Percentages Of Secondary Structure Research Articles

Artificial intelligence analysis of FTIR and CD spectroscopic data for predicting and quantifying the length and content of protein secondary structures

Besides NMR and X-ray crystallography, FTIR and CD spectroscopy are widely considered to be useful for determining protein secondary structure. These techniques can be used to obtain data in few minutes, using small quantities of proteins, which make them amenable for proteomics research. Here we explore the possibility of using artificial intelligence techniques to simultaneously analyse both FTIR and CD spectroscopic data for an identical set of proteins. Neural network analysis was carried out on normalised regions of FTIR (1700-1600 cm−1) and CD (180-259 nm) spectral data both with and without boxcar averaging in order to quantify the average length and percentages of secondary structures. A hybrid genetic algorithm/neural network approach, that automatically selects structure-sensitive wavelength/frequency, was used for the quantification of the protein secondary structure. Using this algorithm we also successfully identified the region of the CD spectrum that contains the most structure-sensitive information. This was located between 214-251 nm, suggesting that this region alone may be sufficient to rapidly determine the secondary structure content from CD spectral data. Overall, CD spectroscopic analysis produced better results compared to FTIR spectroscopy when selected wavelengths were used, although FTIR was better when the entire region between 1700-1600 cm−1 (FTIR), and 180-259 nm (CD), was subjected to neural network analysis. Application of Adaptive Neuro-Fuzzy Inference System (ANFIS) with fuzzy subtractive clustering for the analysis of the spectral data led to a slightly better prediction of the average helix/sheet length for FTIR spectroscopy compared to CD. Our findings reveal the potential of using artificial intelligence techniques for not only extracting structural information but also for better understanding of the relationship between complex spectral data and biologically important information.

Effect of Fermentation with Two Molds on Characteristics of Chicken Meat

In the present study, we investigated the characteristics of chicken meat fermented with Penicillium nalgiovense and Penicillium chrysogenum. Hardness and springiness gradually decreased, while gumminess gradually increased during fermentation. Fermentation with P. chrysogenum led to higher hardness and lower gumminess than fermentation with P. nalgiovense. Fermentation with two molds resulted in similar microstructure, such as granule formation and fractured myofibril. The highest percentage of secondary structure was ɑ-helix, and tyrosine residues were buried after fermentation. P. nalgiovense-fermented samples contained more bound water, lower relative content of alkanes, and higher relative content of aldehydes than P. chrysogenum-fermented samples.

Secondary Structural Transformation of Bovine Lactoferricin Affects Its Antibacterial Activity.

Lactoferricin (Lfcin) is a potent antibacterial peptide derived from lactoferrin by pepsin hydrolysis. It was hypothesized that structural transformation of Lfcin could affect its antibacterial function through forming and breaking of intramolecular disulfide bond. To prove this hypothesis, bovine Lfcin (bLfcin) and its two derivatives, bLfcin with a disulfide bond (bLfcin DB) and bLfcin with a mutation C36G (bLfcin C36G), were synthesized, purified, and identified. The circular dichroism (CD) spectra of the peptides were detected in solutions with different ionic and hydrophobic strength. Then, the secondary structure contents of the peptides were calculated on the basis of the CD spectra. The antibacterial activity of the peptides against Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028, Shigella flexneri ATCC 12022, and Staphylococcus aureus ATCC 25923 was evaluated. The results showed that bLfcin and bLfcin C36G had similar percentages of secondary structure in water, while bLfcin and bLfcin DB had similar ratios of secondary structure under less hydrophobic conditions. The synthetic peptides exhibited antibacterial activity against all the tested bacteria, except for S. aureus ATCC 25923. bLfcin demonstrated higher antibacterial activity compared with its derivatives. The results suggested that bLfcin could transform its structure under alterative ionic strengths and hydrophobic conditions, and the transformation of structures was beneficial to enhancing the antibacterial function.

