Promising insights into the kosmotropic effect of magnetic nanoparticles on proteins: The pivotal role of protein corona formation

Increasing concerns about biosafety of nanoparticles (NPs) has raised the need for detailed knowledge of NP interactions with biological molecules especially proteins. Herein, the concentration-dependent effect of magnetic NPs (MNPs) on bovine serum albumin and hen egg white lysozyme was explored. The X-ray diffraction patterns, zeta potential, and dynamic light scattering measurements together with scanning electron microscopy images were employed to characterize MNPs synthesized through coprecipitation method. Then, we studied the behavior of two model proteins with different surface charges and structural properties on interaction with Fe₃O₄. A thorough investigation of protein–MNP interaction by the help of intrinsic fluorescence at different experimental conditions revealed that affinity of proteins for MNPs is strongly affected by the similarity of protein and MNP surface charges. MNPs exerted structure-making kosmotropic effect on both proteins under a concentration threshold; however, binding strength was found to determine the extent of stabilizing effect as well as magnitude of the concentration threshold. Circular dichroism spectra showed that proteins with less resistance to conformational deformations are more prone to secondary structure changes upon adsorption on MNPs. By screening thermal aggregation of proteins in the presence of Fe₃O₄, it was also found that like chemical stability, thermal stability is influenced to a higher extent in more strongly bound proteins. Overall, this report not only provides an integrated picture of protein–MNP interaction but also sheds light on the molecular mechanism underling this process.     

Lyophilization and enhanced stability of fluorescent protein nanoparticles

Protein nanoparticles (PNPs) that are nanostructured biomaterials with intrinsic biological function have been widely employed as three-dimensional nanobiomaterials for sensitive bioassays, MRI contrast, semiconductor devices, template for hybrid materials, etc., and stable and long-term maintenance of PNPs seems to be of crucial importance. We evaluated the stability of PNPs and the efficacy of lyophilization for the long-term stability of PNPs, especially using green fluorescent protein nanoparticles (gFPNPs) as a model PNP. Fluorescence intensities and TEM images of gFPNPs were analyzed to monitor their functional and structural stabilities. Unlike the green fluorescent protein monomers (eGFP) that were gradually inactivated in aqueous solution, gFPNP in the same aqueous solution retained the initial fluorescence activity and spherical nanoparticle structure even for 2 weeks at 4 °C. To ensure stable and long-term maintenance of gFPNPs, gFPNPs in aqueous solution were converted to the dried solid forms through lyophilization. It is notable that fluorescence activity and nanoparticle structure of gFPNPs that were lyophilized with both Tween 80 and sucrose were very stably maintained even for 10 weeks at various storage temperatures (−20 °C, 4 °C, 25 °C, and 37 °C). During the period of 10 weeks, the fluorescence of gFPNP was always more than 80% level of initial fluorescence at a wide range of temperature. Although this stability study was focused on gFPNPs, the developed optimal lyophilization conditions for gFPNPs can be applied in general to stable and long-term maintenance of many other PNP-derived biomaterials.     

Proteomic and biochemical evidence links the callose synthase in Nicotiana alata pollen tubes to the product of the NaGSL1 gene

The NaGSL1 gene has been proposed to encode the callose synthase (CalS) enzyme from Nicotiana alata pollen tubes based on its similarity to fungal 1,3-beta-glucan synthases and its high expression in pollen and pollen tubes. We have used a biochemical approach to link the NaGSL1 protein with CalS enzymic activity. The CalS enzyme from N. alata pollen tubes was enriched over 100-fold using membrane fractionation and product entrapment. A 220 kDa polypeptide, the correct molecular weight to be NaGSL1, was specifically detected by anti-GSL antibodies, was specifically enriched with CalS activity, and was the most abundant polypeptide in the CalS-enriched fraction. This polypeptide was positively identified as NaGSL1 using both MALDI-TOF MS and LC-ESI-MS/MS analysis of tryptic peptides. Other low-abundance polypeptides in the CalS-enriched fractions were identified by MALDI-TOF MS as deriving from a 103 kDa plasma membrane H+-ATPase and a 60 kDa beta-subunit of mitochondrial ATPase, both of which were deduced to be contaminants in the product-entrapped material. These analyses thus suggest that NaGSL1 is required for CalS activity, although other smaller (<30 kDa) or low-abundance proteins could also be involved.     

