Changes In Protein Secondary Structure Research Articles

Can cells maintain their bioactivity in ionic liquids? A novel single‐cell assessment by Raman microspectroscopy

AbstractIonic liquids (ILs) are widely exploited in numerous applications in chemistry, biology, and materials science. Assessment of the toxicity of ILs is thus urgently needed to develop genuinely “green” ILs that do not pose hazards to the environment and humans. Despite considerable efforts mostly with cell viability assays, little has been understood about how, at the molecular level, ILs affect the bioactivity and intracellular chemistry of single individual cells. Here, we report a new method based on Raman microspectroscopy for quantitatively examining the effects of ILs on the bioactivity of single living fission yeast cells beyond just counting viable cells. The ILs studied are two hydrophilic ILs, 2‐hydroxyethyl‐trimethylammonium l‐(+)‐lactate [Cho][Lac] and 1‐ethyl‐3‐methylimidazolium acetate [emim][AcO], and they were mixed with liquid medium. We used a characteristic yeast Raman band at 1,602 cm−1, which sharply reflects the bioactivity of yeast cells. For both ILs, the Raman spectra measured at lipid droplets in IL‐treated yeast cells clearly exhibited the 1,602‐cm−1 band, indicating that yeast cells can indeed maintain their bioactivity in the ILs. Furthermore, we observed that the amide I peak in the cytoplasmic/nuclear Raman spectrum significantly blueshifts in [emim][AcO] compared with that in the normal medium and [Cho][Lac]. This unexpected finding suggests an IL‐induced change in protein secondary structure from α‐helix to β‐sheet and random‐coil structures. The present study not only demonstrates that Raman microspectroscopy utilizing the 1,602‐cm−1 band of yeast is a unique, powerful tool for single‐cell assessment of IL toxicity, but it also calls attention to the effects of ILs on the structure and hence functions of intracellular molecules that might have been overlooked in previous studies.

Effects of formulation development methods on the stability of model protein pharmaceuticals embedded in solid lipid matrices

This study was conducted to investigate the influence of formulation development methods on the stability (secondary structure, aggregation, and biological activity) of protein drugs embedded in lipid matrices. Catalase, horseradish peroxidase, and α-chymotrypsin were employed as model proteins, while Precirol® AT05 (glyceryl palmitostearate) was used as lipid matrix. Protein-loaded lipid matrices were prepared using melting and mixing and wet granulation methods. Attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy, size exclusion chromatography (SEC) and biological activity analyses were performed. ATR FT-IR analysis indicated significant interference of the lipid with the protein amide-I band, which was eliminated using spectral subtraction. Wet granulation method induced more changes in protein secondary structure compared to melting and mixing method. SEC analysis gave evidence of protein aggregation for catalase upon adopting the wet granulation method. The biological activity of catalase was found to reduce significantly than other two proteins upon using wet granulation method, which might be ascribed to both secondary structure alterations and the formation of aggregates. Horseradish peroxidase and α-chymotrypsin did not form any soluble aggregates. In conclusion, melting and mixing method emerged as a better incorporation method compared to wet granulation because of better stability shown by the formulated proteins.

Effects of ultrasound-assisted freezing at different power levels on the structure and thermal stability of common carp (Cyprinus carpio) proteins

This study investigated the effect of ultrasound-assisted immersion freezing (UIF) at different ultrasonic power levels on the myofibrillar protein primary, secondary and tertiary structures of common carp (Cyprinus carpio). Furthermore, protein thermal stability, electrophoresis pattern, and microstructure of the muscle tissue were also studied. Compared with a control, an ultrasonic power of less than 175 W had no significant negative effect on protein primary structure (P > 0.05), including total sulfhydryl, reactive sulfhydryl, carbonyl groups, free amino groups, dityrosine content, and surface hydrophobicity. UIF at 175 W (UIF-175) minimized the changes in protein secondary and tertiary structures. There were no obvious changes in the SDS-PAGE patterns of the control and frozen sample proteins. Microstructure analysis showed that an appropriate ultrasonic power (UIF-175) promoted the formation of smaller and more uniform ice crystals, reduced the damage of muscle tissue by ice crystals, and maintained the sarcomere integrity. In addition, UIF-175 samples had higher protein thermal stability. Overall, ultrasound treatment at a proper power (UIF-175) effectively minimized the changes in protein structure and protected the protein thermal stability during freezing process.

