The nano-bio interface mapped by oxidative footprinting of the adsorption sites of myoglobin (original) (raw)
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Analytical Chemistry, 2011
For the first time, the possible binding site of nanoparticles on protein was revealed by crosslinking chemistry coupled with mass spectrometry. The peptides locating very close to the polyacrylic acid (PAA)-coated Fe 3 O 4 nanoparticles (NPs) during interaction with human serum albumin (HSA) were crosslinked to the surface of NPs. Following protease digestion, the attached peptides were cleaved off the particle surface, and identified by matrix-assisted laser desorption/ ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The peptides were found to be part of the so-called drug binding site 2 of HSA; and the competitive binding to HSA between the corresponding drug, ibuprofen, and the NPs was observed. Our results demonstrated that crosslinking chemistry coupled with MS was a quick and simple method for locating the possible binding sites of NPs on protein. Information on NP-protein binding interface will benefit study of how the interactions are governed by the physicochemical properties of NPs, for guiding the design of functional bio-nano constructs. It can also help to predict the biological consequence of protein adsorption on NPs, for obtaining more knowledge on nanotoxicity.
Mapping Molecular Structure of Protein Locating on Nanoparticles with Limited Proteolysis
Analytical Chemistry, 2019
The molecular structure of a protein could be altered when attached to nanoparticles (NPs), affecting the performance of NPs present in biological systems. Limited proteolysis coupled with LC-MS/MS could reveal the changes in protein structure when it binds to a variety of entities, including macro-molecules and small drugs, but has not yet been applied to study protein-NP interaction. Herein, adsorption of proteins, transferrin and catalase, on the polystyrene (PS) or iron oxide (IO) NPs was analyzed with this method. Both increased and decreased proteolytic efficiency in certain regions on the proteins were observed. Identification of the peptides affected by protein-NP interaction led to proper prediction of alterations to protein function as well as to colloidal stability of NPs. Overall, the present work has demonstrated the utility of limited proteolysis in helping elucidate the potential biological outcomes of the protein-NP conjugate, obtaining knowledge to guide improvement of the rational design of the protein conjugated NPs for biomedical applications, and to understand the biological behaviors of the engineered NPs. Protein adsorption on the surface of engineered nanoparticles (NPs) unintentionally exposed to biological systems is inevitable. 1-5 On the other hand, intentionally attaching specific proteins (i.e. antibodies) on NPs as the target recognition moieties has also been used to achieve active targeting and assist delivery of NPs for therapeutic purposes. 6-8 While the adsorbed protein corona could be beneficial to the outcomes of the NPs by keeping the NPs from being cleared by the innate immune system; 9-11 structural changes, e.g. unfolding, fibrillation and aggregation, 12-14 could occur to the protein after binding to the NPs, and induce negative impacts on the protein-NP conjugates. 7,15,16 For example, protein unfolding can expose the originally buried cryptic epitopes to induce immune clearance of the NPs and
Mechanism of Myoglobin Molecule Adsorption on Silica: QCM, OWLS and AFM Investigations
International Journal of Environmental Research and Public Health, 2021
Adsorption kinetics of myoglobin on silica was investigated using the quartz crystal microbalance (QCM) and the optical waveguide light-mode spectroscopy (OWLS). Measurements were carried out for the NaCl concentration of 0.01 M and 0.15 M. A quantitative analysis of the kinetic adsorption and desorption runs acquired from QCM allowed to determine the maximum coverage of irreversibly bound myoglobin molecules. At a pH of 3.5–4 this was equal to 0.60 mg m−2 and 1.3 mg m−2 for a NaCl concentration of 0.01 M and 0.15 M, respectively, which agrees with the OWLS measurements. The latter value corresponds to the closely packed monolayer of molecules predicted from the random sequential adsorption approach. The fraction of reversibly bound protein molecules and their biding energy were also determined. It is observed that at larger pHs, the myoglobin adsorption kinetics was much slower. This behavior was attributed to the vanishing net charge that decreased the binding energy of molecules ...
Langmuir : the ACS journal of surfaces and colloids, 2005
The binding of protein to solid surfaces often induces changes in the structure, and to investigate these matters we have selected two different protein-nanoparticle systems. The first system concerns the enzyme human carbonic anhydrase II which binds essentially irreversibly to the nanoparticles, and the second system concerns human carbonic anhydrase I which alternate between the adsorbed and free state upon interaction with nanoparticles. Application of the TROSY pulse sequence has allowed high-resolution NMR analysis for both of the protein-nanoparticle systems. For HCAII it was possible to observe spectra of protein when bound to the nanoparticles. The results indicated that HCAII undergoes large rearrangements, forming an ensemble of molten globule-like structures on the surface. The spectra from the HCAI-nanoparticle system are dominated by HCAI molecules in solution. A comparative analysis of variations in intensity from 97 amide resonances in a 1H-15N TROSY spectrum reveale...
