Protein Secondary Structure Content Research Articles

From Structure to Strength: Analyzing the Impact of Sulfuric Acid on Pig Bone Demineralization Through FTIR, LIBS, and AAS.

The present research aimed to investigate the demineralizing effects of sulfuric acid on pig bone. Alterations in collagen and phosphate contents and changes in the elemental composition of the bone during the 14-day-long immersion in sulfuric acid solutions of different concentrations were estimated using ATR-FTIR, LIBS, and AAS. FTIR spectra at amide I (1800-1600 cm-1) and phosphate ν1/ν3 (PO43-) (1300-900 cm-1) domains were scrutinized using the deconvolution method for monitoring changes in the protein secondary structure and mineral content. The results implicated sulfuric acid as a powerful demineralization agent and effective in targeting mineral components, such as hydroxyapatite, while leaving the collagen matrix relatively preserved with a complex secondary structure. Collagen maturity marker values gave valuable insights into the structural integrity of the bone. LIBS and AAS indicated changes in bone hardness; phosphorous-to-carbon ratio; and calcium, phosphorous, and magnesium content in the solutions left after the immersion period. The changes in the ratio of ionic-to-atomic calcium lines in the LIBS spectra indicated hardening of the bone, with increasing acid concentration and prolonged action, due to the deposition of calcium sulfate on the surface. The calcium concentration in the solutions decreased with increased acid concentration, while the change in phosphorus and magnesium concentrations was reversed.

Effect of stepwise cooking on the water-retention capacity and protein denaturation degree of chicken breast

This study investigated the impact of different center temperatures (60 °C, 65 °C, 70 °C) combined with low temperature long time cooking (cooking at 60 °C for 30 min or 60 min) on the water-retention capacity of chicken breasts. The effects of different heat treatment methods on microstructure, texture parameters, water content, cooking loss, color, pH value, protein denaturation degree and secondary structure content of chicken breast were analyzed. The results revealed that both center temperature and low temperature long times cooking (LTLT cooking) time significantly influenced the water-retention capacity of chicken breasts (P < 0.05). With increasing heat exposure levels, there was a corresponding rise in pH value as well as protein solubility and hydrophobicity in meat. This led to increased alignment of myofibrils along with gradual structural disruption and connective tissue efflux resulting in significant increases in meat hardness, chewiness and cooking loss (P < 0.05). However, when the center temperature reached 70 °C with an LTLT cooking time of 60 min, the muscle fiber structure is almost completely destroyed, causing the connective tissue between the muscle bundles or fibers to refill, resulting in a random coil content increase of about 2%, increased the water content by around 12% and decreased hardness by about 31%. These findings demonstrate that the group subjected to a combination treatment involving a center temperature of 70 °C and an LTLT cooking time of 60 min was effective in enhancing the water content while reducing the hardness of meat.

Ultrasound-assisted improvement of thawing quality of Tibetan pork by inhibiting oxidation

The challenge of meat quality degradation due to transportation difficulties in high-altitude plateaus underscores the importance of an efficient thawing process for Tibetan pork to ensure its quality. This study compared four thawing methods ultrasound thawing (UT), refrigerator thawing (RT), hydrostatic thawing (HT), and microwave thawing (MT) to assess their impact on the quality of Tibetan pork, focusing on thawing loss, tenderness, color variation, and alterations in protein secondary structure and moisture content. Additionally, the study examined the impact of thawing on the metabolites of Tibetan pork using metabolomics techniques. The results indicated that UT yielded the highest quality samples. UT significantly accelerated the thawing rate and had minimal impact on tenderness compared to traditional thawing methods. Moreover, protein and lipid oxidation levels were reduced by UT treatment. Furthermore, it enhanced the binding capacity of protein and water molecules, reduced drip loss, and maintained meat color stability. What’s more, amino acid metabolites such as l-glutamic acid, l-proline, oxidized glutathione, and 1-methylhistidine played a significant role in thawing oxidation in Tibetan pork, exhibiting a positive correlation with protein oxidation. UT resulted in a notable decrease in the levels of hypoxanthine and 2-aminomethylpyrimidine, contributing to the reduction of bitterness in the thawed meat and consequently enhancing the freshness of Tibetan pork. This study offers novel insights into understanding the biological changes occurring during the thawing process, while also furnishing a theoretical framework and technical assistance to improve the quality of Tibetan pork and propel advancements in food processing technology.

