Observing proteins as single molecules encapsulated in surface-tethered polymeric nanocontainers.

Immobilizing biomolecules provides the advantage of observing them individually for extended time periods, which is impossible to accomplish for freely diffusing molecules in solution. In order to immobilize individual protein molecules, we encapsulated them in polymeric vesicles made of amphiphilic triblock copolymers and tethered the vesicles to a cover slide surface. A major goal of this study is to investigate polymeric vesicles with respect to their suitability for protein-folding studies. The fact that polymeric vesicles possess an extreme stability under various chemical conditions is supported by our observation that harsh unfolding conditions do not perturb the structural integrity of the vesicles. Moreover, polymerosomes prove to be permeable to GdnHCl and, thereby, ideally suited for unfolding and refolding studies with encapsulated proteins. We demonstrate this with encapsulated phosphoglycerate kinase, which was fluorescently labeled with Atto655, a dye that exhibits pronounced photoinduced electron transfer (PET) to a nearby tryptophan residue in the native state. Under unfolding conditions, PET was reduced, and we monitored alternating unfolding and refolding conditions for individual encapsulated proteins.

Peptide nanoparticles serve as a powerful platform for the immunogenic display of poorly antigenic actin determinants.

The role of actin in transcription and RNA processing is now widely accepted but the form of nuclear actin remains enigmatic. Monomeric, oligomeric or polymeric forms of actin seem to be involved in nuclear functions. Moreover, uncommon forms of actin such as the "lower dimer" have been observed in vitro. Antibodies have been pivotal in revealing the presence and distribution of different forms of actin in different cellular locations. Because of its high degree of conservation, actin is a poor immunogen and only few specific actin antibodies are available. To unravel the mystery of less common forms of actin, in particular those in the nucleus, we chose to tailor monoclonal antibodies to recognize distinct forms of actin. To increase the immune response, we used a new approach based on peptide nanoparticles, which are designed to mimic an icosahedral virus capsid and allow the repetitive, ordered display of a specific epitope on their surface. Actin sequences representing the highly conserved "hydrophobic loop," which is buried in the filamentous actin filament, were grafted onto the surface of nanoparticles by genetic engineering. After immunization with "loop nanoparticles," a number of monoclonal antibodies were established that bind to the hydrophobic loop both in vitro and in situ. Immunofluorescence studies on cells revealed that filamentous actin filaments were only labeled once the epitope had been exposed. Our studies indicate that self-assembling peptide nanoparticles represent a versatile platform that can easily be customized to present antigenic determinants in repetitive, ordered arrays and elicit an immune response against poor antigens.

Nucleo-copolymers: oligonucleotide-based amphiphilic diblock copolymers.

For the first time, poly(butadiene) has been covalently linked to an oligonucleotide sequence and the resulting nucleo-copolymer exhibits amphiphilic properties in dilute aqueous solution, self-assembling into nanometer-sized vesicular structures.

Dynamic disorder in horseradish peroxidase observed with total internal reflection fluorescence correlation spectroscopy.

This paper discusses the application of objective-type total internal reflection fluorescence correlation spectroscopy (TIR-FCS) to the study of the kinetics of immobilized horseradish peroxidase on a single molecule level. Objective-type TIR-FCS combines the advantages of FCS with TIRF microscopy in a way that allows for simultaneous ultra-sensitive spectroscopic measurements using a single-point detector and convenient localization of single molecules on a surface by means of parallel imaging.

Stochastic approach to data analysis in fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) has emerged as a powerful technique for measuring low concentrations of fluorescent molecules and their diffusion constants. In FCS, the experimental data is conventionally fit using standard local search techniques, for example, the Marquardt-Levenberg (ML) algorithm. A prerequisite for these categories of algorithms is the sound knowledge of the behavior of fit parameters and in most cases good initial guesses for accurate fitting, otherwise leading to fitting artifacts. For known fit models and with user experience about the behavior of fit parameters, these local search algorithms work extremely well. However, for heterogeneous systems or where automated data analysis is a prerequisite, there is a need to apply a procedure, which treats FCS data fitting as a black box and generates reliable fit parameters with accuracy for the chosen model in hand. We present a computational approach to analyze FCS data by means of a stochastic algorithm for global search called PGSL, an acronym for Probabilistic Global Search Lausanne. This algorithm does not require any initial guesses and does the fitting in terms of searching for solutions by global sampling. It is flexible as well as computationally faster at the same time for multiparameter evaluations. We present the performance study of PGSL for two-component with triplet fits. The statistical study and the goodness of fit criterion for PGSL are also presented. The robustness of PGSL on noisy experimental data for parameter estimation is also verified. We further extend the scope of PGSL by a hybrid analysis wherein the output of PGSL is fed as initial guesses to ML. Reliability studies show that PGSL and the hybrid combination of both perform better than ML for various thresholds of the mean-squared error (MSE).

Prism-based multicolor fluorescence correlation spectrometer.

We report the design and application of a prism-based detection system for fluorescence (cross) correlation spectroscopy. The system utilizes a single laser wavelength for the simultaneous excitation of several dyes of different emission spectra. Fluorescence light is spectrally separated with a prismatic setup, and wavelengths are selected by scanning a fiber-coupled avalanche photodiode across the image spots. Multicolor autocorrelations are demonstrated with standard and tandem dyes, and fluorescence cross-correlation measurements of biotinylated nanocontainers and streptavidin are presented. This spectrometer offers high optical stability and no focal volume mismatch for the multicolor detection of molecular dynamics and interactions, with single-molecule sensitivity.

