Random Coil Components Research Articles

Preparation of lentil and quinoa protein complexes through protein–protein interactions and water kefir–assisted fermentation to improve protein quality and functionality

IntroductionIndustrial applications of lentil (LP) and quinoa (QP) proteins are limited due to their relatively poor water solubility. In this study, a combination of protein-protein interaction (PPI) and fermentation was used to improve the functionality and nutritional value of LP by conjugating them with QP. The reaction conditions between LP and QP for producing these conjugates were established.MethodsThe ratio of LP to QP was equal (50:50), and complexation was carried out at 25°C for 60 min. Fermentation of the solubilized LP-QP complexes (1%, w/v) for 5 days at 25°C with water kefir (5%, v/v) was carried out to enhance the protein quality and functionality of the LP-QP complexes.ResultsThe combined technique significantly enhanced protein digestibility, decreased the proportion of α-helices in the protein structure in favor of random coil components, and improved the phenolic content of the LP-QP complexes. Digestibility increased to 87%, up from 76% for unfermented LP-QP. Moreover, the LP-QP complexes produced using the combined technique generated a highly nutritional protein with a reduced saponin content.ConclusionThis research revealed that a combination of PPI and water kefir fermentation significantly enhances the nutritional and functional quality of LP, creating new opportunities for leveraging the growing popularity of plant-based proteins into high-value industrial applications.

Asymmetric Polymerization-Induced Crystallization-Driven Self-Assembly of Helical, Rod-Coil Poly(aryl isocyanide) Block Copolymers

Polymerization-induced crystallization-driven self-assembly (PI-CDSA) is combined, for the first time, with helical, rod-coil block copolymer (BCP) self-assembly to enable scalable and controllable in situ synthesis of chiral nanostructures of variable shape, size, and dimensionality. Herein, we report newly developed asymmetric PI-CDSA (A-PI-CDSA) methodologies in the synthesis and in situ self-assembly of chiral, rod-coil BCPs composed of poly(aryl isocyanide) (PAIC) rigid-rod and poly(ethylene glycol) (PEG) random-coil components. Using PEG-based nickel(II) macroinitiators, the construction of PAIC-BCP nanostructures with variable chiral morphologies is accomplished at solids contents ranging 5.0-10 wt %. At low core-to-corona ratios for PAIC-BCPs, we demonstrate the scalable formation of chiral one-dimensional (1D) nanofibers via "living" A-PI-CDSA whose contour lengths can be tuned through alterations to unimer-to-1D seed particle ratio. At high core-to-corona ratios, A-PI-CDSA was implemented for the rapid fabrication of molecularly thin, uniform hexagonal nanosheets via spontaneous nucleation and growth aided by vortex agitation. Investigations into 2D seeded, living A-PI-CDSA revealed a brand-new paradigm in the context of CDSA where hierarchically chiral, M helical spirangle morphologies (i.e., hexagonal helicoids) are size-tuned in three dimensions (i.e., heights and areas) via alterations to unimer-to-seed ratio. These unique nanostructures are formed in situ at scalable solids contents up to 10 wt % via rapid crystallization about screw dislocation defect sites in an enantioselective fashion. The liquid crystalline nature of PAIC blocks dictates the hierarchical assembly of these BCPs, with chirality translated across length scales and in multiple dimensions affording large amplifications in chiroptical activity with g-factors reaching -0.030 for spirangle nanostructures.

Binding Investigation of Some Important Metal Ions Copper (I), Nickel (II), and Aluminium (III) with Bovine Serum Albumin Using Valid Spectroscopic Techniques

