Pressure-tuning of the secondary structures of proteins: Fourier transform infrared studies in the diamond anvil cell

Abstract

A self-deconvolution and fitting procedure is presented that allows the determination of pressure-induced changes in the secondary structure of proteins starting from the infrared spectrum. The method takes into account the elastic as well as the possible conformational shift of the spectral bands. Applications are presented on pressure-induced changes in bovine pancreatic trypsin inhibitor and in gramicidin incorporated into lipid bilayers.     

Influence of membrane organization on the dimerization ability of ToxR from Photobacterium profundum under high hydrostatic pressure

High hydrostatic pressure (HHP) is suggested to influence bacterial physiology by changing the structure and function of membranes and/or integral membrane proteins. In this work, the HHP-modulated dimerization behavior of the transmembrane regulatory protein ToxR from Photobacterium profundum SS9 was investigated, in response to changes in membrane organization induced by temperature and addition of phenethyl alcohol, in a background of different organisms (Escherichia coli and P. profundum) and mutants deficient in unsaturated fatty acid synthesis. Reporter strains were constructed by chromosomal integration of an ompL-promoter lacZ fusion cassette. Arabinose-controlled ToxR expression was achieved by plasmid pBADK-ToxR-his. The results demonstrate that changes in the lipid environment have a marginal effect on the function of ToxR; instead, ToxR activity appears to be largely determined by the properties of the protein itself.     

Theoretical study on interaction of cytochrome f and plastocyanin complex by a simple coarse-grained model with molecular crowding effect

ABSTRACT

We present a simple coarse-grained model with the molecular crowding effect in solvent to investigate the structure and dynamics of protein complexes including association and/or dissociation processes and investigate some physical properties such as the structure and the reaction rate from the viewpoint of the hydrophobic intermolecular interactions of protein complex. In the present coarse-grained model, a function depending upon the density of hydrophobic amino acid residues in a binding area of the complex is introduced, and the function involves the molecular crowding effect for the intermolecular interactions of hydrophobic amino acid residues between proteins. We propose a hydrophobic intermolecular potential energy between proteins by using the density-dependent function. The present coarse-grained model is applied to the complex of cytochrome f and plastocyanin by using the Langevin dynamics simulation to investigate some physical properties such as the complex structure, the electron transfer reaction rate constant from plastocyanin to cytochrome f and so on. We find that for proceeding the electron transfer reaction, the distance between metals in their active sites is necessary within about 18 Å. We discuss some typical complex structures formed in the present simulation in relation to the molecular crowding effect on hydrophobic interactions.     

Critical damage to the cellular organelles of Saccharomyces cerevisiae under sublethal conditions upon high pressure carbon dioxide treatment

ABSTRACT

High pressure carbon dioxide treatment is a non-thermal pasteurization technique. However, critical damage, resulting from the treatment, to microbial cells has not been observed directly, and the detailed mechanism of the microbicidal activity is not understood. In this study, we analyzed the damage to Saccharomyces cerevisiae organelles, which were visualized using organelle-specific GFP fusion proteins. Yeast strains were subjected to high pressure carbon dioxide treatments at 30°C and 4.0?MPa for 2–8?h. Reduction in yeast cell viability was accompanied by the disruption of endoplasmic reticulum, nuclear membrane, Golgi body, and nucleolus. However, visible damage to the cell membrane was not observed. Fluorescence microscopy was utilized to confirm that high pressure carbon dioxide treatment damaged membranes of major organelles, but not the cell membrane.     

Influence of high-intensity ultrasound on the IgE binding capacity of Act d 2 allergen,secondary structure,and In-vitro digestibility of kiwifruit proteins

Kiwifruit can trigger allergic reactions that can lead to death, causing public health concerns worldwide. In the present study, we treated kiwifruit samples with high-intensity ultrasound (20 kHz, 400 W, 50% duty cycle) for 0 to 16 min to evaluate its effect on the IgE binding capacity of kiwifruit allergen Act d 2, secondary structure and in-vitro digestibility of kiwifruit proteins. The changes in the protein solubility and microstructures of kiwifruit were also analyzed. The results showed that treatment with powerful ultrasound caused a significant disruption in the microstructure of kiwifruit tissues, leading to the changes in the secondary structures of proteins, including a loss of alpha-helixes and an increase in beta-sheet structures. These structural changes were due to the ultrasound treatment, especially 16 min of treatment, resulted in a 50% reduction in Act d 2 allergen content and significantly improved in-vitro digestibility up to 62% from the initial level of 35%. Furthermore, the solubility of the total proteins present in kiwifruit samples was significantly decreased by 20% after 16-min ultrasound processing. The results of this work showed that high-intensity ultrasound treatment has a potential application in reducing the allergenicity of kiwifruit or related products.     

