Imaging proteins at the single-molecule level.

Imaging single proteins has been a long-standing ambition for advancing various fields in natural science, as for instance structural biology, biophysics, and molecular nanotechnology. In particular, revealing the distinct conformations of an individual protein is of utmost importance. Here, we show the imaging of individual proteins and protein complexes by low-energy electron holography. Samples of individual proteins and protein complexes on ultraclean freestanding graphene were prepared by soft-landing electrospray ion beam deposition, which allows chemical- and conformational-specific selection and gentle deposition. Low-energy electrons do not induce radiation damage, which enables acquiring subnanometer resolution images of individual proteins (cytochrome C and BSA) as well as of protein complexes (hemoglobin), which are not the result of an averaging process.

Tumor pH-Responsive Albumin/Polyaniline Assemblies for Amplified Photoacoustic Imaging and Augmented Photothermal Therapy.

Tumor-microenvironment-responsive theranostics have great potential for precision diagnosis and effective treatment of cancer. Polyaniline (PANI) is the first reported pH-responsive organic photothermal agent and is widely used as a theranostic agent. However, tumor pH-responsive PANI-based theranostic agents are not explored, mainly because the conversion from the emeraldine base (EB) to emeraldine salt (ES) state of PANI requires pH < 4, which is lower than tumor acidic microenvironment. Herein, a tumor pH-responsive PANI-based theranostic agent is designed and prepared for amplified photoacoustic imaging guided augmented photothermal therapy (PTT), through intermolecular acid-base reactions between carboxyl groups of bovine serum albumin (BSA) and imine moieties of PANI. The albumin/PANI assemblies (BSA-PANI) can convert from the EB to ES state at pH < 7, accompanied by the absorbance redshift from visible to near-infrared region. Both in vitro and in vivo results demonstrate that tumor acidic microenvironment can trigger both the photoacoustic imaging (PAI) signal amplification and the PTT efficacy enhancement of BSA-PANI assemblies. This work not only highlights that BSA-PANI assemblies overcome the limitation of low-pH protonation, but also provides a facile assembly strategy for a tumor pH-responsive PANI-based nanoplatform for cancer theranostics.

Development and In Vitro Release of Isoniazid and Rifampicin-Loaded Bovine Serum Albumin Nanoparticles.

BACKGROUND Bovine serum albumin nanoparticles loaded with isoniazid and rifampicin (INH-RFP-BSA-NPs) were prepared and their release characteristics were studied in vitro. MATERIAL AND METHODS The INH-RFP-BSA-NPs were prepared by a modified self-emulsion solvent diffusion method, with albumin and polylactic acid used as carriers and to form the nanoparticles structure. Transmission electron microscopy was used to observe the morphology of the INH-RFP-BSA-NPs. The size distribution of the INH-RFP-BSA-NPs were assessed using a submicron particle-size analyzer for drug loadings, and the coating rate of the INH-RFP-BSA-NPs was measured by high-performance liquid chromatography. A dynamic membrane dialysis method was used to study the in vitro release characteristics of the INH-RFP-BSA-NPs. RESULTS The INH-RFP-BSA-NPs were smooth, sphere-like, relatively uniform in size, and well-dispersed, and the average diameter was 60.5±4.6 nm. Drug loading and entrapment efficiencies were high, at 19.8% and 87.8% for isoniazid, respectively, and 20.1% and 98.0% for rifampicin, respectively. Drug release was slow and sustained with 97.02% INH cumulative release at 6 days, and full release of RFP requiring 5 days. CONCLUSIONS INH-RFP-BSA-NPs exhibit uniform NP diameter, good dispersion, high drug loading and encapsulation rates, and have sustained release properties.

Digestibility of Bovine Serum Albumin and Peptidomics of the Digests: Effect of Glycation Derived from α-Dicarbonyl Compounds.

α-Dicarbonyl compounds, which are widely generated during sugar fragmentation and oil oxidation, are important precursors of advanced glycation end products (AGEs). In this study, the effect of glycation derived from glyoxal (GO), methylglyoxal (MGO) and diacetyl (DA) on the in vitro digestibility of bovine serum albumin (BSA) was investigated. Glycation from α-dicarbonyl compounds reduced digestibility of BSA in both gastric and intestinal stage of digestion according to measurement of degree of hydrolysis. Changes in peptide composition of digests induced by glycation were displayed, showing absence of peptides, occurrence of new peptides and formation of peptide-AGEs, based on the results obtained using liquid chromatography electron-spray-ionization tandem mass spectrometry (LC-ESI-MS/MS). Crosslinked glycation structures derived from DA largely reduced the sensitivity of glycated BSA towards digestive proteases based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results. Network structures were found to remain in the digests of glycated samples by transmission electron microscope (TEM), thus the impact of AGEs in unabsorbed digests on the gut flora should be an interest for further studies.