Studies based on the interaction of metals with proteins resulted in the development of promising metal-based compounds with encouraging medicinal potential. This study was aimed to utilize FT-IR and UV-Vis spectroscopic techniques to analyze the interactions of biologically significant metal ions, such as Al3+, Ni+2, and Cu+, with bovine serum albumin (BSA). Different concentrations of metal ions were interacted with BSA, and the complexes were analyzed using the two techniques. The change in the BSA secondary structure components such as β-sheet, β-antiparallel, α-helix, β-turn, and random coil were analyzed using second derivative resolution enhancement. The FT-IR spectroscopy suggested a marked decrease in the C=O stretching (corresponding to amide I) and C=N stretching (corresponding to amide II) intensities. Interestingly, upon complexation, a marked reduction (22.58–29.03%) in the α-helical component was observed with a considerable increase in the random coil component. The intensity of the absorption peak of BSA obtained using UV was observed to increase consecutively as the concentration of Cu+, Al3+, and Ni2+ ions increased. The binding constants for the BSA-Cu+, BSA-Ni+2, and BSA-Al+3 complexes were calculated to be 3.46 × 104 M−1, 1.28 × 104 M−1, and 2.08 × 104 M−1, respectively. It was concluded that the binding interaction decreased in the order Cu+ > Al3+ > Ni2+. These findings were similar to our previous findings using affinity capillary electrophoresis (ACE). Therefore, it can be inferred that the FT-IR and UV techniques might be utilised effectively to assess the metal-protein interaction and can have wide application in routine analysis. These techniques have several advantages in being simple, easy-to-perform, rapid, and affordable over other high-end techniques.

Fungal fermentation inducing improved nutritional qualities associated with altered secondary protein structure of soybean meal determined by FTIR spectroscopy

Abstract A FTIR spectro-chemometric measurement was first-ever made available to monitor chemical and molecular structural changes during the fungal fermentation of soybean meal. The samples of soybean meal were fermented for 72 hr using single Aspergillus ficuum or combined Aspergillus ficuum + Aspergillus niger inoculants. Nutrient contents determined by chemical methods on three independent sub-samples taken at 24 hr intervals were predicted by a method of FTIR. Furthermore, secondary protein components were quantified by FTIR spectra deconvolution method. Fungal fermentation caused enriched soybean meal with protein and organic acids with lowered dietary fibre, tannin, phytic acid and urease contents, while there was increased trypsin inhibitors. The chemometric model excellently predicted the nutrient contents with high accuracy (99% recovery rate) and precision (R2 = 0.98), except for the crude ash and total carbohydrate. Fermented soybean meal has a re-organised secondary protein structure: a complete degradation of α-helix component caused to an increased β-sheet (6%), β-turn (15%) and random coil component (100%). It is recommended that infrared spectroscopic monitoring of fermentation process can replace the chemical methods due to its low cost and robustness.

Effects of high pressure homogenize treatment on the physicochemical and emulsifying properties of proteins from scallop (Chlamys farreri)

In this study, the effects of high pressure homogenization (HPH) (0, 20, 40, 60, 80 and 100 MPa) on physicochemical and emulsifying properties of scallop (Chlamys farreri) protein (CFP) solutions were investigated. With the increasing of homogenize pressure, CFP solutions have better solubility. HPH could significantly decrease the particle size, which is distributed mainly in 300–600 nm. Compared with other treatments, HPH at 100 MPa showed highest surface hydrophobicity index (H0) (131.22 ± 2.22), higher nitrogen soluble index (NSI) (92.72 ± 0.53%) and higher free sulfhydryl group (3.97 ± 0.20 μmol/g protein). Circular dichroism (CD) inspections showed that 100 MPa of high pressure homogenization could make the random coil component decrease and the β-sheet component increase. The adsorbed proteins (AP %) of the emulsions progressively increased from 39.69 ± 0.11% to 61.89 ± 1.01% with the pressure of homogenization increasing from 20 to 100 MPa. Most of the proteins in the emulsions were adsorbed after HPH. Laser scanning confocal microscopy (LSCM) showed stability of the emulsion was increased by HPH. This work showed that HPH treatment has positive effects on physiochemical and emulsifying properties of CFP for innovative technologies for the potential industrial applications.