Effect of salting and dehydration treatments on the physicochemical and gel properties of hen and duck egg yolks, plasma and granules.

Salted hen egg yolks are less oily and less flavorful than salted duck egg yolks. However, hen eggs have a more adequate market supply and have a broader application prospect than duck eggs. In the present study, egg yolks, plasma, and granules were dehydrated by adding 1% NaCl to simulate traditional curing process of salted egg yolk. The changes in the pickling process of hen egg yolks (HEY) and duck egg yolks (DEY) plasma and granules were compared to reveal the gelation mechanism and the underlying causes of quality differences in salted HEY and DEY. Salted HEY can be compared with the changes in DEY during the pickling process to provide a theoretical basis for the quality improvement of salted HEY to salted DEY. The results showed that both plasma and granules were involved in gel formation, but exhibited different aggregation behaviors. Based on the intermolecular forces, the HEY proteins achieved aggregation mainly through hydrophobic interactions and DEY proteins mainly through covalent binding. According to spin-spin relaxation time, HEY gels immobilized a large amount of lipid and interacted strongly with lipids. DEY gels showed much free lipid and had weak interaction with lipid. The microstructure showed that HEY proteins were easily unfolded to form a homogeneous three-dimensional gel network structure after salting, whereas heterogeneous aggregates were formed to hinder the gel development in DEY. Changes in protein secondary structure content showed that pickling can promote the transformation of the α-helices to β-sheets structure in HEY gels, whereas more α-helices structure was formed in DEY gels. The present study has demonstrated that different gelation behaviors of hen and duck egg yolk proteins (especially in plasma) through salting treatment led to the difference in the quality of salted HEY and DEY. © 2024 Society of Chemical Industry.

Use of Synchrotron Radiation Circular Dichroism to Analyze the Interaction and Insertion of Proteins into Bacterial Outer Membrane Vesicles.

Circular dichroism (CD) is a spectroscopic technique commonly used for the analysis of proteins. Particularly, it allows the determination of protein secondary structure content in various media, including the membrane environment. In this chapter, we present how CD applications can be used to analyze the interaction of proteins with bacterial outer membrane vesicles (OMVs). Most CD studies characterizing the structure of proteins inserted into membranes rely on artificial lipid bilayers, mimicking natural membranes. Nevertheless, these artificial models lack the important features of the true membrane, especially for the outer membrane of Gram-negative bacteria. These features include lipid diversity, glycosylation, and asymmetry. Here, we show how to analyze the interactions of proteins, either integral or peripheral, with OMVs in solution and with supported membranes of OMVs, using conventional CD and orientated circular dichroism (OCD). We explain how to decipher the spectroscopic signals to obtain information on the molecular structure of the protein upon its interaction with an OMV and through its potential insertion into an OMV membrane.

‘Synthesis, antiviral activity, molecular docking, and molecular dynamics studies of ethoxy phthalimide pyrazole derivatives against Cytomegalovirus and Varicella-Zoster virus: potential consequences and strategies for developing new antiviral treatments’

Substituted ethoxy phthalimide pyrazole derivatives (6a–e) have been produced using a one-pot synthesis technique. Spectral analysis was used to establish the molecular structure of the synthesized compounds, and they were examined in silico and in vitro for their ability to bind to and inhibit replication of the AD-169 strain, the Davis strain of CMV, the OKA strain and the 07/1 strain of Varicella-Zoster virus (VZV). Molecular Docking was used to estimate the binding mechanism and energy of compounds 4, 6a–e to their respective target proteins, thymidine kinase (TK), Varicella-Zoster protease (VZP) of VZV and tegument protein pp71 (TPpp71) of Cytomegalovirus (CMV). The MIC50 and EC50 were utilized to evaluate the antiviral and cytotoxic activities of test compounds in human embryonic lung (HEL) cells against the two reference medicines, Ganciclovir and Acyclovir. The chemicals studied showed a high affinity for binding sites and near binding sites of target proteins by generating H-bonds, carbon-hydrogen bonds, π-anion, π-sulfur, π-sigma, alkyl and π-alkyl interactions. All of the test compounds (6a–e) had higher binding energy than the standard medications. The ADME/T data suggests that these potential inhibitors are less toxic. Drug-protein complexes are structurally compact and demonstrate minimal conformational change in molecular dynamics (MDs) simulations, indicating stability and stiffness. MM-PBSA and post-simulation analysis can predict lead compound active cavity binding stability. By inhibiting multitargeted proteins, these synthetic compounds may improve antiviral therapy. Our research suggests that these unique synthesized chemicals may be useful and accessible adjuvant antiviral therapy for Varicella Zoster and CMV. Highlights Two components synthesis of substituted ethoxy phthalimide pyrazole derivatives (6a–e). Tested compounds (6a–e) have antiviral and cytotoxicity activity against CMV and Varicella-Zoster virus (VZV) in HEL cells. Compounds bind to TK, Varicella-Zoster protease (VZP) of VZV, and modeled TPpp71 of Cytomegalovirus (CMV). In comparison to reference drugs, compounds have strong binding free energy and interactions with VZV and CMV protein complexes. The RMSD, RMSF, Rg, residual correlative motion (RCM), No. of hydrogen bonds, protein secondary structure content, per-residue protein secondary structure and MM/PBSA energy calculated for the selected compound with thymidine kinase (TK), VZP of VZV, and modeled tegument protein pp71 (TPpp71) of CMV through MD simulation studies for 50 ns. In comparison to the two reference drugs, ligands/compounds were found to meet the Lipinski rule of five and to have strong biological activity. Communicated by Ramaswamy H. Sarma