Conformational analysis of proteins with a dual polarisation silicon microring.

Optical microresonator biosensors have proven to be a valid tool to perform affinity analysis of a biological binding event. However, when these microresonators are excited with a single optical mode they can not distinguish between a thin dense layer of biomolecules or a thick sparse layer. This means the sensor is "blind" to changes in shape of bound biomolecules. We succeeded in exciting a Silicon-on-Insulator (SOI) microring with TE and TM polarisations simultaneously by using an asymmetrical directional coupler and as such were able to separately determine the thickness and the density (or refractive index) of a bound biolayer. A proof-of-concept is given by determining both parameters of deposited dielectric layers and by analysing the conformational changes of Bovine Serum Albumin (BSA) proteins due to a change in pH of the buffer.

Morphological effect of gold nanoparticles on the adsorption of bovine serum albumin.

Various properties of gold nanoparticles (GNPs) are found to play crucial roles in their biological activity. Among them, the morphology and surface chemistry are extremely important. This is because of differences in surface energies of various crystal facets arising from a large fraction of edges, corners and vertices. In the present work, we provide a comparative study on the adsorption and binding affinities of bovine serum albumin (BSA) onto triangular gold nanoplates (TGNP) and gold nanorods (GNR). The results were compared with similar size of both CTAB and citrate stabilized spherical GNPs. Our data suggested stronger binding of BSA on citrate stabilized spherical GNPs whereas TGNP shows the weakest binding among all the GNPs. A blue shift of approximately 20 nm in tryptophan fluorescence was observed for all CTAB stabilized GNPs, indicating the local dielectric changes surrounding the tryptophan residue. Loss of the secondary structure was also observed for all CTAB stabilized GNPs. No spectral shift was observed for citrate stabilized spherical GNPs though maximum quenching of fluorescence and minimum structural loss was observed. With the help of molecular simulation recently developed by our group, a binding model is proposed to explain all the above experimental results.

Spectroscopic studies of interaction between biologically synthesized silver nanoparticles and bovine serum albumin.

Binding interaction of biologically synthesized silver nanoparticles with bovine serum albumin (BSA) has been investigated by UV-Vis and fluorescence spectroscopic techniques. UV-Vis analysis implies the formation of the ground state complex between BSA and silver nanoparticles. The analysis of fluorescence spectrum and fluorescence intensity indicates that silver nanoparticles (SNP) have a strong ability to quench the intrinsic fluorescence of BSA by dynamic quenching mechanisms. The number of binding sites 'n' and binding constants 'K' were determined at different temperatures based on fluorescence quenching. The thermodynamic parameters namely deltaH, deltaG, and deltaS were calculated at different temperatures (20, 30, and 40 degrees C) and the results indicate that both hydrophobic and electrostatic interactions were predominantly present in the SNP-BSA complex. Negative deltaG values imply that the binding process is spontaneous.

Optical trapping of a single protein.

We experimentally demonstrate the optical trapping of a single bovine serum albumin (BSA) molecule that has a hydrodynamic radius of 3.4 nm, using a double-nanohole in an Au film. The strong optical force in the trap not only stably traps the protein molecule but also unfolds it. The unfolding of the BSA is confirmed by experiments with changing optical power and with changing solution pH. The detection of the trapping event has a signal-to-noise ratio of 33, which shows that the setup is extremely sensitive to detect the presence of a protein, even at the single molecule level.

Increasing the X-ray diffraction power of protein crystals by dehydration: the case of bovine serum albumin and a survey of literature data.