Binding of peanut allergen Ara h 2 with Vaccinium fruit polyphenols

The potential for 42 different polyphenols found in Vaccinium fruits to bind to peanut allergen Ara h 2 and inhibit IgE binding epitopes was investigated using cheminformatics techniques. Out of 12 predicted binders, delphinidin-3-glucoside, cyanidin-3-glucoside, procyanidin C1, and chlorogenic acid were further evaluated in vitro. Circular dichroism, UV–Vis spectroscopy, and immunoblotting determined their capacity to (i) bind to Ara h 2, (ii) induce protein secondary structural changes, and (iii) inhibit IgE binding epitopes. UV–Vis spectroscopy clearly indicated that procyanidin C1 and chlorogenic acid interacted with Ara h 2, and circular dichroism results suggested that interactions with these polyphenols resulted in changes to Ara h 2 secondary structures. Immunoblotting showed that procyanidin C1 and chlorogenic acid bound to Ara h 2 significantly decreased the IgE binding capacity by 37% and 50%, respectively. These results suggest that certain polyphenols can inhibit IgE recognition of Ara h 2 by obstructing linear IgE epitopes.

Infrared and Raman Spectroscopic Studies of Molecular Disorders in Skin Cancer.

To investigate the molecular structural disorders of cancerous skin. Human malignant melanoma and basal cell carcinoma biopsies were used for the investigation. Fourier transform infrared (FT-IR), Raman spectroscopy, and scanning electron microscopy were utilized. Spectral differences between healthy, basal cell carcinoma and melanoma tissues were recorded. The FT-IR bands of vasCH2, vsCH2 and Raman vsCH3 of cell membrane lipids were increased in intensity in melanoma due to an increased lipophilic environment. The FT-IR band at 1,744 cm-1 assigned to malondialdehyde can be used as a band diagnostic of cancer progression. The amide I bands at 1,654 cm-1 and 1,650 cm-1 for Raman and FT-IR, respectively were broader in spectra from melanoma, reflecting changes of protein secondary structure from α-helix to β-sheet and random coil. The intensity of the FT-IR band at 1,046 cm-1 was increased in melanoma, suggesting glycosylation of the skin upon cancer development. Another band that might be considered as diagnostic was found at about 815 cm-1 in melanoma and was attributed to Z-DNA configuration. As far as we know, this is the first time that scanning electron microscopy revealed that metal components of titanium alloys from tooth implants were transferred to melanoma tissue taken from the back of one patient. Vibrational spectroscopy highlighted increased glycosylation in melanoma.

Effect of Temperature on Oxymyoglobin and Metmyoglobin Denaturation Properties

ObjectivesPremature browning is a condition where the interior of patty/steak will appears fully cooked before the temperature necessary to kill foodborne pathogens is reached. Previous research reported that approximately 50% of ground beef retailed in the US is susceptible to premature browning. Myoglobin form present in the interior of steak or patties determines the cooked color appearance. Although previous studies noted that myoglobin denaturation is primarily responsible for the cooked color appearance, limited knowledge is currently available about the effect of temperature on oxymyoglobin and metmyoglobin denaturation properties. The objective of the current study was to determine the effects of myoglobin forms on thermal stability using circular dichroism spectroscopy.Materials and MethodsOxymyoglobin and metmyoglobin solutions at pH 5.6 in 50 mM sodium phosphate buffer were incubated in a continuous heat increment water bath for 10 min. At specific temperature points (65, 71, 73, and 76°C), myoglobin denaturation was determined by changes in myoglobin concentration and by protein unfolding (fluorescence and absorbance) methods. The myoglobin thermal stability was also determined by circular dichroism spectroscopy. Changes in secondary protein structure were determined every 2°C from 52 to 92°C. The data were analyzed as completely randomized using the Mixed Procedure of SAS. A significance level of 0.05 was used to determine differences between means.ResultsOxymyoglobin had greater (p < 0.05) unfolding (as indicated by absorbance changes) than metmyoglobin at all temperatures. However, at 65, 71, and 73°C there were no differences (p > 0.05) in fluorescence intensities between myoglobin forms. Circular dichroism spectroscopy indicates that oxymyoglobin is more heat labile than metmyoglobin.ConclusionThe results indicate that oxymyoglobin had greater denaturation and unfolding than metmyoglobin. Use of appropriate myoglobin denaturation quantification technique will help characterize premature browning.