Determination of Local Structure of 13C Selectively Labeled 47-mer Peptides as a Model for Gly-Rich Region of Nephila clavipes Dragline Silk Using a Combination of 13C Solid-State NMR and MD Simulation

For the first time, we elucidate the complex structure of the Gly-rich regions in Nephila clavipes dragline silk through synergistic experimental and theoretical studies. First, the 13C selectively labeled 47-mer peptides selected from the glycine (Gly)-rich region of N. clavipes dragline silk were synthesized. The 13C CP/MAS NMR spectra were analyzed to determine the fractions of the conformations of individual Gly and Ala residues through 13C conformation-dependent chemical shifts and peak deconvolution. By comparing the 13C solid-state NMR spectra of several simple model peptides, the presence of 31 helix in the 47-mer peptides was disproved, and the (Ala)6 regions were shown to form β-sheet structure in the staggered arrangement. Although the fraction of β-sheet components tended to increase and the fraction of random coil component decrease toward both chain ends, significant change in the fractions was observed depending on the amino acid position. These results were successfully rationalized throug...

Structural Analysis of Hand Drawn Bumblebee Bombus terrestris Silk.

Bombus terrestris, commonly known as the buff-tailed bumblebee, is native to Europe, parts of Africa and Asia. It is commercially bred for use as a pollinator of greenhouse crops. Larvae pupate within a silken cocoon that they construct from proteins produced in modified salivary glands. The amino acid composition and protein structure of hand drawn B. terrestris, silk fibres was investigated through the use of micro-Raman spectroscopy. Spectra were obtained from single fibres drawn from the larvae salivary gland at a rate of 0.14 cm/s. Raman spectroscopy enabled the identification of poly(alanine), poly(alanine-glycine), phenylalanine, tryptophan, and methionine, which is consistent with the results of amino acid analysis. The dominant protein conformation was found to be coiled coil (73%) while the β-sheet content of 10% is, as expected, lower than those reported for hornets and ants. Polarized Raman spectra revealed that the coiled coils were highly aligned along the fibre axis while the β-sheet and random coil components had their peptide carbonyl groups roughly perpendicular to the fibre axis. The protein orientation distribution is compared to those of other natural and recombinant silks. A structural model for the B. terrestris silk fibre is proposed based on these results.

A Conventional and 2DCOS Infrared Study of Amyloid Fibril Formation

Amyloidosis is characterized by the abnormal self-assembly and deposition of proteinaceous material into insoluble ordered aggregates. There are several pathologies associated with this aggregates, known as “protein misfolding” disorders. In many proteins, when heated at high temperature and low pH, a series of structural changes resulting in the formation of fibrillar structures are produced. Insulin is a model of fibril formation that has produced a wealth of biochemical and structural data. The time-course of fibril formation can be followed by infrared spectroscopy looking at the appearance of a characteristic band in the amide I region. The kinetics is triggered by temperature at 70 °C and pH at 2.3. The infrared spectrum shows, that after a lag time (concentration-dependent), α-helix and β-turns bands decrease first whereas the random coil component increases. Later, a band at 1626 cm-1, associated with extended chains, replaces the random coil component. Infrared 2D-COS has been applied to different stages of the process. Maps have been formed at different incubation times: before random coil formation and at different stages in the random coil-fibril exchange. The synchronous maps do not give in principle much information because they are dominated by the appereance of the band due to the fibril. In order to extract such information we have developed an approach in which we take a window and move it along the perturbation (2D-moving lapse). This, together with the asynchronous maps indicate a two-step process with a first stage associated with an opening of the protein driven by destabilization of β-turns, located in the outer part of the protein, and a second part where the fibril is formed.This work was supported in part by the Spanish Ministerio de Educacion y Ciencia (grant No. BFU 2010-22103).