Serum albumin is one of the most widely studied proteins. It is the most abundant protein in plasma with a typical concentration of 5 g/100 mL and the principal transporter of fatty acids in plasma. While the crystal structures of human serum albumin (HSA) free and in complex with fatty acids, hemin, and local anesthetics have been characterized, no crystallographic models are available on bovine serum albumin (BSA), presumably because of the poor diffraction power of existing hexagonal BSA crystals. Here, the crystallization and diffraction data of a new BSA crystal form, obtained by the hanging drop method using MPEG 5K as precipitating agent, are presented. The crystals belong to space group C2, with unit-cell parameters a = 216.45 Å, b = 44.72 Å, c = 140.18 Å, ß = 114.5°. Dehydration was found to increase the diffraction limit of BSA crystals from ~8 Å to 3.2 Å, probably by improving the packing of protein molecules in the crystal lattice. These results, together with a survey of more than 60 successful cases of protein crystal dehydration, confirm that it can be a useful procedure to be used in initial screening as a method of improving the diffraction limits of existing crystals.

Fabrication of gold nanorods-doped, bovine serum albumin microstructures via multiphoton excited photochemistry.

In this study, three-dimensional (3D) crosslinked bovine serum albumin (BSA) microstructures containing gold nanorods (AuNRs) were fabricated via multiphoton excited photochemistry using Rose Bengal (RB) as the photoactivator. To retain AuNRs in the 3D crosslinked BSA microstructures, the laser wavelength was chosen for two-photon RB absorption for improved two-photon crosslinking efficiency, but not for enhancing the longitudinal plasmon resonance of AuNRs which may result in photothermal damage of AuNRs. Furthermore, with two-photon excitation of RB via AuNRs plasmonics, the laser power can be reduced by about 30%. As a result, 3D BSA microstructures containing AuNRs can be successfully fabricated. The AuNRs-doped BSA microstructures can be applied in biomedical scaffolds with plasmonic properties such as two-photon luminescence imaging and photothermal therapy.

The importance of protein-protein interactions on the pH-induced conformational changes of bovine serum albumin: a small-angle X-ray scattering study.

The combined effects of concentration and pH on the conformational states of bovine serum albumin (BSA) are investigated by small-angle x-ray scattering. Serum albumins, at physiological conditions, are found at concentrations of approximately 35-45 mg/mL (42 mg/mL in the case of humans). In this work, BSA at three different concentrations (10, 25, and 50 mg/mL) and pH values (2.0-9.0) have been studied. Data were analyzed by means of the Global Fitting procedure, with the protein form factor calculated from human serum albumin (HSA) crystallographic structure and the interference function described, considering repulsive and attractive interaction potentials within a random phase approximation. Small-angle x-ray scattering data show that BSA maintains its native state from pH 4.0 up to 9.0 at all investigated concentrations. A pH-dependence of the absolute net protein charge is shown and the charge number per BSA is quantified to 10(2), 8(1), 13(2), 20(2), and 26(2) for pH values 4.0, 5.4, 7.0, 8.0, and 9.0, respectively. The attractive potential diminishes as BSA concentration increases. The coexistence of monomers and dimers is observed at 50 mg/mL and pH 5.4, near the BSA isoelectric point. Samples at pH 2.0 show a different behavior, because BSA overall shape changes as a function of concentration. At 10 mg/mL, BSA is partially unfolded and a strong repulsive protein-protein interaction occurs due to the high amount of exposed charge. At 25 and 50 mg/mL, BSA undergoes some re-folding, which likely results in a molten-globule state. This work concludes by confirming that the protein concentration plays an important role on the pH-unfolded BSA state, due to a delicate compromise between interaction forces and crowding effects.

L-Arginine reduces thioflavin T fluorescence but not fibrillation of bovine serum albumin.

This work examines the effects of L-arginine (L-Arg) on the aggregation and amyloid fibrillation of bovine serum albumin (BSA). We demonstrate that L-Arg dose-dependently reduces thioflavin T (ThT) fluorescence of BSA within the L-Arg concentration range used (0-1.4 M). However, as revealed by electron microscopy, size exclusion chromatography, and dynamic light scattering results, L-Arg does not prevent amyloid-like fibril formation by BSA. We conclude that L-Arg competes against ThT for binding sites on BSA amyloid-like fibrils, leading to biased results in ThT fluorescence measurements. Moreover, the use of ThT fluorescence assay to screen for potential inhibitors against amyloid fibrillation can give misleading results.

Toxic effects of ethanol on bovine serum albumin.

