Comparative Analysis of Au and Au@SiO2 Nanoparticle-Protein Interactions for Evaluation as Platforms in Theranostic Applications.

Gold nanoparticles are utilized in a variety of sensing and detection technologies because of their unique physiochemical properties. Their tunable size, shape, and surface charge enable them to be used in an array of platforms. The purpose of this study is to conduct a thorough spectroscopic characterization of Au and functionalized hybrid Au@SiO2 nanoparticles under physiological conditions and in the presence of two proteins known to be abundant in serum, bovine serum albumin and human ubiquitin. The information obtained from this study will enable us to develop design principles to synthesize an array of surface-enhanced Raman spectroscopy-based nanoparticles as platforms for theranostic applications. We are particularly interested in tailoring the surface chemistry of the Au@SiO2 nanoparticles for applications in theranostic technologies. We employ common spectroscopic techniques, with particular emphasis on circular dichroism and heteronuclear single quantum correlation nuclear magnetic resonance (HSQC NMR) spectroscopy, as combinatorial tools to understand protein conformational dynamics, binding site interactions, and protein corona for the design of nanoparticles capable of reaching their intended target in vivo. Our results conclude that protein adsorption onto the nanoparticle surface prevents nanoparticle aggregation. We observed that varying the ionic strength and type of ion influences the aggregation and aggregation rate of each respective nanoparticle. The conformation of proteins and the absorption of proteins on the surface of Au nanoparticles are also influenced by ionic strength. Using two-dimensional [15N-1H]-HSQC NMR experiments to compare the interactions of Au and Au@SiO2 nanoparticles with 15N-ubiquitin, we observed small chemical shift perturbations in some amino acid peaks and differences in binding site interactions with ubiquitin and respective nanoparticles.

Cold adaptation shapes the robustness of metabolic networks in Drosophila melanogaster.

When ectotherms are exposed to low temperatures, they enter a cold-induced coma (chill coma) that prevents resource acquisition, mating, oviposition, and escape from predation. There is substantial variation in time taken to recover from chill coma both within and among species, and this variation is correlated with habitat temperatures such that insects from cold environments recover more quickly. This suggests an adaptive response, but the mechanisms underlying variation in recovery times are unknown, making it difficult to decisively test adaptive hypotheses. We use replicated lines of Drosophila melanogaster selected in the laboratory for fast (hardy) or slow (susceptible) chill-coma recovery times to investigate modifications to metabolic profiles associated with cold adaptation. We measured metabolite concentrations of flies before, during, and after cold exposure using nuclear magnetic resonance (NMR) spectroscopy to test the hypotheses that hardy flies maintain metabolic homeostasis better during cold exposure and recovery, and that their metabolic networks are more robust to cold-induced perturbations. The metabolites of cold-hardy flies were less cold responsive and their metabolic networks during cold exposure were more robust, supporting our hypotheses. Metabolites involved in membrane lipid synthesis, tryptophan metabolism, oxidative stress, energy balance, and proline metabolism were altered by selection on cold tolerance. We discuss the potential significance of these alterations.

Identification of isethionic acid and other small molecule metabolites of Fragilariopsis cylindrus with nuclear magnetic resonance.

Nuclear magnetic resonance (NMR) spectroscopy has been used to obtain metabolic profiles of the polar diatom Fragilariopsis cylindrus, leading to the identification of a novel metabolite in this organism. Initial results from an ongoing metabolomics study have led to the discovery of isethionic acid (2-hydroxyethanesulfonic acid, CAS: 107-36-8) as a major metabolite in F. cylindrus. This compound is being produced by the organism under normal culture conditions. This finding is the first report of a diatom producing isethionic acid. In addition to isethionic acid, four other metabolites, dimethylsulfoniopropionate (DMSP), betaine, homarine, and proline were present and may serve as osmoprotectants in F. cylindrus. NMR-based metabolite profiles of F. cylindrus were obtained along a growth curve of the organism. The relative concentration levels of the five metabolites were monitored over a growth period of F. cylindrus from 18 to 25 days. All showed an increase in relative concentration with time, except for proline, which began to decrease after day 21.

NMR-based microbial metabolomics and the temperature-dependent coral pathogen Vibrio coralliilyticus.

Coral bleaching occurs when the symbioses between coral animals and their zooxanthellae is disrupted, either as part of a natural cycle or as the result of unusual events. The bacterium Vibrio coralliilyticus (type strain ATCC BAA-450) has been linked to coral disease globally (for example in the Mediterranean, Red Sea, Indian Ocean, and Great Barrier Reef) and like many other Vibrio species exhibits a temperature-dependent pathogenicity. The temperature-dependence of V. corallillyticus in regard to its metabolome was investigated. Nuclear magnetic resonance (NMR) spectra were obtained of methanol-water extracts of intracellula rmetabolites (endometabolome) from multiple samples of the bacteria cultured into late stationary phase at 27 degrees C (virulent form) and 24 degrees C (avirulent form). The spectra were subjected to principal components analysis (PCA), and significant temperature-based separations in PC1, PC2, and PC3 dimensions were observed. Betaine, succinate, and glutamate were identified as metabolites that caused the greatest temperature-based separations in the PC scores plots. With increasing temperature, betaine was shown to be down regulated, while succinate and glutamate were up regulated.

NMR-derived folate-bound structure of dihydrofolate reductase 1 from the halophile Haloferax volcanii.

Folate binds to dihydrofolate reductase (DHFR) to form a binary complex whose structure maintains the overall configuration of the enzyme; however, some significant changes are evident when a comparison is made to the enzyme. The structure of DHFR1 from the halophilic Halopherax volcanii was solved in its folate-bound form using nuclear magnetic resonance spectroscopy. NOE data obtained from the (15)N-NOESY-HSQC and (13)C-NOESY-HSQC experiments of the triply labeled ((1)H, (13)C, and (15)N) binary complex were used as input for the structure calculation with the Crystallography and Nuclear Magnetic Resonance System program. The resulting family of structures was compared with the enzyme solved by both nuclear magnetic resonance and X-ray crystallography and also to the mesophilic folate-bound enzyme from Escherichia coli. The binary complex exhibited less convergence of structure in the alpha2-helix and differences in the hinge residues D39 and A94. In comparison to the previously reported mesophilic binary complex solved by X-ray crystallography, the halophilic binary complex reported here does not agree with the convergence of the M20 loop to a single structure. The corresponding L21 loop of the halophilic binary complex family of structures solved by nuclear magnetic resonance indicates variability in this region.

Structure in an extreme environment: NMR at high salt.

Proteins from halophiles have adapted to challenging environmental conditions and require salt for their structure and function. How halophilic proteins adapted to a hypersaline environment is still an intriguing question. It is important to mimic the physiological conditions of the archae extreme halophiles when characterizing their enzymes, including structural characterization. The NMR derived structure of Haloferax volcanii dihydrofolate reductase in 3.5 M NaCl is presented, and represents the first high salt structure calculated using NMR data. Structure calculations show that this protein has a solution structure which is similar to the previously determined crystal structure with a difference at the N terminus of beta3 and the type of beta-turn connection beta7 and beta8.

NMR assignments of the binary hvDHFR1:folate complex.

A better understanding of how salt affects enzyme activity can be gained via NMR studies of binary hvDHFR1:folate complex. Chemical shift assignments of the 17.9 kDa enzyme with bound substrate prepare the way for ongoing research of the effects of salt on enzyme flexibility through relaxation studies.