A 2DCOS Infrared Study of Fibril Formation

Amyloid fibrils are proteinaceous aggregates that can be formed in the process of degenerative diseases. Insulin is a model of fibril formation that has produced a wealth of biochemical and structural data. The time-course of fibril formation can be followed by infrared spectroscopy looking at the appearance of a characteristic band in the lower region of extended structure. The kinetics is triggered by temperature at 70 °C and pH 2.3.The infrared spectrum shows, that after a lag time (concentration-dependent), the α-helical band decreases and the random coil component increases subsequently. Random coil increases up to a percentage and later a band at 1626 cm-1, associated with extended chains, replaces the random coil component. Infrared 2D-COS has been applied to different stages of the process. Maps have been formed at different incubation times: before random coil formation and at different stages in the random coil-fibril change. Synchronous two-dimensional IR map shows that the process occurs in a two step mode. At pH 7.0, heating of insulin for long periods does not produce the random coil structure and subsequently no fibrils are formed. Human insulin, with a different aminoacid in the N-terminal segment, forms a fibril formation in a lower time than bovine or porcine. The bandshape corresponding to the fibril is different if bovine insulin is compared with human and porcine; what can be associated with a difference in aminoacids 8 and 10 that are located in the intrachain disulfide bond loop. The results show that small changes in protein sequence makes the kinetics different. Lipids have been proposed as one factor influencing fibril formation. The effect of different lipid composition, including anionic lipids, sphingomyelin and cholesterol has also been studied to see changes in kinetics looking at the lipid charge.

Preparation of regenerated Antheraea yamamai silk fibroin film and controlled‐molecular conformation changes by aqueous ethanol treatment

AbstractThe methods of dissolving Antheraea yamamai (A. yamamai) silk fibroin fibers and preparing the regenerated silk fibroin were studied. The molecular conformations of A. yamamai silk fibroin film treated by different concentration ethanol solution were investigated by XRD, FTIR, and Raman spectra. The results indicated that A. yamamai silk fibroin fibers could be completely dissolved in dense lithium thiocyanate solution at about 35°C for 1 h. The initial regenerated film consisted of α‐helix and random coil components. The aqueous ethanol treatment of the film resulted in significant increase in β‐sheet component and improvement of water resistance of the film. This effect was strongly dependent on ethanol concentration, and 60–80% (w/w) ethanol was most effective. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010

An Infrared 2D-COS Study of Fibril Formation

Amyloid fibrils are proteinaceous aggregates that can be formed in the process of degenerative diseases. Insulin is a model of fibril formation that has produced a wealth of biochemical and structural data. The time-course of fibril formation can be followed by infrared spectroscopy looking at the appearance of a characteristic band in the lower region of extended structure. The kinetics is triggered by a temperature at 70°C and a pH 2.3.The infrared spectrum shows, that after a lag time (concentration-dependent), the α-helical band decreases and the random coil component increases subsequently. Random coil increases up to a percentage and later a band at 1626 cm−1, associated with extended chains, replaces the random coil component. Infrared 2D-COS has been applied to different stages of the process. Maps have been formed at different incubation times: before random coil formation and at different stages in the random coil-fibril change. Synchronous two-dimensional IR map shows that the process occurs in a two step mode. At pH 7.0, heating of insulin for long periods does not produce the random coil structure and subsequently no fibrils are formed. Human insulin, with a different aminoacid in the N-terminal segment, forms a fibril formation in a lower time than bovine or porcine. The bandshape corresponding to the fibril is different if bovine insulin is compared with human and porcine; what can be associated with a difference in aminoacids 8 and 10 that are located in the intrachain disulfide bond loop. The results show that small changes in protein sequence makes the kinetics different. Lipids have been proposed as one factor influencing fibril formation. The effect of different lipid composition, including anionic lipids, sphingomyelin and cholesterol has also been studied to see changes in kinetics looking at the lipid charge.

Controlling molecular conformation of regenerated wild silk fibroin by aqueous ethanol treatment

AbstractRegenerated Antheraea pernyi silk fibroin film was prepared by dissolution of native silk fiber in aqueous lithium thiocyanate. The influence of aqueous ethanol treatment of the dried regenerated film on molecular conformation was studied by X‐ray diffraction, infrared spectroscopy, differential scanning calorimetry, and thermogravimetric analysis. While the initial regenerated film consisted of α‐helix and random coil components, aqueous ethanol treatment of the film resulted in significant increase in β‐sheet component and improvement of water resistance of the film. This effect was strongly dependent on ethanol concentration, and 40–60% (w/w) ethanol was most effective due to balance of hydrophilic/hydrophobic action of the solvent. Copyright © 2003 John Wiley & Sons, Ltd.

Centrin: its secondary structure in the presence and absence of cations.

