmission electron (TEM) and optical microscopies. The CD results show that fibrils with a clockwise arrangement of the bile salt derivative are present in the samples at room temperature. When the tubule formation starts, evolutions of the CD and SLS profiles are observed indicating that the formation process begins with the aggregation of the fibrils accompanied by a simultaneous peculiar reciprocal reorientation of the surfactant molecules. After that, as pointed out by the long time evolution of the curves, a slow transformation towards the final well defined tubules occurs, involving an adjustment of the molecular packing. In the meanwhile, the slow ordering of the tubule walls in well spaced layers takes place, as inferred by SAXS. The TEM images show that short disordered tubules are formed, because of the aggregation of fibrils, in the beginning. Moreover they highlight a final elongation of the tubules taking place without a further aggregation of fibrils. Optical microscopy frames, collected during the process, point out that the tubules grow singly even at quite a high concentration, thus supporting the data interpretation

We report a study on the unfolding behavior of the most abundant protein contained in plasma, human serum albumin. The unfolding mechanisms in denaturing conditions induced by urea are studied for the defatted form (HSA) and for the palmitic acid:albumin (HSAPalm) complex. We employed the singular value decomposition 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-ray scattering patterns and are correlated with the parameters obtained from static and dynamic light scattering experiments. The unfolding process is pointed out by both ab initio and rigid body fitting methods that highlight a stepwise evolution of the protein structure toward open conformations. The superimpositions of the 3D structures provided independently by the two methods show very good agreements. The hydrodynamic radii estimated 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 suggest a multistep unfolding pathway. In particular, a scheme in which domains I and II are opened in sequence and the presence of two intermediates are evidenced is presented. The opening sequence is different from that found using guanidine hydrochloride as denaturant agent. The stabilizing role of the fatty acids in the urea denaturation process is evident. The palmitic acid ligand strongly stabilizes the protein, which remains in the native form up to high denaturant concentrations. In this case, the unfolding process is characterized by a single-step mechanism.

We report here the low-resolution structure of the complex formed by the endo-polygalacturonase from Fusarium phyllophilum and one of the polygalacturonase-inhibiting protein from Phaseolus vulgaris after chemical cross-linking as determined by small-angle x-ray scattering analysis. The inhibitor engages its concave surface of the leucine-rich repeat domain with the enzyme. Both sides of the enzyme active site cleft interact with the inhibitor, accounting for the competitive mechanism of inhibition observed. The structure is in agreement with previous site-directed mutagenesis data and has been further validated with structure-guided mutations and subsequent assay of the inhibitory activity. The structure of the complex may help the design of inhibitors with improved or new recognition capabilities to be used for crop protection.