Effect of intramolecular disulfide bond of bovine lactoferricin on its molecular structure and antibacterial activity against Trueperella pyogenes separated from cow milk with mastitis

BackgroundLactoferricin (Lfcin) is an antimicrobial activity center of lactoferrin, produced by hydrolysis from the N-terminal of lactoferrin. It was hypothesized that the intramolecular disulfide bond in Lfcin could affect its antibacterial function through influencing its molecular structure. To prove this hypothesis, bovine Lfcin (bLfcin) and its two derivatives, bLfcin with an intramolecular disulfate bond (bLfcin DB) and bLfcin with a mutation C36G (bLfcin C36G), were synthesized, purified, and identified. The circular dichroism spectra of the peptides were detected in solutions with different ionic and hydrophobic strength. The antibacterial activity of the peptides against Trueperella pyogenes, separated from cow milk with mastitis, were determined.ResultsThe secondary structure of bLfcin DB showed more β-turn and less random coil than the other peptides in H2O, similar ratios of secondary structures with bLfcin and bLfcin C36G under ionic conditions, and close percentages of secondary structure with bLfcin under hydrophobic conditions. The synthetic peptides exhibited strong antimicrobial activity against T. pyogenes isolates, T. pyogenes ATCC 19,411, and E. coli ATCC 25,922. The antimicrobial activities of the three peptides were greater against T. pyogenes than against E. coli, and bLfcin DB exhibited higher antibacterial activity compared with its derivatives.ConclusionsThe intramolecular disulfide bond could change the molecular structure of bLfcin under alternative ionic strengths and hydrophobic effects, and the formation of the disulfide bond is beneficial to executing the antibacterial function of bLfcin.

The inhibitory effect of farnesiferol C against catalase; Kinetics, interaction mechanism and molecular docking simulation

Farnesiferol C (FC) is a natural sesquiterpene coumarin, which includes a widely range of biological activities. In this work, effects of FC on the structure and catalytic function of bovine liver catalase (BLC) was assessed by various spectroscopic and theoretical methods. Kinetic studies showed that FC has a remarkable inhibitory activity on BLC via mixed-type inhibition. The IC50 value as the inhibitory strength of FC was evaluated 1.5μM. Fluorescence spectroscopy, synchronous fluorescence, CD spectroscopy and UV–vis absorption studies revealed conformational changes in the tertiary and secondary structure of BLC as well as the position of the heme group in the presence of different concentrations of FC. Fluorescence studies revealed that FC quenches intrinsic emission of catalase via static quenching process. The binding constants at 298 and 310K were calculated 1.17×105M−1 and 1.0×105M−1, respectively. Thermodynamic data suggested that hydrophobic interactions play a major role in the binding reaction of FC on BLC. Structural studies indicated that the binding FC to the enzyme is responsible for the changes of the percentage of secondary structures' elements especially α-helix. From the simulation data, the role of Arg353 residue in the mechanism of catalase inhibition has been recognized.

Functional properties of sonicated and non-sonicated extracted leaf protein concentrate from Diplazium esculentum

ABSTRACTLeaf protein concentrate of edible fern Diplazium esculentum was extracted using sonication and non-sonication methods and its functional properties were evaluated. The leaf protein concentrate contained 34.28 and 9.89% protein for sonicated and non-sonicated extractions, respectively. Sonication yielded (36.36%) better emulsion activity over non-sonication (31.10%). Foaming capacity was also found better in sonication (7.27%) over non-sonication (6.99%), and oil absorption capacity also improved in sonication (7.55 g of oil/g) over its counterpart (7.41 g of oil/g). Contact angle experiments with the leaf protein concentrate evidenced the enhancement of more hydrophilicity of the sonicated sample. Thermal properties of the leaf protein concentrate with sonication evidenced more stability (55.58 and 95.95°C) over the non-sonicated (46.75 and 75.60°C). Fourier transform infrared spectral analysis with the leaf protein concentrate of Diplazium esculentum was used to assess the secondary structural data such as α-helix and β-sheets. The various percentages of secondary structures of protein in sonicated and non-sonicated samples were 49.06 and 69.16% for α-helix, 33.59% and 17.02% for β-sheet and 42.02 and 35.68% for turn, respectively. The results of the present investigation showed credible evidence to support that sonication method of extraction of leaf protein concentrate was better in terms of protein yield and functional properties as compared to the non-sonication. The sonicated extracted leaf protein concentrate has the potential for developing value added products.