Selected expression profiling of full-length proteins and their variants in human plasma

With increased interest in clinical proteomics—the comparative investigation of differential protein expression patterns for use in the diagnostic and prognostic assessment of disease states—the demand for techniques that can readily identify changes in select proteome components is greater than ever before. This article describes a targeted proteomics approach to recover and quantify C-reactive protein (CRP) directly from human plasma. CRP, a putative biomarker for cardiac health, was isolated from microliter volumes of human plasma by using novel proteomics tools that combine micro-scale affinity capture with matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) detection. Native CRP was analyzed along with serum amyloid P component (SAP) and retinol binding protein (RBP), that were intentionally targeted to generate a selected protein expression profile. A number of qualitative changes were readily observed within these profiles, including micro heterogeneity in the SAP glycan, C-terminally truncated versions of RBP, and detection of a novel truncated variant of CRP. After quantitative validation of increasing plasma CRP concentrations, the approach was applied to the analysis of eight plasma samples obtained from individuals with known medical histories. The result of the analyses are eight protein profiles, revealing increasing CRP levels that can be associated with individuals ailing from post-surgery inflammation, chronic rheumatoid arthritis, and recent acute myocardial infarction. The technique described in this article lays the foundation for selected protein profiling for use in biomarker discovery, as well as in clinical and diagnostic applications.     

Experimental study and mathematical modeling for encapsulation of fentanyl citrate drug in nanostructured lipid carrier

Abstract

The aim of this study is to prepare a nanostructured lipid carrier (NLC) containing Fentanyl Citrate drug. The materials were selected in a way to achieve a nanostructure with lower particle size and higher drug entrapment efficiency. For this purpose, we used two mathematical models, Van Krevelen-Hoftyze and Hoy’s methods, which are based on the calculation of solubility parameters. Various NLC formulations are prepared experimentally to validate the mathematical modeling results. Hot homogenization method was used for NLC preparation. DLS, HPLC, TEM and DSC analyses are performed to calculate the size, drug entrapment efficiency, morphology and thermal behavior of particles, respectively. Experimental results suggest that the best NLC formulation has a particle size of 90?nm with a spherical morphology and drug entrapment efficiency of about 82%. A comparison of the mathematical and experimental results exhibits that Van Krevelen-Hoftyzer method is unable to provide an accurate estimation of the decreasing trend of particle size by chaining the components of NLC. However, Hoy’s method seems to be suitable for this purpose. Moreover, both mathematical methods could successfully estimate variation trend of drug entrapment efficiency by chaining the NLC components. Results show that surfactants-lipids solubility parameter has a bearing on the nanoparticle size while drug-lipid solubility parameter affects drug entrapment efficiency.

Communicated by Ramaswamy H. Sarma     

利用基因组学和MALDI-TOF MS技术鉴定放线菌纲细菌的核糖体蛋白质标志物

【目的】基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)法基于微生物的特征蛋白指纹图谱鉴定菌种,本研究利用基因组学和MALDI-TOFMS技术鉴定放线菌纲细菌的核糖体蛋白质标志物。【方法】从MALDI-TOF MS图谱数据库选取放线菌纲代表菌种,在基因组数据库检索目标菌种,获取目标菌株或其参比菌株的核糖体蛋白质序列,计算获得分子质量理论值,用于注释目标菌株MALDI-TOFMS指纹图谱中的核糖体蛋白质信号。【结果】从8目,24科,53属,114种,142株放线菌的MALDI-TOFMS图谱中总共注释出31种核糖体蛋白质。各菌株的指纹图谱中核糖体蛋白质信号数量差异显著。各种核糖体蛋白质信号的注释次数差异显著。总共15种核糖体蛋白质在超过半数图谱中得到注释,注释次数最高的是核糖体大亚基蛋白质L36。【结论】本研究找到了放线菌纲细菌MALDI-TOF MS图谱中常见的15种核糖体蛋白质信号,可为通过识别核糖体蛋白质的质谱特征峰鉴定放线菌的方法建立提供依据。     