Evaluation of the secondary structures of protein in the extracellular polymeric substances extracted from activated sludge by different methods

The changes of protein secondary structures in the extracellular polymeric substances (EPS) extracted from activated sludge by four different methods were studied by analyzing the amide I region (1700–1600 cm−1) of the Fourier transform infrared spectra and model protein test. The results showed the molecular weight distribution of organic matter extracted by centrifugation, heating and cation exchange resin (CER) was similar, while the EPS extracted by centrifugation (Control) and CER had similar fluorescent organic matter. The protein secondary structures of extracted EPS by the four methods were different. The similarities of protein secondary structures between the EPS extracted by CER with the Control were the highest among the four extracted EPS. Although the EPS yield extracted by formaldehyde + NaOH method were the highest, its protein secondary structures had the lowest similarity with those extracted by the Control. Additionally, the effects of centrifugation and CER extraction on the secondary structures of bovine serum albumin were also lower than that of other extraction processes. CER enables the second maximum extraction of EPS and maximum retention of the original secondary structure of proteins.

FTIR and Proteome Analysis of Sunflower Seedling (Helianthus annuus L.) under Lead Stress

The effects of lead stress on the dynamic changes of protein secondary structure and differential protein expression of sunflower seedlings in soil were studied using Fourier transform infrared spectroscopy (FTIR) and two-dimensional (2D) electrophoresis. Under lead stress treatment, the α-helices and β-sheets of protein secondary structure for sunflower seedlings were changed. The content changes, 110% increased in a-helices of roots and 40% declined in β-sheets of leaves, were observed, respectively. Furthermore, 11 differential protein spots were obtained by analyzing 2-DE gel images, while No. 9 were identified by MALDI-TOF/TOF MS, successfully.

Insights into protein recognition for γ-lactone essences and the effect of side chains on interaction via microscopic, spectroscopic, and simulative technologies.

The binding properties between γ-lactone essences and HSA were investigated to explore interactional mechanism and influence of ligand side chains on binding via computer simulations, microscopy, and multiple-spectroscopies. Docking and molecular dynamics presented protein recognition mode with low fluctuations. NMR analysis revealed that the lactone ring of ligands was the main group bound to HSA. UV-vis and lifetime results revealed that the combination was static quenching mechanism with binding constants of 102-103 L/mol. FTIR and CD spectra showed conformational changes in the protein secondary structure induced by ligands. Side chains affect the binding process through steric hindrance and hydrophobicity. AFM images showed the four compounds caused different effects on molecular size of HSA. In conclusion, the binding ability and the protein secondary structure changes had a positive correlation with the length of side chain. These studies are beneficial for understanding the safety and biological action of γ-lactone essences.

FTIR study of Achilles tendinopathy: protein secondary structure changes in tendon post injury

Abstract Introduction Tendinopathy, a disease involving tendon inflammation and damage, results in considerable productivity and economic losses for the patient and the society. Currently available diagnosis/ treatment monitoring modalities are less efficacious and highly subjective, underlying the need for better tools. Previously, we have shown that Fourier Transform Infrared (FTIR) spectroscopy has potential in tendinopathy diagnosis/ monitoring, when combined with multivariate statistical analysis. The current study explores the spectral range that gave the best results in statistical analysis, to discover protein secondary structures associated with tendon injury and recovery that can act as markers of disease. Methods Animals (n=60) underwent a surgery in which Achilles tendon were injured by dropping a 20g weight. Rats were divided into three groups (n=20) – control (C), Achilles tendon injury (I) and Achilles tendon injury treated with amniotic membrane fragment treated (T). FTIR spectra were obtained from each group 3, 7, 14, and 28 days post injury/ treatment. Results Triple helix, β-turn, and disordered structure levels differ between control, injured and treated tendons over the time period studied. Parallel β-sheets increase steadily over time in treated tendons compared to control and injured. Conclusion Combined analysis of Triple helix, β- sheets, β-turn, and disordered structure levels may be useful for tendinopathy diagnosis and treatment monitoring. However, further studies in this area are required to confirm the findings.