The toxic effects of ethanol on bovine serum albumin (BSA) were measured by resonance light scattering (RLS), fluorescence spectroscopy, ultraviolet spectrophotometry (UV), circular dichroism (CD), and transmission electron microscopy (TEM). The results indicated that ethanol had toxic effects on BSA, which led to protein denaturation and the effects increased with the ethanol dose. By means of RLS, BSA was found to aggregate in the presence of ethanol and particles smaller than 100 nm were observed from TEM. The fluorescence spectra showed that the intensity of the characteristic peak of BSA decreased and blue shifted, because of changes in the BSA skeleton structure, as well as alteration of the microenvironment of tryptophan (Trp) residues. The conformation changes of BSA were also shown by UV and CD spectrometry.

Chip-based enantioselective open-tubular capillary electrochromatography using bovine serum albumin-gold nanoparticle conjugates as the stationary phase.

In this study, chip-based enantioselective open-tubular CEC (OT-CEC) was developed employing BSA-gold nanoparticle (GNP) conjugates as a chiral stationary phase. An immobilization procedure was realized by prederivatization of the glass microchannel with (3-mercaptopropyl)-trimethoxysilane to provide thiol groups, which linked the BSA-GNP conjugates on the inner surface of the microchannels. Incorporation of GNPs into immobilization of BSA selectors greatly increased the BSA phase ratio and favored the BSA stationary phase generated sufficient EOF. Good resolutions of FITC-labeled ephedrine and norephedrine isomers were achieved with 36 mm effective separation channel length within 250 s. The constructed OT-CEC microdevice exhibited good repeatabilities for run-to-run enantioseparations and kept an enantioselective lifetime of more than 1 month. The effects of pH values and concentrations of a running buffer on the selectivity and resolution of enantioseparations were investigated.

Detailed insight into the formation of protein corona: Conformational change, stability and aggregation.

In a physiological fluid (e.g., blood), nanomaterials will strongly interact with proteins to form "protein corona". The structure and aggregation of protein corona around the nanoparticles are of vital importance in the safe application of nanomaterials in living organisms. Here, we combined systematic methods, including transmission electron microscopy, scanning electron microscopy equipped with energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, hydrogen/deuterium exchange techniques and fluorescence quenching to explore the conformational change, stability and aggregation of protein corona bound on magnetic nanoparticles. For the first time we observed that the conformational change of protein corona could induce proteins to aggregate. We believe that these findings will deepen our understanding of the protein corona.

Revisiting the conformational state of albumin conjugated to gold nanoclusters: A self-assembly pathway to giant superstructures unraveled.

Bovine serum albumin (BSA) is often employed as a proteinaceous component for synthesis of luminescent protein-stabilized gold nanoclusters (AuNC): intriguing systems with many potential applications. Typically, the formation of BSA-AuNC conjugate occurs under strongly alkaline conditions. Due to the sheer complexity of intertwined chemical and structural transitions taking place upon BSA-AuNC formation, the state of albumin enveloping AuNCs remains poorly characterized. Here, we study the conformational properties of BSA bound to AuNCs using an array of biophysical tools including vibrational spectroscopy, circular dichroism, fluorescence spectroscopy and trypsin digestion. The alkaline conditions of BSA-AuNC self-assembly appear to be primary responsible for the profound irreversible disruption of tertiary contacts, partial unfolding of native α-helices, hydrolysis of disulfide bonds and the protein becoming vulnerable to trypsin digestion. Further unfolding of BSA-AuNC by guanidinium hydrochloride (GdnHCl) is fully reversible equally in terms of albumin's secondary structure and conjugate's luminescent properties. This suggests that binding to AuNCs traps the albumin molecule in a state that is both partly disordered and refractory to irreversible misfolding. Indeed, when BSA-AuNC is subjected to conditions favoring self-association of BSA into amyloid-like fibrils, the buildup of non-native ß-sheet conformation is less pronounced than in a control experiment with unmodified BSA. Unexpectedly, BSA-AuNC reveals a tendency to self-assemble into giant twisted superstructures of micrometer lengths detectable with transmission electron microscopy (TEM), a property absent in unmodified BSA. The process is accompanied by ordering of bound AuNCs into elongated streaks and simultaneous decrease in fluorescence intensity. The newly discovered self-association pathway appears to be specifically accessible to protein molecules with a certain restriction on structural dynamics which in the case of BSA-AuNC arises from binding to metal nanoclusters. Our results have been discussed in the context of mechanisms of protein misfolding and applications of BSA-AuNC.

