Percentage Of Β-sheet Research Articles

Molecular basis of protein structure in proanthocyanidin and anthocyanin-enhanced Lc-transgenic alfalfa in relation to nutritive value using synchrotron-radiation FTIR microspectroscopy: A novel approach

To date there has been very little application of synchrotron radiation-based Fourier transform infrared microspectroscopy (SRFTIRM) to the study of molecular structures in plant forage in relation to livestock digestive behavior and nutrient availability. Protein inherent structure, among other factors such as protein matrix, affects nutritive quality, fermentation and degradation behavior in both humans and animals. The relative percentage of protein secondary structure influences protein value. A high percentage of β-sheets usually reduce the access of gastrointestinal digestive enzymes to the protein. Reduced accessibility results in poor digestibility and as a result, low protein value. The objective of this study was to use SRFTIRM to compare protein molecular structure of alfalfa plant tissues transformed with the maize Lc regulatory gene with non-transgenic alfalfa protein within cellular and subcellular dimensions and to quantify protein inherent structure profiles using Gaussian and Lorentzian methods of multi-component peak modeling. Protein molecular structure revealed by this method included α-helices, β-sheets and other structures such as β-turns and random coils. Hierarchical cluster analysis and principal component analysis of the synchrotron data, as well as accurate spectral analysis based on curve fitting, showed that transgenic alfalfa contained a relatively lower ( P < 0.05) percentage of the model-fitted α-helices (29 vs. 34) and model-fitted β-sheets (22 vs. 27) and a higher ( P < 0.05) percentage of other model-fitted structures (49 vs. 39). Transgenic alfalfa protein displayed no difference ( P > 0.05) in the ratio of α-helices to β-sheets (average: 1.4) and higher ( P < 0.05) ratios of α-helices to others (0.7 vs. 0.9) and β-sheets to others (0.5 vs. 0.8) than the non-transgenic alfalfa protein. The transgenic protein structures also exhibited no difference ( P > 0.05) in the vibrational intensity of protein amide I (average of 24) and amide II areas (average of 10) and their ratio (average of 2.4) compared with non-transgenic alfalfa. Cluster analysis and principal component analysis showed no significant differences between the two genotypes in the broad molecular fingerprint region, amides I and II regions, and the carbohydrate molecular region, indicating they are highly related to each other. The results suggest that transgenic Lc-alfalfa leaves contain similar proteins to non-transgenic alfalfa (because amide I and II intensities were identical), but a subtle difference in protein molecular structure after freeze drying. Further study is needed to understand the relationship between these structural profiles and biological features such as protein nutrient availability, protein bypass and digestive behavior of livestock fed with this type of forage.

Stability, Unfolding, and Structural Changes of Cofactor-Free 1H-3-Hydroxy-4-oxoquinaldine 2,4-Dioxygenase

Stability, unfolding mechanism, spectroscopic, densimetric, and structural characteristics of the oxidatively stable C69S variant (HodC) of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) have been determined by classical and pressure modulation scanning calorimetry (DSC and PMDSC, respectively), circular dichroism (CD) spectroscopy, differential scanning densimetry (DSD), and dynamic light scattering measurements. At 25 degrees C, hexahistidine-tagged HodC has a hydrodynamic radius of 2.3 nm and is characterized by an unusually high degree of alpha-helical structure of approximately 60%, based on deconvolution of CD spectra. The percentage of beta-sheets and -turns is expected to be relatively low in view of its sequence similarity to proteins of the alpha/beta-hydrolase fold superfamily. His6HodC exhibits three-state unfolding (N <--> I <--> D) with an intermediate state I that exhibits at the transition temperature a volume larger than that of the native or denatured state. The intermediate state I is also associated with the highest isothermal expansion coefficient, alphaP, of the three states and exhibits a significantly lower percentage of alpha-helical structure than the native state. The stability difference between the native and intermediate state is rather small which makes I a potential candidate for reactions with various ligands, particularly those having a preference for the apparently preserved beta-type motifs.