Metabolic adaptation of a Chlamydomonas acidophila strain isolated from acid mine drainage ponds with low eukaryotic diversity

The diversity and biological characteristics of eukaryotic communities within acid mine drainage (AMD) sites is less well studied than for prokaryotic communities. Furthermore, for many eukaryotic extremophiles the potential mechanisms of adaptation are unclear. This study describes an evaluation of eight highly acidic (pH 1.6–3.1) and one moderately acidic (pH 5.6) metal-rich acid mine drainage ponds at a disused copper mine. The severity of AMD pollution on eukaryote biodiversity was examined, and while the most species-rich site was less acidic, biodiversity did not only correlate with pH but also with the concentration of dissolved and particulate metals. Acid-tolerant microalgae were present in all ponds, including the species Chlamydomonas acidophila, abundance of which was high in one very metal-rich and highly acidic (pH 1.6) pond, which had a particularly high PO4-P concentration. The C. acidophila strain named PM01 had a broad-range pH tolerance and tolerance to high concentrations of Cd, Cu and Zn, with bioaccumulation of these metals within the cell. Comparison of metal tolerance between the isolated strain and other C. acidophila strains previously isolated from different acidic environments found that the new strain exhibited much higher Cu tolerance, suggesting adaptation by C. acidophila PM01 to excess Cu. An analysis of the metabolic profile of the strains in response to increasing concentrations of Cu suggests that this tolerance by PM01 is in part due to metabolic adaptation and changes in protein content and secondary structure.

Effects of sonication on the physicochemical and functional properties of walnut protein isolate

The objective of this study was to investigate the impact of high-intensity ultrasound treatment (sonication) on the molecular, physicochemical, and functional properties of walnut protein isolate. Aqueous walnut protein suspensions were sonicated at varying power levels (200, 400 or 600 W) and times (15 or 30 min), and then any alternations in protein structure and properties were determined. SDS-PAGE demonstrated that there were no changes in protein electrophoretic patterns, indicating that sonication did not break covalent bonds. Circular dichroism spectroscopy indicated a small change in protein secondary structure after sonication, with a decrease in α-helix and increase in β-sheet, β-turn, and random coil content. There was an increase in surface free sulfhydryl (SH) groups and a decrease in fluorescence intensity after sonication, indicating that appreciable changes in tertiary structure occurred. Ultrasound reduced the size of the particles in aqueous walnut protein dispersions as confirmed by static light scattering and scanning electron microscopy, suggesting that sonication dissociated protein aggregates. Moreover, the water-solubility (+22%), emulsifying activity index (+26%), and emulsifying stability index (+41%) all increased after sonication. These results suggest that sonication is a valuable tool for improving the functional attributes of walnut proteins.

Hempseed meal protein isolates prepared by different isolation techniques. Part I. physicochemical properties

Protein isolates from hemp seed meal were prepared using alkaline extraction/isoelectric precipitation (HPI) and micellization (HMI) procedures and compared in terms of their physicochemical properties and functionality. The micellization technique resulted in lower protein recovery than the isoelectric precipitation technique. Both HPI and HMI proteins had protein contents higher than 90%. The HMI protein powders were lighter in colour than the corresponding HPI isolates due to higher content of co-extracted polyphenols for the latter. The electrophoretic mobility and subunit composition, as well as amino acid composition of the isolates were not affected by the extraction procedure, indicative of similar protein composition. The HPI exhibited minimum protein solubility at pH 5.0, while for HMI it was shifted to pH 6.0. Differential scanning calorimetry indicated that highly alkaline conditions during HPI extraction led to partial protein denaturation which is reflected in lower transition enthalpy of HPI than HMI. FTIR spectra have also confirmed changes in HPI protein secondary structure, i.e. lower intensity of the peak (1634 cm−1) corresponding to native protein structural elements such as intramolecular β-sheets and higher intensities of peaks (1618 cm−1, 1683 cm−1 and 1694 cm−1) indicating enhanced protein aggregation compared to HMI. Protein conformational changes during alkali extraction resulted in higher water retention capacities of HPI in comparison to HMI.