Investigation of the manganese stress on wheat plant by attenuated total reflectance Fourier transform infrared spectroscopy

ABSTRACT

The present paper deals with the investigation of the changes in chemical contents of the leaf of wheat seedling stressed by excess manganese using attenuated total reflectance Fourier transform infrared spectroscopy (FTIR) technique. The infrared spectra of the leaves of control and manganese-treated wheat plants have been recorded in the spectral region 485–4000?cm^?1 at a resolution of 4?cm^?1. The recorded spectrum has been analyzed with the help of curve-fitting method for the quantitative estimation of chemical contents and conformational changes. The study indicated changes arising in the polysaccharide, lignin, amino acid, secondary structure of protein, and lipid in the leaves of control and manganese-treated wheat plants. Manganese treatment increased the amount of cellulose, lignin, and amide II till 200?μM concentration, while a decrease was observed at 1000?µM concentration. Manganese induced conformational changes in the secondary structure of protein which was indicated by shifting of the bands to the higher wavenumber and change in the β sheet/α helix ratio. In addition, manganese stress decreased lipid content in the leaves of the wheat seedlings. The study demonstrates the potential of attenuated total reflectance FTIR for the non-invasive and rapid monitoring of the plants stressed with heavy metals.     

The Possible Contribution of Isotope Effects on Ester- and Aldol Condensations to Isotope Discriminations and Isotopic Patterns of Natural Compounds

Abstract

Acetyl-CoA provides the C-2-units for the formation of many secondary compounds. The primary pattern of this intermediate is originating from glucose, further modifications are introduced by isotope effects on the pyruvate-dehydrogenase-reaction and on ester- and aldol-reactions in the course of the biosynthesis of secondary products. The overlap of these various effects is demonstrated through the pattern of some natural products.

The partial pattern of limonene and pulegone, respectively, products from different origin are not identical, this is interpreted as a consequence of individual relative contributions of the above mentioned effects in the plants in question.

On the other hand the distinct relative depletion in a position of sinigrin, a glucosinolate from Brassica nigra, originating from CH₃ of acetate can be assigned to an isotope effect of the aldol reaction between a precursor and acetyl-CoA. An corresponding depletion has been measured in position 5 of citrate. Therefore the kinetic isotope effect on the citrate synthase reaction is studied in vitro at present.     

Interactive Effects of Pressure,Temperature and Time on the Molecular Structure of Ovalbumin,Lysozyme and β-Lactoglobulin

The structure of three food proteins, ovalbumin, lysozyme and g -lactoglobulin were investigated when subjected to pressure, temperature and holding time. Structural effects were determined by the examination of circular dichroism spectra. Experiments were performed using pressures of up to 105 MPa, temperatures up to 79 °C and holding times of 30 minutes using experimental design methodology and compared with ultra high pressures (600 MPa). Examination of the spectra showed that the structure of the three proteins behaved differently to the processing conditions. g -lactoglobulin was found to be the least stable protein while lysozyme was the most stable protein. The higher pressure of 105 MPa was not sufficient to cause structural change when used at ambient conditions but when used in conjunction with raised temperatures and holding time, the applied energy was found to be sufficient to disrupt the protein structure.     

