Ultraviolet Resonance Raman Spectra of Serum Albumins.

The ultraviolet resonance Raman (UVRR) spectra of the two proteins bovine serum albumin (BSA) and human serum albumin (HSA) in an aqueous solution are compared with the aim to distinguish between them based on their very similar amino acid composition and structure and to obtain signals from tryptophan that has only very few residues. Comparison of the protein spectra with solutions of tryptophan, tyrosine, and phenylalanine in comparative ratios as in the two proteins shows that at an excitation wavelength of 220 nm, the spectra are dominated by the strong resonant contribution from these three amino acids. While the strong enhancement of two and one single tryptophan residue in BSA and HSA, respectively, results in pronounced bands assigned to fundamental vibrations of tryptophan, its weaker overtones and combination bands do not play a major role in the spectral range above 1800 cm-1. There, the protein spectra clearly reveal the signals of overtones and combination bands of phenylalanine and tyrosine. Assignments of spectral features in the range of Raman shifts from 3800 to 5100 cm-1 to combinations comprising fundamentals and overtones of tyrosine were supported by spectra of amino acid mixtures that contain deuterated tyrosine. The information in the high-frequency region of the UVRR spectra could provide information that is complementary to near-infrared absorption spectroscopy of the proteins.

Surface enhanced Raman scattering for probing cellular biochemistry.

Surface enhanced Raman scattering (SERS) from biomolecules in living cells enables the sensitive, but also very selective, probing of their biochemical composition. This minireview discusses the developments of SERS probing in cells over the past years from the proof-of-principle to observe a biochemical status to the characterization of molecule-nanostructure and molecule-molecule interactions and cellular processes that involve a wide variety of biomolecules and cellular compartments. Progress in applying SERS as a bioanalytical tool in living cells, to gain a better understanding of cellular physiology and to harness the selectivity of SERS, has been achieved by a combination of live cell SERS with several different approaches. They range from organelle targeting, spectroscopy of relevant molecular models, and the optimization of plasmonic nanostructures to the application of machine learning and help us to unify the information from defined biomolecules and from the cell as an extremely complex system.

Correlated electric field modulation of electron transfer parameters and the access to alternative conformations of multifunctional cytochrome c.

Cytochrome c (Cytc) is a multifunctional protein that, in its native conformation, shuttles electrons in the mitochondrial respiratory chain. Conformational transitions that involve replacement of the heme distal ligand lead to the gain of alternative peroxidase activity, which is crucial for membrane permeabilization during apoptosis. Using a time-resolved SERR spectroelectrochemical approach, we found that the key physicochemical parameters that characterize the electron transfer (ET) canonic function and those that determine the transition to alternative conformations are strongly correlated and are modulated by local electric fields (LEF) of biologically meaningful magnitude. The electron shuttling function is optimized at moderate LEFs of around 1 V nm-1. A decrease of the LEF is detrimental for ET as it rises the reorganization energy. Moreover, LEF values below and above the optimal for ET favor alternative conformations with peroxidase activity and downshifted reduction potentials. The underlying proposed mechanism is the LEF modulation of the flexibility of crucial protein segments, which produces a differential effect on the kinetic ET and conformational parameters of Cytc. These findings might be related to variations in the mitochondrial membrane potential during apoptosis, as the basis for the switch between canonic and alternative functions of Cytc. Moreover, they highlight the possible role of variable LEFs in determining the function of other moonlighting proteins through modulation of the protein dynamics.

Protein corona on biogenic silver nanoparticles provides higher stability and protects cells from toxicity in comparison to chemical nanoparticles.

The development of environmentally friendly new procedures for the synthesis of metallic nanoparticles is one of the main goals of nanotechnology. Proteins and enzymes from plants, filamentous fungi, yeast, and bacteria to produce nanoparticles are both valuable and viable alternatives to conventional synthesis of nanomaterials due to their high efficiency and the low cost to scale up and generate large quantities. The aim of this work is to compare biogenic silver nanoparticles (AgNPs) obtained from cell-free filtrates from the fungus Macrophomina phaseolina to conventional chemical AgNPs, in biocidal activity and toxicity. Our results show that bio-AgNPs displayed similar bactericidal activity than chemical AgNPs, but less toxicity in the model organism Caenorhabditis elegans. We employed biochemical and proteomic techniques to profile the unique surface chemistry of the capping in the bio-AgNPs and therefore to identify the proteins involved in their synthesis and stability. These results not only suggest that the proteins involved in the synthesis of the nanoparticles and corona formation in the bio-AgNPs are responsible for keeping the silver core preserved making them more stable in time, but also masking and protecting eukaryotic cells from metal toxicity.