Study of Changes of Protein Secondary Structure in Fertilized and Un-Fertilized Hatching Eggs during Storage and Incubation

This study was carried out at Poultry Researches Station – Livestock Department in the Agricultural Researches Directorate in Baghdad Province/ Abu Gharib from 28/12/2015 to 26/1/2016, The aim of the study is to find out an important chemical changes in fertilized and unfertilized eggs during storage and incubation, the eggs were used in experiment from a local laying hens flock of the same station. 360 fertilized egg and 360 un-fertilized egg had been used and distributed into four treatments, 180 eggs for each treatment. The eggs of each treatment had been labeled and weighted, then two groups of fertilized and un-fertilized eggs had been stored for 7 days while others two groups for 4 days on the temperature 15-18 and 70-75% relative humidity. The eggs of treatments had been carried into the hatchery and incubated on 37.8 (99.5 Fahrenheit) and 60-65% relative humidity. The results display that the effect of store 4 and 7 days were not different significantly in the values of (pH) for eggs whites and yolks, the results of (FTIR) showed certain changes in the minor formation of Protein in the fertilized egg yolks and whites in processing of store period because of protein change into the form which could be utilized by living creature.

Structural changes and energy absorption mechanisms during fracture of thermoplastic protein blends using synchrotron FTIR

Novatein thermoplastic protein embrittles quickly after processing due to desorption of plasticizer, leading to poor energy absorbing properties such as impact strength. However, impact modified Novatein blends, containing functionalized polyethylene or nano‐scale core‐shell particles exhibit increased energy absorbing properties. The phase distribution of reinforced blends, and the protein secondary structure changes as a result of blending and fracture was investigated using synchrotron FT‐IR microspectroscopy. Morphological changes in Novatein/polyethylene blends were responsible for variations in mechanical properties, rather than changes in secondary structure. In contrast, a greater impact strength was correlated to an increase of disordered secondary structures in core‐shell particle reinforced Novatein. Blends which exhibited greater elongation or yielding during fracture showed a significant difference between protein secondary structure in the bulk matrix and the yielded material after fracture. It was concluded that the change in conformation of protein structures during fracture is an energy absorbing mechanism that acts in conjunction with the free elongation of the matrix due to decreasing interparticle distance for materials reinforced with nano‐particles. POLYM. ENG. SCI., 58:E124–E134, 2018. © 2017 Society of Plastics Engineers

Serum albumin interaction with xanthine drugs at nano-bio interfaces: A combined multi-spectroscopic and molecular modelling approach

Theophylline (THP), theobromine (THB) and caffeine (CAF) belong to the xanthine group of drugs and are principle component of tea or coffee with important pharmacological activities and clinical applications. The interaction between these drugs and serum albumin proteins obtained from bovine and human (BSA and HSA, respectively) in the presence and absence of silver as well as gold nanoparticles (Ag and AuNP, respectively) was investigated for the first time by using a series of biophysical techniques and molecular modelling calculations. Time resolved fluorescence measurements confirm that all the drugs quench the intrinsic tryptophan fluorescence of the proteins through a static mechanism irrespective of the absence or presence of NPs. However, the binding constant of the proteins towards the drugs changes considerably in presence of NPs. Addition of drugs decreases α-helicity of the proteins both in the absence and presence of NPs; however, no substantial change in protein secondary structure is perceived only with the NPs. The results show that the drug carrying capacity of the albumins can be significantly modulated with metal NPs towards desired pharmacokinetic and therapeutic applications without compromising the basic structure; and therefore, the function of the proteins.

Alternative stable conformation capable of protein misinteraction links tRNA synthetase to peripheral neuropathy.

While having multiple aminoacyl-tRNA synthetases implicated in Charcot-Marie-Tooth (CMT) disease suggests a common mechanism, a defect in enzymatic activity is not shared among the CMT-causing mutants. Protein misfolding is a common hypothesis underlying the development of many neurological diseases. Its process usually involves an initial reduction in protein stability and then the subsequent oligomerization and aggregation. Here, we study the structural effect of three CMT-causing mutations in tyrosyl-tRNA synthetase (TyrRS or YARS). Through various approaches, we found that the mutations do not induce changes in protein secondary structures, or shared effects on oligomerization state and stability. However, all mutations provide access to a surface masked in the wild-type enzyme, and that access correlates with protein misinteraction. With recent data on another CMT-linked tRNA synthetase, we suggest that an inherent plasticity, engendering the formation of alternative stable conformations capable of aberrant interactions, links the tRNA synthetase family to CMT.