ACS Nano, 2012
Fully understanding the influence of blood proteins on the assembly structure and dynamics within nanoparticles is difficult because of the complexity of the system and the difficulty in probing the diverse elements and milieus involved. Here we show the use of site-specific labeling with spin probes and fluorophores combined with electron paramagnetic resonance (EPR) spectroscopy and fluorescence resonance energy transfer (FRET) measurements to provide insights into the molecular architecture and dynamics within nanoparticles. These tools are especially useful for determining nanoparticle stability in the context of blood proteins and lipoproteins, and have allowed us to quantitatively analyze the dynamic changes in assembly structure, local stability and cargo diffusion of a class of novel telodendrimer-based micellar nanoparticles. When combined with human plasma and individual plasma components, we find that non-crosslinked nanoparticles immediately lose their original assembly structure and release their payload upon interaction with lipoproteins. In contrast, serum albumins and immunoglobulin gamma have moderate affects on the integrity of the nanoparticles. Disulfide crosslinked nanoparticles show minimal interaction with lipoproteins, and can better retain their assembly structure and payload in vitro and in vivo. We further demonstrate how the enhanced stability and release property of disulfide crosslinked nanoparticles can be reversed in reductive conditions. These findings identify factors are crucial to the performance of nanomedicines and provide design modes to control their interplay with blood factors.
Advances in Colloid and Interface Science, 2007
The major aim of our current work is to develop a deep understanding of biological effects of nanoparticles and how these effects are mediated by proteins that are adsorbed on the nanoparticles under different biological circumstances. Due to their small size, nanoparticles have distinct properties compared to the bulk form of the same materials, and these properties are rapidly revolutionizing many areas of medicine and technology. However, relatively little is known about the interaction of nanoscale objects with biological systems, as this requires quite different concepts from more established nanoscience. Thus, we have argued that in a biological fluid, proteins associate with nanoparticles, and it is the amount and presentation of the proteins on the surface rather than the particles themselves that are the cause of numerous biological responses. It is this outer layer of proteins that is seen by the biological cells, and leads to their responses. We are developing novel techniques to identify and quantify the proteins that are consistently associated with nanoparticles, as a function of the nanoparticle size, shape, and surface properties, and to correlate the adsorbed protein identities with their association and dissociation rates to and from the nanoparticles. We also seek to understand the degree of conformational change that they undergo upon adsorption to the nanoparticles. In essence, we wish to create "epitope maps" of the protein corona that surrounds nanoparticles in biological solutions, as it is the particle-protein complex that is the biologically active entity.
Structure and Catalytic Behavior of Myoglobin Adsorbed onto Nanosized Hydrotalcites
Langmuir, 2009
The adsorption of myoglobin (Mb) onto nanosized nickel aluminum hydrotalcite (NiAl-HTlc) surface was studied, and the structural properties of the resulting protein layer were analyzed by using FT-IR, Raman, and fluorescence spectroscopies. Upon adsorption onto the nanoparticle surface, the protein molecules maintained their secondary structure, while the tertiary structure was altered. The fluorescence spectra and anisotropy values of adsorbed Mb revealed that the emitting amino acid residues are affected by different microenvironments when compared to the native protein behavior. Moreover, the decrease of fluorescence decay times of tryptophan indicated the occurrence of interactions among the fluorophores and the constituents of the nanoparticles, such as the metal cations, which can take place when conformational changes of Mb occur. Raman spectra indicated that the interaction of Mb molecules with NiAl-HTlc nanoparticles modified the porphyrin core, changing the spin state of the heme iron from high spin (HS) to low spin (LS). The enzymatic activity of the nanostructured biocomposite was evaluated in the oxidation of 2-methoxyphenol by hydrogen peroxide and discussed on the basis of structural properties of adsorbed myoglobin.
Protein-nanoparticle interactions
Nano Today, 2008
The key role of protein-nanoparticle interactions in nanomedicine and nanotoxicity has begun to emerge recently with the development of the idea of the nanoparticle-protein 'corona'. This dynamic layer of proteins (and other biomolecules) adsorbs to nanoparticle surfaces immediately upon contact with living systems. While within the biomaterials field the role of adsorbed molecules in cellular responses is acknowledged, there are several new issues at stake where nanoparticles are concerned. We show here that highly selective protein adsorption, added to the fact that particles can reach subcellular locations, results in significant new potential impacts for nanoparticles on protein interactions and cellular behavior. This article does not seek to provide a comprehensive review of all articles published on protein-nanoparticle interactions, rather it seeks to provide a snapshot of the range of activities and to highlight some of the key research directions and paradigms that are emerging and evolving in this exciting arena. An important example includes the recent discovery that nanoparticles can impact protein fibrillation processes.
Proceedings of the National Academy of Sciences, 2007
Due to their small size, nanoparticles have distinct properties compared with the bulk form of the same materials. These properties are rapidly revolutionizing many areas of medicine and technology. Despite the remarkable speed of development of nanoscience, relatively little is known about the interaction of nanoscale objects with living systems. In a biological fluid, proteins associate with nanoparticles, and the amount and presentation of the proteins on the surface of the particles leads to an in vivo response. Proteins compete for the nanoparticle ''surface,'' leading to a protein ''corona'' that largely defines the biological identity of the particle. Thus, knowledge of rates, affinities, and stoichiometries of protein association with, and dissociation from, nanoparticles is important for understanding the nature of the particle surface seen by the functional machinery of cells. Here we develop approaches to study these parameters and apply them to plasma and simple model systems, albumin and fibrinogen. A series of copolymer nanoparticles are used with variation of size and composition (hydrophobicity). We show that isothermal titration calorimetry is suitable for studying the affinity and stoichiometry of protein binding to nanoparticles. We determine the rates of protein association and dissociation using surface plasmon resonance technology with nanoparticles that are thiol-linked to gold, and through size exclusion chromatography of protein-nanoparticle mixtures. This method is less perturbing than centrifugation, and is developed into a systematic methodology to isolate nanoparticle-associated proteins. The kinetic and equilibrium binding properties depend on protein identity as well as particle surface characteristics and size.