Centrin is a low molecular mass (20 kDa) protein that belongs to the EF-hand superfamily of calcium-binding proteins. Local and overall changes were investigated for interactions between cations and Chlamydomonas centrin using Fourier transform infrared (FT-IR) and circular dichroic (CD) spectroscopies. FT-IR spectral features studied included the amide I' band and the side-chain absorbances for aspartate residues located almost exclusively at the calcium-binding sites in the spectral region of 1700-1500 cm(-1). The amide I' band is exquisitely sensitive to changes in protein secondary structure and is observed to shift from 1626.5 to 1642.7 cm(-1) in the presence and absence of calcium. These spectral bands are complex and were further studied using two-dimensional Fourier transform infrared (2D-FT-IR) correlation along with curve-fitting routines. Using these methods the secondary structure contributions were determined for holocentrin and apocentrin. The alpha-helical content in centrin was determined to be 60%-53% in the presence and absence of cations, respectively. Furthermore, the beta-strand content was determined to be 12%-36%, while the random coil component remained almost constant at 7%-13.5% in the presence and absence of cations, respectively. Changes in the side-chain band are mostly due to the monodentate coordination of aspartate to the cation. A shift of approximately 4 cm(-1) (for the COO- antisymmetric stretch in Asp) from 1565 to 1569 cm(-1) is observed for apocentrin and holocentrin, respectively. Thermal dependence revealed reversible conformational transition temperatures for apocentrin at 37 degrees C and holocentrin at 45 degrees C, suggesting greater stability for holocentrin.

Induction of Secondary Structure in a COOH-terminal Peptide of Histone H1 by Interaction with the DNA: AN INFRARED SPECTROSCOPY STUDY

We have studied the conformation of the peptide Ac-EPKRSVAFKKTKKEVKKVATPKK (CH-1), free in solution and bound to the DNA, by Fourier-transform infrared spectroscopy. The peptide belongs to the COOH-terminal domain of histone H1(0) (residues 99-121) and is adjacent to the central globular domain of the protein. In aqueous (D(2)O) solution the amide I' is dominated by component bands at 1643 cm(-1) and 1662 cm(-1), which have been assigned to random coil conformations and turns, respectively. In accordance with previous NMR results, the latter component has been interpreted as arising in turn-like conformations in rapid equilibrium with unfolded states. The peptide becomes fully structured either in 90% trifluoroethanol (TFE) solution or upon interaction with the DNA. In these conditions, the contributions of turn (1662 cm(-1)) and random coil components virtually disappear. In TFE, the spectrum is dominated by the alpha-helical component (1654 cm(-1)). The band at 1662 cm(-1) shifts to 1670 cm(-1), and has been assigned to the COOH-terminal TPKK motif in a more stable turn conformation. A band at 1637 cm(-1), also present in TFE, has been assigned to 3(10) helical structure. The amide I' band of the complexes with the DNA retains the components that were attributed to 3(10) helix and the TPKK turn. In the complexes with the DNA, the alpha-helical component observed in TFE splits into two components at 1657 cm(-1) and 1647 cm(-1). Both components are inside the spectral region of alpha-helical structures. Our results support the presence of inducible helical and turn elements, both sharing the character of DNA-binding motifs.

Orientation of rhodopsin alpha-helices in in retinal rod outer segment membranes studied by infrared linear dichroism.

Frog retinal rod outer segments, oriented by a magentic field, were shown to contain rhodopsin alpha-helical segments preferentially aligned perpendicular to the plane of the disc membrane, by the technique of infrared linear dichroism. Infrared absorption parallel and perpendicular to the rod axes by peptide C parallel to O groups, whose absorption band contains alpha-helical and random coil components at slightly different frequencies, showed positive dichroism centered on the alpha-helix frequence. We conclude that the alpha-helical portion of the protein has an average orientation in the transmembrane direction. Furthermore, infrared spectra of rods in 2H2O Ringer's solution exhibit two distinct peptide amino group absorption bands: the unexchanged N-2H band, which is nondichroic. This implies that the oriented part of the protein is in the lipid bilayer, supporting a model for rhodopsin with a hydrophobic core containing partially oriented alpha-helices and hydrophilic ends consisting of unoriented polypeptide.