Encapsulation of fluorescent molecules by functionalized polymeric nanocontainers: investigation by confocal fluorescence imaging and fluorescence correlation spectroscopy.

Nanocontainers (NCs) were prepared from amphiphilic triblock copolymers, having an average molecular weight of around 8000 g/mol, by using previously published preparation methods consisting of dispersing the polymer in an aqueous buffer solution containing molecules for encapsulation. A small molecular weight fluorophore, sulforhodamine B, as well as the fluorescent protein avidin labeled with Alexa 488 were encapsulated, and the resulting nanocontainers were characterized using fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS). Nanocontainer size determination by FCS is very robust and compares well with results obtained from photon correlation spectroscopy: the measured diameters of the polymeric nanocontainers vary between 140 and 172 nm. Encapsulation of fluorescent molecules was determined by evaluating the molecular brightness of nanocontainers with an encapsulated fluorescently labeled protein (avidin-Alexa 488). Results indicate that the number of encapsulated avidin-Alexa 488 molecules corresponds well with the initial concentration of the fluorescently labeled protein and the encapsulated volume. A nanocontainer binding assay was developed using biotinylated fluorescently labeled nanocontainers. Binding of biotinylated nanocontainers to fluorescently labeled streptavidin was followed by fluorescence cross-correlation spectroscopy. The intrinsic dissociation constant, K(d), of labeled streptavidin to the ligand-modified nanocontainers is 1.7 +/- 0.4 x 10(-8) M, and about 1921 +/- 357 molecules of labeled streptavidin are bound to each nanocontainer.

Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low background and high count-rate per molecule.

We designed a fluorescence correlation spectroscopy (FCS) system for measurements on surfaces. The system consists of an objective-type total internal reflection fluorescence (TIRF) microscopy setup, adapted to measure FCS. Here, the fluorescence exciting evanescent wave is generated by epi-illumination through the periphery of a high NA oil-immersion objective. The main advantages with respect to conventional FCS systems are an improvement in terms of counts per molecule (cpm) and a high signal to background ratio. This is demonstrated by investigating diffusion as well as binding and release of single molecules on a glass surface. Furthermore, the size and shape of the molecule detection efficiency (MDE) function was calculated, using a wave-vectorial approach and taking into account the influence of the dielectric interface on the emission properties of fluorophores.

Controlled immobilization of membrane proteins to surfaces for fourier transform infrared investigations.

We show that it is possible to immobilize membrane proteins uniformly and reversibly as self-assembled (sub)monolayers on nitrilotriacetic acid-covered sensor surfaces via hexahistidine sequences present either in the protein or in lipid membranes. Fourier transform infrared spectra of such self-assembled (sub)monolayers deliver important structural information of the membrane proteins and are suited to screen the function of cellular receptors.

Downscaling Fourier transform infrared spectroscopy to the micrometer and nanogram scale: secondary structure of serotonin and acetylcholine receptors.

High signal-to-noise Fourier transform infrared (FTIR) spectra of the 5-hydroxytryptamine (serotonin) receptor (5-HT(3)R) and the nicotinic acetylcholine receptor (nAChR) were obtained by microscope FTIR spectroscopy using micrometer-sized, fully hydrated protein films. Because this novel procedure requires only nanogram quantities of membrane proteins, which is 4-5 orders of magnitude less than the amount of protein typically used for conventional FTIR spectroscopy, it opens the possibility to access the structure and dynamics of many important mammalian receptor proteins. The secondary structure of detergent-solubilized 5-HT(3)R determined by curve fitting of the amide I band yielded 36% alpha-helix, 33% beta-strand, 15% beta-turn, and 16% nonregular structures, which remained unchanged upon reconstitution in lipid membranes. From hydrogen-deuterium exchange, the secondary structure of the water-accessible part of 5-HT(3)R was determined as 14% alpha-helix, 16% beta-strand, 26% beta-turn, and 14% nonregular structures. Interestingly, we found that both the overall and the water-accessible nAChR secondary structures were nearly identical to those of 5-HT(3)R, in agreement with predicted structures of this class of receptors. This is the first time that structural investigations were obtained for two closely related ligand-gated ion channels under strictly identical experimental conditions.

Reversible immobilization of peptides: surface modification and in situ detection by attenuated total reflection FTIR spectroscopy.

A generic method is described for the reversible immobilization of polyhistidine-bearing polypeptides and proteins on attenuated total reflecting (ATR) sensor surfaces for the detection of biomolecular interactions by FTIR spectroscopy. Nitrilotriacetic acid (NTA) groups are covalently attached to self-assembled monolayers of either thioalkanes on gold films or mercaptosilanes on silicon dioxide films deposited on germanium internal reflection elements. Complex formation between Ni2+ ions and NTA groups activates the ATR sensor surface for the selective binding of polyhistidine sequences. This approach not only allows a stable and reversible immobilization of histidine-tagged peptides (His-peptides) but also simultaneously allows the direct in situ quantification of surface-adsorbed molecules from their specific FTIR spectral bands. The surface concentrations of both NTA and His-peptide on silanized surfaces were determined to be 1.1 and 0.4 molecules nm-2, respectively, which means that the surface is densely covered. A comparison of experimental FTIR spectra with simulated spectra reveals a surface-enhancement effect of one order of magnitude for the gold surfaces. With the presented sensor surfaces, new ways are opened up to investigate, in situ and with high sensitivity and reproducibility, protein-ligand, protein-protein, protein-DNA interactions, and DNA hybridization by ATR-FTIR spectroscopy.