Streptavidin reduces oxygen quenching of biotinylated ruthenium(II) and palladium(II) complexes.

Transition metal complexes such as biotinylated ruthenium(II) tris(bipyridyl) and palladium(II) porphyrin show an increase in luminescence intensity and lifetime upon binding to streptavidin in aqueous solution. Here we show that this increase of luminescence lifetime and intensity are caused by the shielding of the transition metal complexes from dissolved oxygen through streptavidin rather than by hydrophobicity effects as recently claimed.

Shaping emission spectra of fluorescent molecules with single plasmonic nanoresonators.

We show that plasmonic nanoresonators composed of two gold nanoparticles change not only the intensity but also the spectral shape of the emission of fluorescent molecules. The plasmonic resonance frequency can be tuned by varying the distance between the nanoparticles, which allows us to selectively favor transitions of a fluorescent molecule to a specific vibrational ground state. Experimental data from correlated scattering and fluorescence microscopy agree well with calculations in the framework of generalized Mie theory. Our results show that the widely used description of a dye molecule near a metal surface as a mere two-level system is inadequate.

Moving nanoparticles with Raman scattering.

We show how to change optically the distance between two protein-linked gold nanoparticles by Raman-induced motion of the linker protein. Rayleigh scattering spectroscopy of the coupled-particle plasmon allows us to compare the inter-nanoparticle distance of individual protein-linked gold nanoparticle dimers before and after surface-enhanced Raman scattering (SERS). We find that low-intensity (50 microW/microm2) laser light in resonance with the nanoparticle-dimer plasmon provokes a change of the inter-nanoparticle distance on the order of 0.5 nm whenever SERS from the proteins connecting the nanoparticles can be observed.

Electron density imaging of protein films on gold-particle surfaces with transmission electron microscopy.

BACKGROUND: Surface bound proteins on colloid particles are widely used in biotechnological applications such as diagnostics or separation. Analysis of colloid surfaces by imaging methods provides information on the structure of these protein films, and an understanding of the functional relationships of biomolecules immobilised on solid surfaces. METHODS: In order to visualise protein molecules organised in films on surfaces of nano-sized gold-particles, an electron-microscopic approach based on the scattering absorption contrast of the specimen was applied. RESULTS: Analysing protein conjugated gold particles with a transmission electron microscope, protein films on gold particle surfaces cause a significant scattering absorption contrast based on the materials' electron density. Thus, the thickness of such films becomes directly measurable in planar projection and the shape of these films are visualised without negative staining methods. The insertion of Ruthenium-labelled antibodies instead of non-labelled antibodies as a marker with increased electron-density in these films yields a contrast enhancement of the whole film. Additional labelling with anti-Mouse IgG Gold conjugates localises the position of the surface bound antibodies in such protein films. CONCLUSIONS: The power of transmission electron microscopy to resolve protein-films on colloid surfaces without staining or labelling as a sample preparation procedure has been demonstrated. Thus, this direct method provides an analytical tool for studying protein films and their structural features on particle surfaces.

Folding and association of beta-Galactosidase.

beta-D-Galactosidase from Escherichia coli is one of the largest tetrameric enzymes known at present. Although its physiological importance, the regulation of its synthesis, its enzymatic properties and its structure are well established, little is known about the stability and the folding pathway of this enzyme. Here we show that the overall folding mechanism of chemically denatured beta-galactosidase consists of three stages: (i) formation of elements of secondary structure; (ii) collapse to subdomains and structured monomers; (iii) association to the native quaternary structure via dimeric intermediates. The first rate-limiting step is the association of structured monomers to form dimers in a bi-molecular reaction, with a rate constant of 4.3x10(3) M-1 s-1 at 20 degreesC. The second rate-limiting uni-molecular folding step leads to dimers which are competent for further association, with a rate constant of 0.5x10(-3) s-1 at 20 degreesC. Tetramers form from these dimers in a fast reaction. By determining a similar mechanism for alpha-complementation of beta-galactosidase fragments it could be confirmed that beta-galactosidase follows a consecutive bi-uni-molecular mechanism of folding and association.

Stabilization of proteins and peptides in diagnostic immunological assays by the molecular chaperone Hsp25.

Diagnostic assays for proteins devoid of enzymatic activity are becoming increasingly important. Antibodies generated against the respective proteins are used for their detection in enzyme-linked immunosorbent assay or patient sera are used to monitor disease-related antibodies against recombinantly produced antigens. A problem frequently encountered with these assays is that the proteins or fragments thereof used as standards have a limited shelf life. A similar problem arises when activities of labile enzymes are used for diagnostic detection. Here, we present a novel approach to 'stabilizing' enzymatic activity and antigenicity of proteins used for immunogenic detection by molecular chaperones. We have exploited the ability of molecular chaperones to keep proteins in their active conformation to overcome the biotechnological problems encountered in protein-based diagnostics of heart attack, stroke, and viral infections such as hepatitis C. We show that Hsp25, a member of the family of small heat shock proteins, known to act as a molecular chaperone in protein folding reactions, can stably bind labile standard proteins. Complex formation does not interfere with immunogenic detection and, importantly, antigenic as well as enzymatic activity remains constant for weeks. This strategy seems to be applicable to a wide range of assays involving unstable proteins, including the generation of vaccines.