Approaches for increasing the solution stability of proteins

Stabilization of proteins through proper formulation is an important challenge for the pharmaceutical industry. Two approaches for stabilization of proteins in solution are discussed. First, work describing the effect of additives on the thermally induced denaturation and aggregation of low molecular weight urokinase is presented. The effects of these additives can be explained by preferential exclusion of the solute from the protein, leading to increased thermal stability with respect to denaturation. Diminished denaturation leads to reduced levels of aggregation. The second approach involves stoichiometric replacement of polar counter ions (e.g., chloride, acetate, etc.) with anionic detergents, in a process termed hydrophobic ion pairing (HIP). The HIP complexes of proteins have increased solubility in organic solvents. In these organic solvents, where the water content is limited, the thermal denautration temperatures greatly exceed those observed in aqueous solution. In addition, it is possible to use HIP to selectively precipitate basic proteins from formulations that contain large amounts of stabilizers, such as human serum albumin (HSA), with a selectivity greater than 2000-fold. This has been demonstrated for various mixtures of HSA and interleukin-4. (c) 1995 John Wiley & Sons, Inc.     

The Study on the Entrapment Efficiency and In Vitro Release of Puerarin Submicron Emulsion

The entrapment efficiency (EE) and release in vitro are very important physicochemical characteristics of puerarin submicron emulsion (SME). In this paper, the performance of ultrafiltration (UF), ultracentrifugation (UC), and microdialysis (MD) for determining the EE of SME were evaluated, respectively. The release study in vitro of puerarin from SME was studied by using MD and pressure UF technology. The EE of SME was 86.5%, 72.8%, and 55.8% as determined by MD, UF, and UC, respectively. MD was not suitable for EE measurements of puerarin submicron oil droplet, which could only determine the total EE of submicron oil droplet and liposomes micelles, but it could be applied to determine the amount of free drug in SMEs. Although UC was the fastest and simplest to use, its results were the least reliable. UF was still the relatively accurate method for EE determination of puerarin SME. The release of puerarin SME could be evaluated by using MD and pressure UF, but MD seemed to be more suitable for the release study of puerarin emulsion. The drug release from puerarin SME at three drug concentrations was initially rapid, but reached a plateau value within 30 min. Drug release of puerarin from the SME occurred via burst release.     

Structural basis for unfolding pathway‐dependent stability of proteins: Vectorial unfolding versus global unfolding

Point mutations in proteins can have different effects on protein stability depending on the mechanism of unfolding. In the most interesting case of I27, the Ig‐like module of the muscle protein titin, one point mutation (Y9P) yields opposite effects on protein stability during denaturant‐induced “global unfolding” versus “vectorial unfolding” by mechanical pulling force or cellular unfolding systems. Here, we assessed the reason for the different effects of the Y9P mutation of I27 on the overall molecular stability and N‐terminal unraveling by NMR. We found that the Y9P mutation causes a conformational change that is transmitted through β‐sheet structures to reach the central hydrophobic core in the interior and alters its accessibility to bulk solvent, which leads to destabilization of the hydrophobic core. On the other hand, the Y9P mutation causes a bend in the backbone structure, which leads to the formation of a more stable N‐terminal structure probably through enhanced hydrophobic interactions.     

Direct electrochemistry and electrocatalysis of heme proteins immobilized on gold nanoparticles stabilized by chitosan

Three heme proteins, myoglobin, hemoglobin, and cytochrome c, have been adsorbed onto chitosan-stabilized gold nanoparticles (Chit-Aus) modified Au electrode via a molecule bridge like cysteine. UV-vis spectra indicated that the proteins on Chit-Aus films retained near-native secondary structures. The fabricated procedures and electrochemical behaviors of proteins on such an interface were characterized with electrochemical impedance spectra and cyclic voltammetric techniques. It was demonstrated that Chit-Aus film could not only offer a friendly environment to immobilize protein molecules but also enhance the electron transfer ability between protein molecules and underlying electrode. The effects of scan rate and pH on the electrochemical behaviors of each heme protein are discussed in detail. The resultant electrode displayed an excellent electrocatalytic response to the reduction of H(2)O(2), long-term stability, and good reproducibility.     

Local database and the search program for proteomic analysis of sperm proteins in the ascidian Ciona intestinalis

Separation of proteins by two-dimensional electrophoresis and following mass spectrometry (MS) is now a conventional technique for proteomic analysis. For proteomic analysis of a certain tissue with a limited information of primary structures of proteins, we have developed an analytical system for peptide mass fingerprinting in gene products in the testis of the ascidian Ciona intestinalis. Ciona sperm proteins were separated by two-dimensional gel electrophoresis and the tryptic fragments were subjected to MALDI-TOF/MS. The mass pattern was searched against on-line databases but resulted in less identification of these proteins. We have constructed a MS database from Ciona testis ESTs and the genome draft sequence, along with a newly devised, perl-based search program PerMS for peptide mass fingerprinting. This system could identify more than 80% of Ciona sperm proteins, suggesting that it could be widely applied for proteomic analysis for a limited tissue with less genomic information.     