Protein-inspired multilayer nanofilms: science, technology and medicine

The field of polypeptide multilayer nanofilm research flourishes where study of protein structure and function shares a border with development of polyelectrolyte multilayers. The soil is fertile for creative input and promises a harvest of interesting results: the structure of a film can be predetermined on a layer-by-layer (LBL) basis, a huge variety of polypeptide sequences can be realized in large quantities by modern methods of synthesis, and the fabrication process is environmentally benign. In electrostatic LBL assembly, multilayer film assembly is driven primarily by coulombic interactions, but hydrophobic interactions and hydrogen bonds also contribute to film formation and stability, the amount depending on polypeptide design. Most peptides suitable for LBL assembly form films with a large percentage of β-sheet at neutral pH; it would appear that β-sheet is favored over α-helix in this context by the contribution to entropy of the number of ways of forming a β-sheet from a single polypeptide chain. Film thickness and roughness depend rather substantially on amino acid composition. Promising applications of the polypeptide multilayer film platform technology include coatings for medical implant devices, scaffolds for tissue engineering, coatings for targeted drug delivery, artificial cells for oxygen therapeutics, and artificial viruses for immunization. In each case peptide structure is tailored to the application. Here we summarize recent results of experimental studies and computational work from our laboratory, showing how the study of protein structure has inspired the design of polypeptide films and pointing out new opportunities for technology development. This work also provides a brief introduction to polypeptide structure and multilayer films.

Use of synchrotron-based FTIR microspectroscopy to determine protein secondary structures of raw and heat-treated brown and golden flaxseeds: A novel approach

The objectives of the study were to use synchrotron Fourier transform infrared microspectroscopy (S-FTIR) as a novel approach to: (1) reveal ultra-structural chemical features of protein secondary structures of flaxseed tissues affected by variety (golden and brown) and heat processing (raw and roasted), and (2) quantify protein secondary structures using Gaussian and Lorentzian methods of multi-component peak modeling. By using multi-component peak modeling at protein amide I region of 1700–1620 cm-1, the results showed that the golden flaxseed contained relatively higher percentage of α-helix (47.1 vs. 36.9%), lower percentage of β-sheet (37.2 vs. 46.3%) and higher (P &lt; 0.05) ratio of α-helix to β-sheet than the brown flaxseed (1.3 vs. 0.8). The roasting reduced (P &lt; 0.05) percentage of α-helix (from 47.1 to 36.1%), increased percentage of β-sheet (from 37.2 to 49.8%) and reduced α-helix to β-sheet ratio (1.3 to 0.7) of the golden flaxseed tissues. However, the roasting did not affect percentage and ratio of α-helix and β-sheet in the brown flaxseed tissue. No significant differences were found in quantification of protein secondary structures between Gaussian and Lorentzian methods. These results demonstrate the potential of highly spatially resolved S-FTIR to localize relatively pure protein in the tissue and reveal protein secondary structures at a cellular level. The results indicated relative differences in protein secondary structures between flaxseed varieties and differences in sensitivities of protein secondary structure to the heat processing. Further study is needed to understand the relationship between protein secondary structure and protein digestion and utilization of flaxseed and to investigate whether the changes in the relative amounts of protein secondary structures are primarily responsible for differences in protein availability. Key words: Synchrotron, FTIR microspectrosopy, flaxseeds, intrinsic structural matrix, protein secondary structures, protein nutritive value

Protein secondary structures (α-helix and β-sheet) at a cellular level and protein fractions in relation to rumen degradation behaviours of protein: a new approach