Silkworm sericin peptides alleviate the hardening of soy protein bars during early storage

In this study, the innovative use of sericin peptide (SRP) as an anti-hardening ingredient was studied and investigated for the anti-hardening effect on high protein nutrition bars (HPNBs) made from soybean protein isolate (SPI). Texture analysis showed that the addition of SRP to the SPI-HPNBs alleviated the hardening of the samples during early storage (72 h). The mobility of water and small molecules in the samples were improved by the addition of SRP. CLSM, SEM and AFM showed that SRP slowed down the phase separation in the samples and maintained the microstructure. The addition of SRP resulted in changes in ζ-potential, protein secondary structure content and surface hydrophobicity, which prevented self-aggregation of proteins. Overall, SRP could effectively hinder the phase separation of SPI-HPNBs caused by water migration and protein self-aggregation during early storage and alleviate the hardening, and this effect increased with the increase of SRP concentration. These results suggest that SRP can be used as a promising anti-hardening ingredient in the food industry to improve the texture of food products.

Minimising sample volume and run times for circular dichroism spectroscopy.

Circular dichroism (CD) spectroscopy is widely used to characterise chiral structures in solution, including to determine protein secondary structure content. However, protein CD experiments typically require 10-20 μg of protein. An additional challenge is that some proteins change their structure when their concentration is changed (they unfold or aggregate) so, particularly for protein biopharmaceuticals, it is attractive to have a range of different path lengths to hand. This paper reports the validation of demountable DMV Bio-cells of path lengths 0.5 mm, 0.2 mm and 0.125 mm, which have approximately 1 mm diameter windows, for CD spectroscopy. When complemented with a 10 cm focal length focusing lens positioned 9.5 cm in front of the sample, the signal : noise ratio of the final spectrum is equivalent to data collected on a standard cell wide enough to allow the full light beam to pass through. The DMV Bio-cells require 2-3% of the sample needed for a standard full-width cuvette and can be assembled without bubbles with the nominal path length. In addition, they are easy to clean and less likely to be chipped or broken than standard cuvettes which, together with the sample saved, compensates for their much higher price. For smaller path lengths the Jasco MSD-462 microsampling disk enabled fairly reproducible assembly at 4-7 μm depending on the sample viscosity and similarly high quality data.

Searching for a Better Match between Protein Secondary Structure Definitions and Protein FTIR Spectra.

Obtaining protein secondary structure content from high-resolution structures requires definitions and thresholds for the various parameters involved, typically hydrogen bond energy or length/angle and backbone φ/ψ angles. Several definitions are currently used and can have a profound impact on secondary structure content. Fourier transform infrared (FTIR) spectroscopy has its own sensitivity to molecular geometry. It is, therefore, important to select a set of definitions that matches this sensitivity. Here, we used a new protein set consisting of 92 proteins designed for the calibration of spectroscopic methods. Spectra have been obtained from protein microarrays in a high throughput process. The potential for improving secondary structure predictions from FTIR spectra has been tested using 71 structures determined according to different definitions. The paper demonstrates that different secondary structure definitions result in large variations in secondary structure content that are not equivalent in view of the protein FTIR spectra. The prediction quality factor ζ can be improved by ca. 20-50% by selecting an adequate definition set. The results also indicate that the dictionary of secondary structure of proteins (DSSP) algorithm, which is currently widely used to evaluate protein secondary structure content, is a good choice when dealing with FTIR spectra.