Effective hard particle model for the osmotic pressure of highly concentrated binary protein solutions.

The experimentally measured concentration dependence of the osmotic pressure of an equimolar mixture of hen egg ovalbumin and bovine serum albumin at pH 7.0 and 25 degrees C in the presence of 0.15 M NaCl is shown to be quantitatively accounted for by a model in which each protein species is represented by an effective hard sphere. The size of this sphere is determined by analysis of the concentration dependence of the osmotic pressure of the isolated protein.

Aggregation and fibrillation of bovine serum albumin.

The all-alpha helix multi-domain protein bovine serum albumin (BSA) aggregates at elevated temperatures. Here we show that these thermal aggregates have amyloid properties. They bind the fibril-specific dyes Thioflavin T and Congo Red, show elongated although somewhat worm-like morphology and characteristic amyloid X-ray fiber diffraction peaks. Fibrillation occurs over minutes to hours without a lag phase, is independent of seeding and shows only moderate concentration dependence, suggesting intramolecular aggregation nuclei. Nevertheless, multi-exponential increases in dye-binding signal and changes in morphology suggest the existence of different aggregate species. Although beta-sheet content increases from 0 to ca. 40% upon aggregation, the aggregates retain significant amounts of alpha-helix structure, and lack a protease-resistant core. Thus BSA is able to form well-ordered beta-sheet rich aggregates which nevertheless do not possess the same structural rigidity as classical fibrils. The aggregates do not permeabilize synthetic membranes and are not cytotoxic. The ease with which a multidomain all-alpha helix protein can form higher-order beta-sheet structure, while retaining significant amounts of alpha-helix, highlights the universality of the fibrillation mechanism. However, the presence of non-beta-sheet structure may influence the final fibrillar structure and could be a key component in aggregated BSA's lack of cytotoxicity.

Novel scaffolds fabricated from protein-loaded microspheres for tissue engineering.

Biodegradable scaffolds play an important role in tissue engineering by providing physical and biochemical support for both differentiated and progenitor cells. Here, we describe a novel method for incorporating proteins in 3D biodegradable scaffolds by utilizing protein-loaded microspheres as the building blocks for scaffold formation. Poly(l,d-lactic-co-glycolic acid) (PLGA) microspheres containing bovine serum albumin (BSA) were fused into scaffolds using dichloromethane vapor for various time intervals. Microspheres containing 0, 0.4, 1.5, 4.3% BSA showed that increased protein loading required increased fusion time for scaffold fabrication. Protein release from the scaffolds was quantified in vitro over 20 days and compared to that of loose microspheres. Scaffolds had a slightly lower (up to 20%) release over the first 10 days, however, the cumulative release from both microspheres and scaffolds at the end of the study was not statistically different and the rate of release was the same, indicating that microsphere release can be predictive of scaffold kinetics. Scaffolds fused from larger (113.3 +/- 58.0 microm) rather than smaller (11.15 +/- 11.08 microm) microspheres, generated pores on the order of 200 microm as compared to 20 microm, respectively, showing control over pore size. In addition, four dyes (carbon black, acid green, red 27, and fast green FCF) were encapsulated in PLGA microspheres and fused into homogeneous and partitioned scaffolds, indicating control over spatial distribution within the scaffold. Finally, the scaffolds were seeded with fibroblast cells, which attached and were well spread over the polymer surface after 4h of incubation. These results highlight the versatility of this simple scaffold fusion method for incorporating essentially any combination of loaded microspheres into a 3D structure, making this a powerful tool for tissue engineering and drug delivery applications.

Electrostatic repulsion as a mechanism in fouling of ultrafiltration membranes.

Studies of electrostatic repulsion in ultrafiltration membranes are limited to applications of different organic compounds carrying a set of unique characteristics, or to changes of general water parameters such as ionic strength and pH. The proposed method of deliberate alteration of surface charge of organic molecule by succinylation or by guanidination provides an opportunity to selectively investigate the electrostatic mechanism without changing size or hydrophobic properties of investigated molecule. The approach was successfully implemented on BSA protein, and new inside into the mechanism of electrostatic mechanism was obtained. The electrostatic repulsion becomes important when zeta potential of the protein exceeded 20 mV, when before the threshold the interactions were mainly governed by size exclusion.