Probing the Intracellular Bio-Nano Interface in Different Cell Lines with Gold Nanostars.

Gold nanostars are a versatile plasmonic nanomaterial with many applications in bioanalysis. Their interactions with animal cells of three different cell lines are studied here at the molecular and ultrastructural level at an early stage of endolysosomal processing. Using the gold nanostars themselves as substrate for surface-enhanced Raman scattering, their protein corona and the molecules in the endolysosomal environment were characterized. Localization, morphology, and size of the nanostar aggregates in the endolysosomal compartment of the cells were probed by cryo soft-X-ray nanotomography. The processing of the nanostars by macrophages of cell line J774 differed greatly from that in the fibroblast cell line 3T3 and in the epithelial cell line HCT-116, and the structure and composition of the biomolecular corona was found to resemble that of spherical gold nanoparticles in the same cells. Data obtained with gold nanostars of varied morphology indicate that the biomolecular interactions at the surface in vivo are influenced by the spike length, with increased interaction with hydrophobic groups of proteins and lipids for longer spike lengths, and independent of the cell line. The results will support optimized nanostar synthesis and delivery for sensing, imaging, and theranostics.

Intracellular optical probing with gold nanostars.

Gold nanostars are important nanoscopic tools in biophotonics and theranostics. To understand the fate of such nanostructures in the endolysosomal system of living cells as an important processing route in biotechnological approaches, un-labelled, non-targeted gold nanostars synthesized using HEPES buffer were studied in two cell lines. The uptake of the gold nanostructures leads to cell line-dependent intra-endolysosomal agglomeration, which results in a greater enhancement of the local optical fields than those around individual nanostars and near aggregates of spherical gold nanoparticles of the same size. As demonstrated by non-resonant surface-enhanced Raman scattering (SERS) spectra in the presence and absence of aggregation, the spectroscopic signals of molecules are of very similar strength over a wide range of concentrations, which is ideal for label-free vibrational characterization of cells and other complex environments. In 3T3 and HCT-116 cells, SERS data were analyzed together with the properties of the intracellular nanostar agglomerates. Vibrational spectra indicate that the processing of nanostars by cells and their interaction with the surrounding endolysosomal compartment is connected to their morphological properties through differences in the structure and interactions in their intracellular protein corona. Specifically, different intracellular processing was found to result from a different extent of hydrophobic interactions at the pristine gold surface, which varies for nanostars of different spike lengths. The sensitive optical monitoring of surroundings of nanostars and their intracellular processing makes them a very useful tool for optical bionanosensing and therapy.

Relating the composition and interface interactions in the hard corona of gold nanoparticles to the induced response mechanisms in living cells.

Understanding the formation of the intracellular protein corona of nanoparticles is essential for a wide range of bio- and nanomedical applications. The innermost layer of the protein corona, the hard corona, directly interacts with the nanoparticle surface, and by shielding the surface, it has a deterministic effect on the intracellular processing of the nanoparticle. Here, we combine a direct qualitative analysis of the hard corona composition of gold nanoparticles with a detailed structural characterization of the molecules in their interaction with the nanoparticle surface and relate both to the effects they have on the ultrastructure of living cells and the processing of the gold nanoparticles. Cells from the cell lines HCT-116 and A549 were incubated with 30 nm citrate-stabilized gold nanoparticles and with their aggregates in different culture media. The combined results of mass spectrometry based proteomics, cryo soft X-ray nanotomography and surface-enhanced Raman scattering experiments together revealed different uptake mechanisms in the two cell lines and distinct levels of induced cellular stress when incubation conditions were varied. The data indicate that the different incubation conditions lead to changes in the nanoparticle processing via different protein-nanoparticle interfacial interactions. Specifically, they suggest that the protein-nanoparticle surface interactions depend mainly on the surface properties of the gold nanoparticles, that is, the ζ-potential and the resulting changes in the hydrophilicity of the nanoparticle surface, and are largely independent of the cell line, the uptake mechanism and intracellular processing, or the extent of the induced cellular stress.