Effect of salts and nonionic surfactants on thermal characteristics of egg white proteins

Effect of salts (Sodium chloride (NaCl), Sodium sulfate (Na2SO4), Ammonium sulfate ((NH4)2SO4), and nonionic surfactants (glycerol, tween20, tween80) on thermal properties of egg white proteins as a whole were investigated. Egg white solutions with additive (0, 0.5 and 1%) were collected after 0, 1 and 2min heat treatment. Physico-chemical properties of egg white proteins were evaluated by measuring heat coagulation time, solubility and turbidity of solution. Adding glycerol caused the most significant decrease in turbidity and increase in heat coagulation time and solubility of egg white, although Sodium Chloride had the least positive impact on physico-chemical properties of egg white under heat treatment. The Fourier transform infrared (FT-IR) spectroscopy analysis of heat treated egg white proteins as a whole with additives demonstrated changes in secondary protein structure, which are presented regarding the shape, intensity and position of FT-IR band. Meanwhile, it showed a good correlation with the physico-chemical properties consequences. Generally, the effect of nonionic surfactants were more noticeable than that of salts in preventing of egg white proteins aggregation under heat treatment. By improving thermal stability of egg white proteins, its usage in thermal processing industry can be evolved.

Evaluation of in silico ADMET analysis and human serum albumin interactions of a new lanthanum(III) complex by spectroscopic and molecular modeling studies

A new lanthanum(III) complex, [La(Phe)3(H2O)2] (Phe=phenylalanine), was synthesized. The complex was characterized by elemental analysis, conductivity measurement, FT-IR, UV/Vis and 1H NMR techniques. Drug-like properties of this complex was evaluated through in silico ADMET predictions. The reactivity of this complex to human serum albumin (HSA) under simulated physiological conditions was studied by spectroscopic and molecular docking analysis. The fluorescence quenching of protein by addition of La(III) complex was due to static quenching mechanism. The thermodynamic parameters show that the binding process was spontaneous and that hydrogen bonds and hydrophobic forces play a major role in the association of the HSA–La(III) complex. Binding parameters between La(III) complex and protein such as binding sites number (n ∼1) and binding constant (Ka ∼104M−1) were determined. The conformational changes of protein secondary structure in the presence of above complex were demonstrated by FT-IR, three-dimensional fluorescence and UV–Vis absorption techniques. Molecular docking of La(III) complex relating to HSA was investigated and confirmed the experimental results. All these experimental and computational results clarified that La(III) complex could bind with HSA effectively, which could be a useful guideline for efficient drug design.

Synthesis, Characterization, Cytotoxicity and Detailed HSA Interaction of New Zinc(II) Complexes Containing Dithiocarbamate and Heterocyclic N-donor Ligands

ABSTRACT[Zn(ph-dtc)(bpy)]Cl (1) and [Zn(ph-dtc)(phen)]Cl (2) (where bpy = 2,2′-bipyridine, phen = 1,10-phenanthroline and ph-dtc = phenylacetichydrazidedithiocarbamate) were synthesized and characterized by elemental analysis and spectroscopic methods (FT–IR, UV–Vis and 1H NMR). Zn(II) complexes were examined in biological tests in vitro using MCF-7 breast cancer cell line. Both complexes showed significant cytotoxic activity against human breast cancer MCF-7 cells. The interaction of above compounds with Human Serum Albumin (HSA) was investigated by means of various spectroscopic (at pH ∼ 7.4 in Tris–HCl buffer medium) and molecular docking methods. The fluorescence data showed that 1 and 2 quench the intrinsic fluorescence of HSA through a static quenching procedure. The binding constants (Kb) and the number of binding sites (n ∼ 1) were calculated. The thermodynamic analysis suggested that hydrophobic interaction played major roles in the binding of 1 or 2 to HSA. The distance r between protein and the above-mentioned compounds was obtained according to fluorescence resonance energy transfer. The conformational changes of protein secondary structure in the presence of Zn(II) complexes were proven using UV–Vis absorption and circular dichroism techniques. Also, docking results confirmed the spectroscopic results.