Over-expression and characterization of NS3 and NS5A of Hepatitis C virus genotype 3a

BackgroundHepatitis C virus (HCV) is a common and leading cause for liver cirrhosis and hepatocellular carcinoma. Current therapies to treat HCV infection are shown to be partially effective and poorly tolerated. Therefore, ample efforts are underway to rationally design therapies targeting the HCV non-structural proteins. Most of the work carried out in this direction has been focusing mainly on HCV genotype 1. Two direct-acting antiviral agents (DAAs) Telaprevir and Boceprevir are being used against genotype 1a infection in combination therapy with interferon and ribavirin. Unfortunately these DAAs are not effective against genotype 3a. Considering the wide spread infection by HCV genotype 3a in developing countries especially South Asia, we have focused on the recombinant production of antiviral drug targets NS3 and NS5A from HCV genotype 3a. These protein targets are to be used for screening of inhibitors.ResultsHigh-level expression of NS3 and NS5A was achieved at 25°C, using ~1 and 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG), respectively. Yields of the purified NS3 and NS5A were 4 and 1 mg per liter culture volume, respectively. Although similar amounts of purified NS3 were obtained at 25 and 14°C, specificity constant (Kcat/Km) was somewhat higher at expression temperature of 25°C. Circular dichroism (CD) and Fourier-transform infrared (FT-IR) spectroscopy revealed that both NS3 and NS5A contain a mixture of alpha-helix and beta-sheet secondary structures. For NS3 protein, percentages of secondary structures were similar to the values predicted from homology modeling.ConclusionsNS3 and NS5A were over-expressed and using Nickel-affinity method both proteins were purified to ~ 95% purity. Yield of the purified NS3 obtained is four fold higher than previous reports. CD spectroscopy revealed that difference in activity of NS3 expressed at various temperatures is not related to changes in global structural features of the protein. Moreover, CD and FT-IR analysis showed that NS3 and NS5A contain both alpha-helical and beta-sheet structures and for NS5A, the proportion is almost equal. The production of NS3 and NS5A in milligram quantities will allow their characterization by biophysical and biochemical means that will help in designing new strategies to fight against HCV infection.

DsHsp90 Is Involved in the Early Response of Dunaliella salina to Environmental Stress †

Heat shock protein 90 (Hsp90) is a molecular chaperone highly conserved across the species from prokaryotes to eukaryotes. Hsp90 is essential for cell viability under all growth conditions and is proposed to act as a hub of the signaling network and protein homeostasis of the eukaryotic cells. By interacting with various client proteins, Hsp90 is involved in diverse physiological processes such as signal transduction, cell mobility, heat shock response and osmotic stress response. In this research, we cloned the dshsp90 gene encoding a polypeptide composed of 696 amino acids from the halotolerant unicellular green algae Dunaliella salina. Sequence alignment indicated that DsHsp90 belonged to the cytosolic Hsp90A family. Further biophysical and biochemical studies of the recombinant protein revealed that DsHsp90 possessed ATPase activity and existed as a dimer with similar percentages of secondary structures to those well-studied Hsp90As. Analysis of the nucleotide sequence of the cloned genomic DNA fragment indicated that dshsp90 contained 21 exons interrupted by 20 introns, which is much more complicated than the other plant hsp90 genes. The promoter region of dshsp90 contained putative cis-acting stress responsive elements and binding sites of transcriptional factors that respond to heat shock and salt stress. Further experimental research confirmed that dshsp90 was upregulated quickly by heat and salt shock in the D. salina cells. These findings suggested that dshsp90 might serve as a component of the early response system of the D. salina cells against environmental stresses.

Do Natural Proteins Differ from Random Sequences Polypeptides? Natural vs. Random Proteins Classification Using an Evolutionary Neural Network