Computational studies on non-succinimide-mediated stereoinversion mechanism of aspartic acid residues assisted by phosphate

ABSTRACT

Although nearly all of the amino acids that constitute proteins are l-amino acids, d-amino acid residues in human proteins have been recently reported. d-amino acid residues cause a change in the three-dimensional structure of proteins, and d-aspartic acid (Asp) residues are considered to be one of the causes of age-related diseases. The stereoinversion of Asp residues in peptides and proteins is thought to proceed via a succinimide intermediate; however, it has been reported that stereoinversion can occur even under conditions where a succinimide intermediate cannot be formed. In order to elucidate the non-succinimide-mediated stereoinversion pathway, we investigated the stereoinversion of l-Asp to d-Asp catalysed by phosphate and estimated the activation barrier using B3LYP/6?31+G(d,p) density functional theory (DFT) calculations. For the DFT calculations, a model compound in which the Asp residue is capped with acetyl and methyl-amino groups on the N- and C-termini, respectively, was used. The calculated activation barrier was not excessively high for the stereoinversion to occur in vivo. Therefore, this stereoinversion mechanism may compete with the succinimide-mediated mechanism.     

Inflection point of the fluorescence excitation spectra induced by secondary inner filter effect

Abstract

The secondary inner filter effect on the fluorescence excitation spectra of rhodamine 6G aqueous solutions with concentration was demonstrated in this paper. The peak of fluorescence excitation spectrum stays at 525?nm at low concentrations, while it breaks up and turns into valley at high concentrations. The threshold concentration was determined to be 3.16?mg/L by the second derivative spectroscopy. A mathematical model was proposed to explain the inflection point of fluorescence excitation spectrum caused by the secondary inner filter effect. On the basis of it, the threshold concentration was calculated to be 2.86?mg/L, approaching to the experimental result.     

Annealing effects on the microstructure of sputtered gold layers on oxidized silicon investigated by scanning electron microscopy and scanning probe microscopy

The structure of Au layers deposited by sputtering on oxidized p-type Si(100) substrates is investigated by a combination of scanning electron microscopy and scanning probe microscopy. The effect of the temperature on the grain structure of the layers has been determined, revealing that an annealing temperature of 300° C results in a larger grain size and smoother surfaces but generates some cracks in the film surface. At an annealing temperature of 500° C, further grain growth is observed, but a high density of cracks and voids also results while there is little enhancement regarding the smoothness of the grain surfaces.     

不同尺度的蛋白质在纯石墨烯及氧化石墨烯表面的吸附比较

Using all-atom molecular dynamics (MD) simulations, we have investigated the adsorption stability and conformation change of different proteins on the surface of pristine graphene (PG) and graphene oxide (GO). We find that: (i) with the cooperation of the electrostatic interactions between proteins and oxygen-containing groups, GO shows better adsorption stability than PG; (ii) the peptide loses its secondary structure on both PG and GO surface, and the a-helix structure of the protein fragment is partially broken on PG surface, but is well preserved on GO surface, while the secondary structure of globular protein has no distinct change on both PG and GO surface. In general, GO presents better biocompatibility than PG. Our results are of significant importance to understand the interactions between proteins and PG/GO and the applications of PG/GO in biotechnology and biomedicine.     

脉冲微波辐照影响心肌细胞膜蛋白构象及其机制的研究

应用显微傅里叶变换红外光谱技术研究了脉冲微波辐照对心肌细胞膜蛋白质构象、功能的影响和相关分子机制。结果表明,辐照可对心肌细胞的细胞膜蛋白质结构产生明显影响。细胞膜脂质中—CH₂—、磷脂结构中CO、蛋白质酰胺Ⅰ,Ⅱ带的伸缩振动峰消失或位移。辐照后心肌细胞膜蛋白质二级结构也出现明显变化,α-螺旋和β-折叠结构减少,二级结构无序化程度增加。上述变化均与辐照剂量呈正相关。结果提示受脉冲微波辐照后,心肌细胞膜蛋白构象的完整性受损,膜稳定性及流动性下降,膜上多种生物活性结构被破坏,上述变化构成了细胞膜功能丧失、细胞形态和结构损伤、细胞凋亡等病理学效应的生物化学基础。文章首次从蛋白质构象角度阐述了微波辐照对心肌细胞膜损伤的分子病理机制。     