The alkaline transition of cytochrome c revisited: Effects of electrostatic interactions and tyrosine nitration on the reaction dynamics.

Here we investigated the effect of electrostatic interactions and of protein tyrosine nitration of mammalian cytochrome c on the dynamics of the so-called alkaline transition, a pH- and redox-triggered conformational change that implies replacement of the axial ligand Met80 by a Lys residue. Using a combination of electrochemical, time-resolved SERR spectroelectrochemical experiments and molecular dynamics simulations we showed that in all cases the reaction can be described in terms of a two steps minimal reaction mechanism consisting of deprotonation of a triggering group followed by ligand exchange. The pKaalk values of the transition are strongly modulated by these perturbations, with a drastic downshift upon nitration and an important upshift upon establishing electrostatic interactions with a negatively charged model surface. The value of pKaalk is determined by the interplay between the acidity of a triggering group and the kinetic constants for the forward and backward ligand exchange processes. Nitration of Tyr74 results in a change of the triggering group from Lys73 in WT Cyt to Tyr74 in the nitrated protein, which dominates the pKaalk downshift towards physiological values. Electrostatic interactions, on the other hand, result in strong acceleration of the backward ligand exchange reaction, which dominates the pKaalk upshift. The different physicochemical conditions found here to influence pKaalk are expected to vary depending on cellular conditions and subcellular localization of the protein, thus determining the existence of alternative conformations of Cyt in vivo.

Extracellular biosynthesis of bactericidal Ag/AgCl nanoparticles for crop protection using the fungus Macrophomina phaseolina.

Synthesis of noble metal nanoparticles using natural products and living organisms has drawn a lot of interest owing to economic prospects and potential applicability in different fields. For this work we used the exudate of the soil fungus Macrophomina phaseolina for a low-cost method of green synthesis to obtain stable silver-silver chloride nanoparticles (Ag/AgCl-NPs). Reaction parameters including media and AgNO3 concentration were further optimized for NPs production. Spectral analysis revealed a peak at 420 nm that corresponds to the surface plasmon resonance of silver NPs. Scanning electron microscopy (SEM) analysis unveiled NPs spherical morphology with a size range of 5-30 nm. The crystalline nature of the synthesized NPs was examined by X-ray diffraction (XRD) analysis. The green synthesized NPs showed activity against gram-positive and gram-negative bacteria. No effect in fungi or yeast cells was detected, though a high inhibitory effect was observed on bacteria growth kinetics. Interaction of bacteria with Ag/AgCl-NPs led to cell membrane damage as observed by SEM, followed by an increase in oxidative stress, being this the possible mechanism behind the strong bactericidal activity depicted. In order to test its possible applicability as a seed protection agent the effect of Ag/AgCl-NPs dosage on soybean (Glycine max L.) seed's germination was also examined. Interestingly, not only the germination process was not affected by the NPs dosage or time of seeds incubation, but also no oxidative damage was detected in seeds after exposure to the biogenic nanoparticles.

Silica@proton-alginate microreactors: a versatile platform for cell encapsulation.

As an alternative approach to the well known Ca(ii)-alginate encapsulation process within silica hydrogels, proton-driven alginate gelation was investigated in order to establish its capacity as a culture carrier, both isolated and embedded in an inorganic matrix. Control over the velocity of the proton-gelation front allows the formation of a hydrogel shell while the core remains liquid, allowing bacteria and microalgae to survive the strongly acidic encapsulation process. Once inside the inorganic host, synthesized by a sol-gel process, the capsules spontaneously redissolve without the aid of external complexing agents. The entrapped cells survive the two-step process to a significant extent; culture's growth restores the initial cell count in less than two weeks. Biosynthesis of Au nanoparticles mediated by the entrapped microalgae illustrates the preservation of the biosynthetic abilities supported by this platform.