MS analysis of chondroitin polymerization: effects of Mn2+ ions on the stability of UDP-sugars and chondroitin synthesis

Chondroitin polymerase from Escherichia coli strain K4 (K4CP) synthesizes chondroitin (CH) polysaccharides by the alternate addition of N-acetyl-D-galactosamine (GalNAc) and D-glucuronic acid (GlcA) to acceptor CH oligosaccharides in the presence of Mn(2+) ions. In this study, we applied matrix-assisted laser desorption ionization and time-of-flight mass spectrometry (MALDI-TOF MS) for the further characterization of the products synthesized by K4CP from CH hexasaccharide as an initial acceptor and UDP-GalNAc and UDP-GlcA as donors. The analysis identified individual CH chains of various lengths and enabled the calculation of their average molecular weights. The ion peaks of the CH chains synthesized in the short-time reactions demonstrated not only the alternate addition of GlcA and GalNAc but also the more frequent transfer of GlcA and GalNAc, consistent with our previous kinetic data. In contrast, the MS spectra of the chains synthesized in the long-time reaction showed that CH chains containing GalNAc at the nonreducing ends were more abundant than those containing GlcA. We found that this inconsistency was due to the preferential decomposition of UDP-GlcA by Mn(2+) ions. We defined the optimal conditions to yield further elongation of the CH chains that have nearly equal numbers of GlcA and GalNAc residues at the nonreducing ends.     

X-ray structures of the maltose-maltodextrin-binding protein of the thermoacidophilic bacterium Alicyclobacillus acidocaldarius provide insight into acid stability of proteins

Maltose-binding proteins act as primary receptors in bacterial transport and chemotaxis systems. We report here crystal structures of the thermoacidostable maltose-binding protein from Alicyclobacillus acidocaldarius, and explore its modes of binding to maltose and maltotriose. Further, comparison with the structures of related proteins from Escherichia coli (a mesophile), and two hyperthermophiles (Pyrococcus furiosus and Thermococcus litoralis) allows an investigation of the basis of thermo- and acidostability in this family of proteins.The thermoacidophilic protein has fewer charged residues than the other three structures, which is compensated by an increase in the number of polar residues. Although the content of acidic and basic residues is approximately equal, more basic residues are exposed on its surface whereas most acidic residues are buried in the interior. As a consequence, this protein has a highly positive surface charge. Fewer salt bridges are buried than in the other MBP structures, but the number exposed on its surface does not appear to be unusual. These features appear to be correlated with the acidostability of the A. acidocaldarius protein rather than its thermostability.An analysis of cavities within the proteins shows that the extremophile proteins are more closely packed than the mesophilic one. Proline content is slightly higher in the hyperthermophiles and thermoacidophiles than in mesophiles, and this amino acid is more common at the second position of beta-turns, properties that are also probably related to thermostability. Secondary structural content does not vary greatly in the different structures, and so is not a contributing factor.     

Preparation of regenerable magnetic nanoparticles for cellulase immobilization: Improvement of enzymatic activity and stability

To obtain regenerable magnetic nanoparticles, triethoxy(3-isocyanatopropyl)silane and iminodiacetic acid (IZ) were used as the starting material and immobilized on Fe₃O₄ nanoparticles. Copper ions (Cu²⁺ ions) were loaded on the Fe-IZ nanoparticles and used for cellulase immobilization. The support was characterized by spectroscopic methods (FTIR, NMR) and thermogravimetric analysis, transmission electron microscopy, scanning electron microscope, X-ray diffraction, energy dispersive X-ray analysis, and vibrating sample magnetometer techniques. As a result of experiments, the amount of protein bound to immobilized cellulase (Fe-IZ-Cu-E) and cellulase activity was found to be 33.1 mg/g and 154 U/g at pH 5, 50°C, for 3 h. The results indicated that the free cellulase had kept only 50% of its activity after 2 h, while the Fe-IZ-Cu-E was observed to be around 77%, at 60°C. It was found that the immobilized cellulase maintained 93% of its initial catalytic activity after its sixth use. Furthermore, the Fe-IZ-Cu-E retained about 75% of its initial activity after 28 days of storage. To reuse the support material (Fe-IZ-Cu), it was regenerated by thorough washing with ammonia or imidazole.     