Studying the secondary structure of proteins leads to an understanding of the components that make up a whole protein, and such an understanding of the structure of the whole protein is often vital to understanding its digestive behaviour and nutritive value in animals. The main protein secondary structures are the alpha-helix and beta-sheet. The percentage of these two structures in protein secondary structures influences protein nutritive value, quality and digestive behaviour. A high percentage of beta-sheet structure may partly cause a low access to gastrointestinal digestive enzymes, which results in a low protein value. The objectives of the present study were to use advanced synchrotron-based Fourier transform IR (S-FTIR) microspectroscopy as a new approach to reveal the molecular chemistry of the protein secondary structures of feed tissues affected by heat-processing within intact tissue at a cellular level, and to quantify protein secondary structures using multicomponent peak modelling Gaussian and Lorentzian methods, in relation to protein digestive behaviours and nutritive value in the rumen, which was determined using the Cornell Net Carbohydrate Protein System. The synchrotron-based molecular chemistry research experiment was performed at the National Synchrotron Light Source at Brookhaven National Laboratory, US Department of Energy. The results showed that, with S-FTIR microspectroscopy, the molecular chemistry, ultrastructural chemical make-up and nutritive characteristics could be revealed at a high ultraspatial resolution ( approximately 10 microm). S-FTIR microspectroscopy revealed that the secondary structure of protein differed between raw and roasted golden flaxseeds in terms of the percentages and ratio of alpha-helixes and beta-sheets in the mid-IR range at the cellular level. By using multicomponent peak modelling, the results show that the roasting reduced (P<0.05) the percentage of alpha-helixes (from 47.1 % to 36.1 %: S-FTIR absorption intensity), increased the percentage of beta-sheets (from 37.2 % to 49.8 %: S-FTIR absorption intensity) and reduced the alpha-helix to beta-sheet ratio (from 0.3 to 0.7) in the golden flaxseeds, which indicated a negative effect of the roasting on protein values, utilisation and bioavailability. These results were proved by the Cornell Net Carbohydrate Protein System in situ animal trial, which also revealed that roasting increased the amount of protein bound to lignin, and well as of the Maillard reaction protein (both of which are poorly used by ruminants), and increased the level of indigestible and undegradable protein in ruminants. The present results demonstrate the potential of highly spatially resolved synchrotron-based infrared microspectroscopy to locate 'pure' protein in feed tissues, and reveal protein secondary structures and digestive behaviour, making a significant step forward in and an important contribution to protein nutritional research. Further study is needed to determine the sensitivities of protein secondary structures to various heat-processing conditions, and to quantify the relationship between protein secondary structures and the nutrient availability and digestive behaviour of various protein sources. Information from the present study arising from the synchrotron-based IR probing of the protein secondary structures of protein sources at the cellular level will be valuable as a guide to maintaining protein quality and predicting digestive behaviours.

Application of advanced synchrotron radiation-based Fourier transform infrared (SR-FTIR) microspectroscopy to animal nutrition and feed science: a novel approach

Synchrotron radiation-based Fourier transform IR (SR-FTIR) microspectroscopy has been developed as a rapid, direct, non-destructive and bioanalytical technique. This technique, taking advantage of synchrotron light brightness and a small effective source size, is capable of exploring the molecular chemistry within the microstructures of a biological tissue without the destruction of inherent structures at ultraspatial resolutions within cellular dimensions. This is in contrast to traditional 'wet' chemical methods, which, during processing for analysis, often result in the destruction of the intrinsic structures of feeds. To date there has been very little application of this technique to the study of feed materials in relation to animal nutrient utilisation. The present article reviews four applications of the SR-FTIR bioanalytical technique as a novel approach in animal nutrition and feed science research. Application 1 showed that using the SR-FTIR technique, intensities and the distribution of the biological components (such as lignin, protein, lipid, structural and non-structural carbohydrates and their ratios) in the microstructure of plant tissue within cellular dimensions could be imaged. The implication from this study is that we can chemically define the intrinsic feed structure and compare feed tissues according to spectroscopic characteristics, functional groups, spatial distribution and chemical intensity. Application 2 showed that the ultrastructural-chemical makeup and density of yellow- and brown-seeded Brassica rape could be explored. This structural-chemical information could be used for the prediction of rapeseed quality and nutritive value for man and animals and for rapeseed breeding programmes for selecting superior varieties for special purposes. More research is required to define the extent of differences that exist between the yellow- and brown-seeded Brassica rape. Application 3 showed with the SR-FTIR technique that chemical differences in the ultrastructural matrix of endosperm tissue between Harrington (malting-type) and Valier (feed-type) barley in relation to rumen degradation characteristics could be identified. The results indicated that the greater association of the protein matrix with the starch granules in the endosperm tissue of Valier barley may limit the access of ruminal micro-organisms to the starch granules and thus reduce the rate and extent of rumen degradation relative to that of Harrington barley. It is the first time that the microstructural matrix in the endosperm of barley has been revealed by using the SR-FTIR technique, which makes it possible to link feed intrinsic structures to nutrient utilisation and digestive behaviour in ruminants. Application 4 showed with the SR-FTIR technique that the chemical features of various feed protein (amide I) secondary structures (such as feather, wheat, oats and barley) could be quantified. With a multi-component fitting program (Lorentz function), the results showed feather containing about 88% beta-sheet and 4% alpha-helix, barley containing about 17% beta-sheet and 71% alpha-helix; oats containing about 2% beta-sheet and 92% alpha-helix; and wheat containing about 42% beta-sheet and 50% alpha-helix. The relative percentage of the two may influence protein value. A high percentage of beta-sheet may reduce the access of gastrointestinal digestive enzymes to the protein structure. Further study is required on feed protein secondary structures in relation to enzyme accessibility and digestibility. In conclusion, the SR-FTIR technique can be used for feed science and animal nutrition research. However, the main disadvantage of this technique is the requirement for a special light source; a synchrotron beam.