Far-infrared radiation effect on the structure and properties of proteins

This paper describes the effect of far-infrared radiation on the activity and secondary structure of albumin, alcohol dehydrogenase, peroxidase and trypsin. For the first time the data obtained allowed a conclusion that enzymatic activity changes under the influence of radiation having the wavelength of 90 m. No disruptures of polypeptide chains or oligomerization of proteins were obtained. It has been shown that albumin secondary structure calculated from circular dichroism spectra changes lineary according to dose of laser emission.Abbreviations FIR far-infarred radiation - CD circular dichroism - ADG alcohol dehydrohenase - PXD peroxidase - ANS 1-anilinenaphalene-8-sulfonic acid     

Effect of high-intensity ultrasound on the structural,rheological, emulsifying and gelling properties of insoluble potato protein isolates

The denaturation and lower solubility of commercial potato proteins generally limited their industrial application. Effects of high-intensity ultrasound (HIU) (200, 400, and 600 W) and treatment time (10, 20, and 30 min) on the physicochemical and functional properties of insoluble potato protein isolates (ISPP) were investigated. The results revealed that HIU treatment induced the unfolding and breakdown of macromolecular aggregates of ISPP, resulting in the exposure of hydrophobic and R–SH groups, and reduction of the particle size. These active groups contributed to the formation of a dense and uniform gel network of ISPP gel and insoluble potato proteins/egg white protein (ISPP/EWP) hybrid gel. Furthermore, the increase of solubility and surface hydrophobicity and the decrease of particle size improved the emulsifying property of ISPP. However, excessive HIU treatment reduced the emulsification and gelling properties of the ISPP. Meanwhile, HIU treatment changes the secondary structure of ISPP. It could be speculated that the formation of a stable secondary structure of ISPP initiated by cavitation and shearing effect might play a dominant role on gel strengthens and firmness. Meanwhile, the decrease in relative content of β-turn had a positive effect on the formation of small particle to improve emulsifying property of ISPP.     

High Pressure Effect on Meat and Lupin Protein Digestibility

High pressure treatment is a mild treatment concerning the nutritional characteristics: for instance vitamins content is very few affected by high-pressure treatment. But the impact of high-pressure on protein digestibility remains poorly understood. This work presents effect of high-pressure treatment on in vitro digestibility of meat and lupin proteins. Two high-pressure conditions (200 and 500 MPa 10 min.) and a heat treatment of 95 °C, 30 min were studied. In vitro digestibility was evaluated with a pepsin reaction. Results show than for both type of proteins, samples pressurised at 500 MPa were the most hydrolysed. Meat proteins were less hydrolysed than control either after heat or 200 MPa treatment. Lupin proteins where more hydrolysed than control after heat or 200 MPa treatment. Thus we revealed that high-pressure treatment presents an effect on the in vitro digestibility of proteins, opening a new perspective of investigations.     

de Haas-van Alphen effect and the Fermi surface of PrNi5

The Fermi surface of PrNi₅ has been studied by the measurements of the de Haas-van Alphen (dHvA) effect at temperatures between 0.3 and in magnetic fields up to 12 T. Two dHvA frequencies have been obtained. The electronic structure of PrNi₅ was calculated using the full potential linearized augmented plane wave method. Five sheets of the Fermi surface and the multiple extremal cross sections were determined. First and second sheet have a hole-like structure. The agreement between theory and experiment is obtained by a small downward shift ( 0.1 eV) of the Fermi energy which is probably due to an underestimation of the role of 4 f electrons. Received 9 May 2000 and Received in final form 20 September 2000     

Unfolding and Fibrillogenesis of Insulin: Temperature,Pressure and Chemistry

Protein folding or unfolding can lead to the population of intermediates or partially unfolded conformations that have a high aggregation tendency. Some of these states associate in vivo to form fibrillar structures. These fibrils are the hallmark of molecular diseases such as Alzheimer's disease. It has been suggested that in vitro fibril formation is a generic property of all proteins. Insulin has been chosen as a model protein to study the process of fibrillation with Fourier-transform infrared spectroscopy. It is found that the formation of fibrils is preceded by amorphous aggregation. We also investigated the effect of hydrostatic pressure on insulin fibrils. The observed spectral changes are interpreted in terms of fibril dissociation into protofilaments. Preliminary results indicate that pressure is an interesting tool to characterize the interactions that maintain the fibril structure.