Silkworm Silk Fiber Bundles as Improved In Vitro Scaffolds for Skeletal Muscle.

To mimic skeletal muscle tissues in vitro, native and transgenic spider silk/silkworm silks were seeded with C2C12 myoblasts to observe if these three-dimensional substrates are preferable to a traditional two-dimensional polystyrene cell culture surface. Silks were wound around an acrylic chassis to produce a novel, three-dimensional cell culture device with suspended muscle fibers that genetically and morphologically resemble native skeletal muscle tissue. The transgenic spider silk/silkworm silk has never before been studied for this application. Genetic expression verified skeletal muscle lineage and differentiation, while fluorescent imaging verified contractile protein synthesis. Genetic analysis also revealed an increase in expression of the Myh2 contractile protein gene on silkworm silks, particularly on the transgenic silk. Mechanical properties and protein secondary structure content of the silks indicated correlation between substrate properties and Myh2 gene expression. This increase in contractile protein gene expression suggests that biologically derived silk substrates that are suspended may be a preferable substrate for in vitro muscle modeling because of the proteinaceous character and mechanical flexibility of the silk.

Pulsed electric fields induce modulation of protein liquid-liquid phase separation.

The time-resolved dynamic assembly and the structures of protein liquid dense clusters (LDCs) were analyzed under pulsed electric fields (EFs) applying complementary polarized and depolarized dynamic light scattering (DLS/DDLS), optical microscopy, and transmission electron microscopy (TEM). We discovered that pulsed EFs substantially affected overall morphologies and spatial distributions of protein LDCs and microcrystals, and affected the phase diagrams of LDC formation, including enabling protein solutions to overcome the diffusive flux energy barrier to phase separate. Data obtained from DLS/DDLS and TEM showed that LDCs appeared as precursors of protein crystal nuclei, followed by the formation of ordered structures within LDCs applying a pulsed EF. Experimental results of circular dichroism spectroscopy provided evidence that the protein secondary structure content is changing under EFs, which may consequently modulate protein-protein interactions, and the morphology, dimensions, and internal structure of LDCs. Data and results obtained unveil options to modulate the phase diagram of crystallization, and physical morphologies of protein LDCs and microcrystals by irradiating sample suspensions with pulsed EFs.

Structural and kinetic characterization of recombinant 2-hydroxymuconate semialdehyde dehydrogenase from Pseudomonas putida G7

The first enzyme in the oxalocrotonate branch of the naphthalene-degradation lower pathway in Pseudomonas putida G7 is NahI, a 2-hydroxymuconate semialdehyde dehydrogenase which converts 2-hydroxymuconate semialdehyde to 2-hydroxymuconate in the presence of NAD+. NahI is in family 8 (ALDH8) of the NAD(P)+-dependent aldehyde dehydrogenase superfamily. In this work, we report the cloning, expression, purification and preliminary structural and kinetic characterization of the recombinant NahI. The nahI gene was subcloned into a T7 expression vector and the enzyme was overexpressed in Escherichia coli ArcticExpress as a hexa-histidine-tagged fusion protein. After purification by affinity and size-exclusion chromatography, dynamic light scattering and small-angle X-ray scattering experiments were conducted to analyze the oligomeric state and the overall shape of the enzyme in solution. The protein is a tetramer in solution and has nearly perfect 222 point group symmetry. Protein stability and secondary structure content were evaluated by a circular dichroism spectroscopy assay under different thermal conditions. Furthermore, kinetic assays were conducted and, for the first time, KM (1.3±0.3μM) and kcat (0.9s−1) values were determined at presumed NAD+ saturation. NahI is highly specific for its biological substrate and has no activity with salicylaldehyde, another intermediate in the naphthalene-degradation pathway.

