Unfolding of Green Fluorescent Protein mut2 in wet nanoporous silica gels

Many of the effects exerted on protein structure, stability, and dynamics by molecular crowding and confinement in the cellular environment can be mimicked by encapsulation in polymeric matrices. We have compared the stability and unfolding kinetics of a highly fluorescent mutant of Green Fluorescent Protein, GFPmut2, in solution and in wet, nanoporous silica gels. In the absence of denaturant, encapsulation does not induce any observable change in the circular dichroism and fluorescence emission spectra of GFPmut2. In solution, the unfolding induced by guanidinium chloride is well described by a thermodynamic and kinetic two-state process. In the gel, biphasic unfolding kinetics reveal that at least two alternative conformations of the native protein are significantly populated. The relative rates for the unfolding of each conformer differ by almost two orders of magnitude. The slower rate, once extrapolated to native solvent conditions, superimposes to that of the single unfolding phase observed in solution. Differences in the dependence on denaturant concentration are consistent with restrictions opposed by the gel to possibly expanded transition states and to the conformational entropy of the denatured ensemble. The observed behavior highlights the significance of investigating protein function and stability in different environments to uncover structural and dynamic properties that can escape detection in dilute solution, but might be relevant for proteins in vivo.     

GFP-mut2 proteins in trehalose-water matrixes: spatially heterogeneous protein-water-sugar structures

We report investigations on the properties of nanoenvironments around single-GFP-mut2 proteins in trehalose-water matrixes. Single-GFPmut2 molecules embedded in thin trehalose-water films were characterized in terms of their fluorescence brightness, bleaching dynamics, excited state lifetime, and fluorescence polarization. For each property, sets of approximately 100-150 single molecules have been investigated as a function of trehalose content and hydration. Three distinct and interconverting families of proteins have been found which differ widely in terms of bleaching dynamics, brightness, and fluorescence polarization, whose relative populations sizably depend on sample hydration. The reported results evidence the simultaneous presence of different protein-trehalose-water nanostructures whose rigidity increases by lowering the sample hydration. Such spatial inhomogeneity is in line with the well-known heterogeneous dynamics in supercooled fluids and in nonsolid carbohydrate glasses and gives a pictorial representation of the sharp, sudden reorganization of the above structures after uptake <==>release of water molecules.     

Structural stability of green fluorescent proteins entrapped in polyelectrolyte nanocapsules

Molecules of a green fluorescent protein mutant, GFPmut2, have been immobilized in nanocapsules, assemblies of charged polyelectrolyte multilayers, with the aim to study the effect of protein‐polyelectrolyte interactions on the protein stability against chemical denaturation. GFPmut2 proteins turn out to be stabilized and protected against the denaturating action of small chemical compounds. The nanocapsule protective effect on GFPmut2 is likely due to protein interactions with the negatively charged polymers, that induce an increase in the local rigidity of the protein nano‐environment. This suggestion is supported by Fluorescence Polarization measurements on GFPmut2 proteins bound to the NC layers. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Structure and single crystal spectroscopy of Green Fluorescent Proteins

Usually, spectroscopic data on proteins in solution are interpreted at molecular level on the basis of the three-dimensional structures determined in the crystalline state. While it is widely recognized that the protein crystal structures are reliable models for the solution 3D structures, nevertheless it is also clear that sometimes the crystallization process can introduce some "artifacts" that can make difficult or even flaw the attempt to correlate the properties in solution with those in the crystalline state. In general, therefore, it would be desirable to identify some sort of control. In the case of the spectroscopic properties of proteins, the most straightforward check is to acquire data not only in solution but also on the crystals. In this regard, the Green Fluorescent Protein (GFP) is an interesting case in that a massive quantity of data correlating the spectroscopic properties in solution with the structural information in the crystalline state is available in literature. Despite that, a relatively limited amount of spectroscopic studies on single crystals of GFP or related FPs have been described. Here we review and discuss the main spectroscopic (in solution) and structural (in crystals) studies performed on the GFP and related fluorescent proteins, together with the spectroscopic analyses on various FPs members in the crystalline state. One main conclusion is that "in cristallo" spectroscopic studies are useful in providing new opportunities for gathering information not available in solution and are highly recommended to reliably correlate solution properties with structural features. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.     

The human bocavirus role in acute respiratory tract infections of pediatric patients as defined by viral load quantification

The major objective of this study was to investigate the pathogenic role of human bocavirus (hBoV) in patients hospitalized with acute respiratory tract infection (ARTI). Overall, 685 respiratory samples from 426 patients were examined by PCR for human bocavirus, as well as for other known human respiratory viruses. HBoV was quantified by PCR. Forty/283 (14.1%) pediatric patients, and 2/143 (1.4%) adult patients were found to harbor hBoV for a total of 45 episodes (16 detected as single infection, and 29 as coinfection) of hBoV-associated respiratory infection. HBoV DNA quantification revealed the presence of an NPA viral load > 1.0 x 10(5) DNA copies/ml in respiratory secretions from 17/40 (42.5%) children and 0/2 adults. Below this cut-off, hBoV appeared to be an innocent bystander or a persistent virus. Although hBoV may be frequently detected in children with upper or lower ARTI, in less than 50% young patients it appears to be potentially pathogenic.     

Surface-exposed tryptophan residues are essential for O-acetylserine sulfhydrylase structure,function, and stability

O-Acetylserine sulfhydrylase is a homodimeric enzyme catalyzing the last step of cysteine biosynthesis via a Bi Bi ping-pong mechanism. The subunit is composed of two domains, each containing one tryptophan residue, Trp50 in the N-terminal domain and Trp161 in the C-terminal domain. Only Trp161 is highly conserved in eucaryotes and bacteria. The coenzyme pyridoxal 5'-phosphate is bound in a cleft between the two domains. The enzyme undergoes an open to closed conformational transition upon substrate binding. The effect of single Trp to Tyr mutations on O-acetylserine sulfhydrylase structure, function, and stability was investigated with a variety of spectroscopic techniques. The mutations do not significantly alter the enzyme secondary structure but affect the catalysis, with a predominant influence on the second half reaction. The W50Y mutation strongly affects the unfolding pathway due to the destabilization of the intersubunit interface. The W161Y mutation, occurring in the C-terminal domain, produces a reduction of the accessibility of the active site to acrylamide and stabilizes thermodynamically the N-terminal domain, a result consistent with stronger interdomain interactions.     

On the myoepithelium of human salivary glands. An immunocytochemical study

This study investigated the immunocytochemical characteristics of normal myoepithelial cells (MECs) of human major and minor salivary glands using the LSAB method. Other human exocrine glands were used as controls. Immunoreactivity of MECs was observed exclusively with fully differentiated smooth muscle antibodies (a-SMA; SMMS-1; CALP; hCD) and with epithelial markers (cytokeratins) Ck14 and Ck17. This epithelial-muscular immunophenotype was similarly expressed in the MECs of other human exocrine glands used as control. In the salivary MECs, we did not observe evidence for neuroectodermic phenotype (S-100 protein, GFAP, NSE). On the contrary, positivity was observed for S-100 protein in Mecs of control glands (mammary, bronchial and sweat glands). Immunoreaction for extracellular matrix markers (fibronectin, laminin and collagen IV) and vimentin always were negative. Our data show that in normal "non transformed" MECs of the salivary glands the smooth muscle phenotype is in a state of complete differentiation, while the epithelial phenotype expresses only Ck14 and Ck17. This double and simultaneous immunoreactivity represents a differential marker of MECs from the epithelial basal cell (EBCs).