Disulfide bonds and the stability of globular proteins.

An understanding of the forces that contribute to stability is pivotal in solving the protein-folding problem. Classical theory suggests that disulfide bonds stabilize proteins by reducing the entropy of the denatured state. More recent theories have attempted to expand this idea, suggesting that in addition to configurational entropic effects, enthalpic and native-state effects occur and cannot be neglected. Experimental thermodynamic evidence is examined from two sources: (1) the disruption of naturally occurring disulfides, and (2) the insertion of novel disulfides. The data confirm that enthalpic and native-state effects are often significant. The experimental changes in free energy are compared to those predicted by different theories. The differences between theory and experiment are large near 300 K and do not lend support to any of the current theories regarding the stabilization of proteins by disulfide bonds. This observation is a result of not only deficiencies in the theoretical models but also from difficulties in determining the effects of disulfide bonds on protein stability against the backdrop of numerous subtle stabilizing factors (in both the native and denatured states), which they may also affect.     

全长SOD2重组蛋白的可溶表达、纯化、稳定性与跨膜效应

超氧化物歧化酶(SOD)家族是保护细胞免受正常代谢过程中产生的活性氧(ROS)毒性所必需的,含Mn2+离子的超氧化物歧化酶(Mn-SOD,SOD2)是其中最重要的一种。本研究合成了人源SOD2全基因序列,并将其插入带有GST的原核表达载体p GEX-4T-1中,成功构建了GST-SOD2融合蛋白表达质粒。然后,将重组质粒p GEX-4T-1-SOD2转化大肠杆菌BL21(DE3),用IPTG在25℃下诱导表达融合蛋白,得到可溶性GST-SOD2融合蛋白,经GST亲和树脂纯化得到比活为1 788 U/mg的纯蛋白,分子量约为46 k Da。利用凝血酶切去GST标签后经肝素亲和柱纯化得到了电泳纯的SOD2重组蛋白,该蛋白分子量约为25 k Da,与SOD2全长序列的理论分子量相符,比活为2 000 U/mg。两种重组SOD2蛋白在生理条件下都具有良好的SOD活性,且都具有显著的跨膜能力(P0.05)。这些工作为深入研究两种全长重组SOD2蛋白的结构与生物效应建立了基础。     

Clickable gold nanoparticles for streamlining capture,enrichment and release of alkyne-labelled proteins

Alkyne-labelled proteins are generated as key intermediates in the chemical probe-based approaches to proteomics analysis. Their efficient and selective detection and isolation is an important problem. We designed and synthesized azide-functionalized gold nanoparticles as new clickable capture reagents to streamline click chemistry-mediated capture, enrichment and release of the alkyne-labelled proteins in one-pot to expedite the post-labelling analysis. Because hydrophobic surface functionalities are known to render gold nanoparticles poorly water-dispersible, hydrophilic PEG linkers with two different lengths were explored to confer colloidal stability to the clickable capture reagents. We demonstrated the ability of the capture reagents to conjugate the alkyne containing proteins at a nanomolar concentration via click chemistry, which can be immediately followed by their enrichment and elution. Furthermore, a bifunctional clickable capture reagent bearing sulforhodamine and azide groups was shown to conveniently attach a fluorophore to the alkyne-labelled protein upon click capture, which facilitated their rapid detection in the gel analysis.     

An electrostatic basis for the stability of thermophilic proteins

Two factors provide key contributions to the stability of thermophilic proteins relative to their mesophilic homologues: electrostatic interactions of charged residues in the folded state and the dielectric response of the folded protein. The dielectric response for proteins in a "thermophilic series" globally modulates the thermal stability of its members, with the calculated dielectric constant for the protein increasing from mesophiles to hyperthermophiles. This variability results from differences in the distribution of charged residues on the surface of the protein, in agreement with structural and genetic observations. Furthermore, the contribution of electrostatic interactions to the stability of the folded state is more favorable for thermophilic proteins than for their mesophilic homologues. This leads to the conclusion that electrostatic interactions play an important role in determining the stability of proteins at high temperatures. The interplay between electrostatic interactions and dielectric response also provides further rationalization for the enhanced stability of thermophilic proteins with respect to cold-denaturation. Taken together, the distribution of charged residues and their fluctuations have been shown to be factors in modulating protein stability over the entire range of biologically relevant temperatures.