Label-Free Detection of Protein Secondary Structure Content in Biological Specimen by Fourier-Transform Infrared Spectroscopy

Fourier-transform infrared (FT-IR) spectroscopy, a non-destructive, label-free, and high sensitive and specific analytical method is a vibrational technique that gives information on the chemical composition of a sample of biological specimens. It is also a powerful approach to detect changes in the protein composition and structure of intact living cells. To determine a cellular function or change of biological materials (cells, tissues, microorganisms, etc.), we have studied the amide I band of protein by using both FT-IR spectra based on attenuated total reflection (ATR) and their second derivative spectra. we have performed the breast cancer cells and the pathogenic microorganisms exposed to anticancer and antibiotic drugs, respectively. Based on these studies, we found that the potential to use this technique for the detection and quantification of the protein secondary structure components of biological specimens under drug application

Quantification of protein secondary structure content by multivariate analysis of deep-ultraviolet resonance Raman and circular dichroism spectroscopies

Determination of protein secondary structure (α-helical, β-sheet, and disordered motifs) has become an area of great importance in biochemistry and biophysics as protein secondary structure is directly related to protein function and protein related diseases.

Amino acid transport in thermophiles: Characterization of an arginine-binding protein from Thermotoga maritima. 3. Conformational dynamics and stability

Arginine-binding protein from Thermotoga maritima (TmArgBP) is a 27.7kDa protein possessing the typical two domain structure of the periplasmic binding protein family. The protein is characterized by high specificity and affinity for binding a single molecule of l-arginine.In this work, the effect of temperature and/or guanidine hydrochloride on structure and stability of the protein in the absence and in the presence of l-arginine has been investigated by differential scanning calorimetry, far-UV circular dichroism and intrinsic tryptophan phosphorescence and fluorescence. The results revealed that TmArgBP undergoes an irreversible one-step thermal unfolding process in a cooperative mode. The TmArgBP melting temperature was recorded at 115°C. The presence of l-arginine did not change the protein secondary structure content as well as the intrinsic phosphorescence and fluorescence protein properties, even if it increases the structural stability of the protein.The obtained results are discussed in combination with a detailed inspection of the three-dimensional structure of the protein.

Structural alterations in rat liver proteins due to streptozotocin-induced diabetes and the recovery effect of selenium: Fourier transform infrared microspectroscopy and neural network study

The relation between protein structural alterations and tissue dysfunction is a major concern as protein fibrillation and/or aggregation due to structural alterations has been reported in many disease states. In the current study, Fourier transform infrared microspectroscopic imaging has been used to investigate diabetes-induced changes on protein secondary structure and macromolecular content in streptozotocin-induced diabetic rat liver. Protein secondary structural alterations were predicted using neural network approach utilizing the amide I region. Moreover, the role of selenium in the recovery of diabetes-induced alterations on macromolecular content and protein secondary structure was also studied. The results revealed that diabetes induced a decrease in lipid to protein and glycogen to protein ratios in diabetic livers. Significant alterations in protein secondary structure were observed with a decrease in α-helical and an increase in β-sheet content. Both doses of selenium restored diabetes-induced changes in lipid to protein and glycogen to protein ratios. However, low-dose selenium supplementation was not sufficient to recover the effects of diabetes on protein secondary structure, while a higher dose of selenium fully restored diabetes-induced alterations in protein structure.

Prediction of protein secondary structure from circular dichroism using theoretically derived spectra

Circular dichroism (CD) is a spectroscopic technique commonly used to investigate the structure of proteins. Major secondary structure types, alpha-helices and beta-strands, produce distinctive CD spectra. Thus, by comparing the CD spectrum of a protein of interest to a reference set consisting of CD spectra of proteins of known structure, predictive methods can estimate the secondary structure of the protein. Currently available methods, including K2D2, use such experimental CD reference sets, which are very small in size when compared to the number of tertiary structures available in the Protein Data Bank (PDB). Conversely, given a PDB structure, it is possible to predict a theoretical CD spectrum from it. The methodological framework for this calculation was established long ago but only recently a convenient implementation called DichroCalc has been developed. In this study, we set to determine whether theoretically derived spectra could be used as reference set for accurate CD based predictions of secondary structure. We used DichroCalc to calculate the theoretical CD spectra of a nonredundant set of structures representing most proteins in the PDB, and applied a straightforward approach for predicting protein secondary structure content using these theoretical CD spectra as reference set. We show that this method improves the predictions, particularly for the wavelength interval between 200 and 240 nm and for beta-strand content. We have implemented this method, called K2D3, in a publicly accessible web server at http://www. ogic.ca/projects/k2d3.