Ethanol or/and captopril-induced precipitation and secondary conformational changes of human serum albumin

We determined the secondary structure of solid-state native human serum albumin (HSA) and its precipitates induced by ethanol, captopril, or a captopril/ethanol mixture. A transmission Fourier transform infrared (FT-IR) microspectroscopy equipped with a thermal analyzer was used. The secondary structural composition of solid-state native HSA was 54% α-helices (1655 cm −1), 22% β-turns (1679 cm −1), and 23% β-sheets (1633 cm −1). After ethanol treatment, a new peak was observed at 1690 cm −1, and the peak at 1633 cm −1 was more apparent in the HSA precipitates. The corresponding compositions consisted of 59% α-helices, 17% β-turns, and 24% β-sheets. After treatment with captopril with or without ethanol, the percentage of α-helices and β-turns decreased in both HSA precipitates, but the percentage of β-sheets increased. The temperature-dependent structural transformation from α-helices/random coils to β-sheets for the solid-state HSA samples occurred at markedly different onset temperatures. The onset temperature for native HSA was 85 °C, and that for HSA precipitates obtained from ethanol, captopril, or captopril/ethanol was 100, 48 or 57 °C, respectively. The thermal-induced structural transformation from α-helices/random coils to β-sheets implies a partial unfolding structure in these HSA samples.

Amyloid Formation by Transthyretin: From Protein Stability to Protein Aggregation

In recent years the issues of protein stability, folding and aggregation have become central in several pathological conditions and in particular in amyloid diseases. Here, we review the recent developments on the molecular mechanisms of amyloid formation by transthyretin (TTR), in particular, in what concerns to protein conformational stability, protein folding and aggregation. Transthyretin has been implicated in pathologies such as senile systemic amyloidosis (SSA), familial amyloid polyneuropathy (FAP) and familial amyloid cardiomyopathy (FAC) which are characterized by extracellular deposition of insoluble amyloid fibrils. SSA is generally a mild disorder and affects predominantly individuals over 80 years of age. In contrast, FAP is an autossomal dominant lethal disease, characterized by peripheral neuropathy, which may affect individuals from their twenties. While in SSA WT-TTR and its fragments are the major constituents of the amyloid fibrils, in FAP and FAC the amyloid fibrils are mostly constituted by variants of TTR. Today, more than 80 amyloidogenic TTR mutations throughout the TTR sequence are known. Transthyretin is a homotetrameric protein found in the plasma and in the cerebral-spinal fluid, it is synthesized in the liver and in the choroid plexus of the brain, it has a total molecular mass of 55kDa and a high percentage of β-sheet. Current views on amyloid fibril formation by TTR state that, depending on the protein variant or solution conditions, the native tetrameric protein might dissociate to non-native or partially unfolded monomeric (or even dimeric) species with a high tendency for ordered aggregation into soluble oligomers which grow into insoluble oligomers and eventually mature amyloid fibrils. Thus, issues such as dissociation thermodynamics and dissociation kinetics of the native tetrameric TTR and thermodynamic stability and conformational fluctuations of the non-native TTR molecular species are essential in determining the amyloidogenic potential of different TTR variants. In addition, several other cellular and tissue factors must be involved in modulating the penetrance and age of onset of amyloid pathologies by TTR.

Conformational studies of the trypsin-aluminum(III) complex in solution by Raman and Fourier transform infrared attenuated total reflectance spectroscopy

This paper presents the results of Raman and Fourier transform IR studies investigating the interaction between Al(III) and trypsin, carried out in order to understand better how Al(III) can modify the biological properties of this proteolytic enzyme. The results indicate that Al(III) influences the secondary structure of trypsin. The trypsin–Al(III) complex in aqueous solution presents a molecular conformation similar to that of the trypsin–Al(III) crystal determined by x-ray diffraction analysis, but differs from the structure of both solid trypsin and trypsin in aqueous solution when analyzed in the absence of Al(III). In particular, the interaction of the protein with aluminum in solution induces a decrease in the percentage of α-helix and an increase in the percentage of β-sheet and random coil. Solid trypsin and trypsin in solution also displayed differences in secondary structure in the absence of Al(III). Copyright © 1999 John Wiley & Sons, Ltd.