Histone H4 Acetylation Differentially Modulates Arginine Methylation by an in Cis Mechanism

Histone H4 undergoes extensive post-translational modifications (PTMs) at its N-terminal tail. Many of these PTMs profoundly affect the on and off status of gene transcription. The molecular mechanism by which histone PTMs modulate genetic and epigenetic processes is not fully understood. In particular, how a PTM mark affects the presence and level of other histone modification marks needs to be addressed and is essential for better understanding the molecular basis of histone code hypothesis. To dissect the interplaying relationship between different histone modification marks, we investigated how individual lysine acetylations and their different combinations at the H4 tail affect Arg-3 methylation in cis. Our data reveal that the effect of lysine acetylation on arginine methylation depends on the site of acetylation and the type of methylation. Although certain acetylations present a repressive impact on PRMT1-mediated methylation (type I methylation), lysine acetylation generally is correlated with enhanced methylation by PRMT5 (type II dimethylation). In particular, Lys-5 acetylation decreases the activity of PRMT1 but increases that of PRMT5. Furthermore, circular dichroism study and computer simulation demonstrate that hyperacetylation increases the content of ordered secondary structures at the H4 tail region. These findings provide new insights into the regulatory mechanism of Arg-3 methylation by H4 acetylation and unravel the complex intercommunications that exist between different the PTM marks in cis. The divergent activities of PRMT1 and PRMT5 with respect to different acetyl-H4 substrates suggest that type I and type II protein-arginine methyltransferases use distinct molecular determinants for substrate recognition and catalysis.

Pre-processing of ultraviolet resonance Raman spectra

The application of UV excitation sources coupled with resonance Raman have the potential to offer information unavailable with the current inventory of commonly used structural techniques including X-ray, NMR and IR analysis. However, for ultraviolet resonance Raman (UVRR) spectroscopy to become a mainstream method for the determination of protein secondary structure content and monitoring protein dynamics, the application of multivariate data analysis methodologies must be made routine. Typically, the application of higher order data analysis methods requires robust pre-processing methods in order to standardize the data arrays. The application of such methods can be problematic in UVRR datasets due to spectral shifts arising from day-to-day fluctuations in the instrument response. Additionally, the non-linear increases in spectral resolution in wavenumbers (increasing spectral data points for the same spectral region) that results from increasing excitation wavelengths can make the alignment of multi-excitation datasets problematic. Last, a uniform and standardized methodology for the subtraction of the water band has also been a systematic issue for multivariate data analysis as the water band overlaps the amide I mode. Here we present a two-pronged preprocessing approach using correlation optimized warping (COW) to alleviate spectra-to-spectra and day-to-day alignment errors coupled with a method whereby the relative intensity of the water band is determined through a least-squares determination of the signal intensity between 1750 and 1900 cm(-1) to make complex multi-excitation datasets more homogeneous and usable with multivariate analysis methods.

IFC2: an integrated web-server for improved prediction of protein structural class, fold type, and secondary structure content

Several descriptors of protein structure at the sequence and residue levels have been recently proposed. They are widely adopted in the analysis and prediction of structural and functional characteristics of proteins. Numerous in silico methods have been developed for sequence-based prediction of these descriptors. However, many of them do not have a public web-server and only a few integrate multiple descriptors to improve the predictions. We introduce iFC² (integrated prediction of fold, class, and content) server that is the first to integrate three modern predictors of sequence-level descriptors. They concern fold type (PFRES), structural class (SCEC), and secondary structure content (PSSC-core). The server exploits relations between the three descriptors to implement a cross-evaluation procedure that improves over the predictions of the individual methods. The iFC² annotates fold and class predictions as potentially correct/incorrect. When tested on datasets with low-similarity chains, for the fold prediction iFC² labels 82% of the PFRES predictions as correct and the accuracy of these predictions equals 72%. The accuracy of the remaining 28% of the PFRES predictions equals 38%. Similarly, our server assigns correct labels for over 79% of SCEC predictions, which are shown to be 98% accurate, while the remaining SCEC predictions are only 15% accurate. These results are shown to be competitive when contrasted against recent relevant web-servers. Predictions on CASP8 targets show that the content predicted by iFC² is competitive when compared with the content computed from the tertiary structures predicted by three best-performing methods in CASP8. The iFC² server is available at http://biomine.ece.ualberta.ca/1D/1D.html .