Are extant proteins the exquisite result of natural selection or are they random sequences slightly edited by evolution? This question has puzzled biochemists for long time and several groups have addressed this issue comparing natural protein sequences to completely random ones coming to contradicting conclusions. Previous works in literature focused on the analysis of primary structure in an attempt to identify possible signature of evolutionary editing. Conversely, in this work we compare a set of 762 natural proteins with an average length of 70 amino acids and an equal number of completely random ones of comparable length on the basis of their structural features. We use an ad hoc Evolutionary Neural Network Algorithm (ENNA) in order to assess whether and to what extent natural proteins are edited from random polypeptides employing 11 different structure-related variables (i.e. net charge, volume, surface area, coil, alpha helix, beta sheet, percentage of coil, percentage of alpha helix, percentage of beta sheet, percentage of secondary structure and surface hydrophobicity). The ENNA algorithm is capable to correctly distinguish natural proteins from random ones with an accuracy of 94.36%. Furthermore, we study the structural features of 32 random polypeptides misclassified as natural ones to unveil any structural similarity to natural proteins. Results show that random proteins misclassified by the ENNA algorithm exhibit a significant fold similarity to portions or subdomains of extant proteins at atomic resolution. Altogether, our results suggest that natural proteins are significantly edited from random polypeptides and evolutionary editing can be readily detected analyzing structural features. Furthermore, we also show that the ENNA, employing simple structural descriptors, can predict whether a protein chain is natural or random.

Purification of a lectin with antibacterial activity from Bothrops leucurus snake venom

A novel lectin was isolated from Bothrops leucurus snake venom using a combination of affinity and gel filtration chromatographies. The lectin (BlL) agglutinated glutaraldehyde-treated rabbit and human erythrocytes with preference for rabbit erythrocytes. Galactose, raffinose, lactose, fetal bovine serum and casein inhibited lectin-induced rabbit erythrocyte agglutination. BlL, with a molecular mass of 30 kDa and composed of two subunits of 15 kDa, showed dependence on calcium. BlL is an acidic protein with highest activity over the pH range of 4.0-7.0 and stable under heating to 70°C. Fluorescence emission spectra showed tryptophan residues partially buried within the lectin structure. The percentages of secondary structure revealed by circular dichroism were 1% α-helix, 44% β-sheet, 24% β-turn and 31% unordered. BlL showed effective antibacterial activity against Gram-positive bacteria Staphylococcus aureus, Enterococcus faecalis and Bacillus subtilis with minimal inhibitory concentrations of 31.25, 62.25 and 125 μg/mL, respectively. In conclusion, B. leucurus snake venom contains a galactoside-binding lectin with antibacterial activity.

Biocontrol of Fusarium species by a novel lectin with low ecotoxicity isolated from Sebastiania jacobinensis

A lectin from Sebastiania jacobinensis bark was isolated using a combination of acetone precipitation, ammonium sulphate fractionation, ion exchange and gel filtration chromatographies. The lectin purified, with a molecular mass of 52.0 kDa and composed of two subunits of 24 kDa, is a glycoprotein with a neutral carbohydrate content of 6.94%. The lectin shows maximum activity over the pH range 4.0–7.5 and heat stability up to 70 °C. Our results show that the lectin is an incompetitive inhibitor for trypsin, with a Ki of 0.39 ± 0.02 μM. Fluorescence spectroscopy indicated the existence of a hydrophobic surface. The percentages of secondary structure are 75% α-helix, 10% β-sheet, 5% β-turn and 10% unordered. Lectin inhibits the mycelial growth of Fusarium moniliforme and Fusarium oxysporum with an IC 50 value of 123 ± 0.5 and 303 ± 0.9 μg, respectively. Artemia salina Leach and embryos of Biomphalaria glabrata are not affected by the lectin, indicating low environmental toxicity. Alternative viewpoints are presented that might hopefully help in future efforts to develop safer and more effective microbial control agents.

Crystal Structure of a Two-domain Multicopper Oxidase

The two-domain multicopper oxidases are proposed to be key intermediates in the evolution of three-domain multicopper oxidases. A number of two-domain multicopper oxidases have been identified from genome sequences and are classified as type A, type B, or type C on the basis of the predicted location of the type 1 copper center. The crystal structure of blue copper oxidase, a type C two-domain multicopper oxidase from Nitrosomonas europaea, has been determined to 1.9 A resolution. Blue copper oxidase is a trimer, of which each subunit comprises two cupredoxin domains. Each subunit houses a type 1 copper site in domain 1 and a type 2/type 3 trinuclear copper cluster at the subunit-subunit interface. The coordination geometry at the trinuclear copper site is consistent with reduction of the copper ions. Although the overall architecture of blue copper oxidase is similar to nitrite reductases, detailed structural alignments show that the fold and domain orientation more closely resemble the three-domain multicopper oxidases. These observations have important implications for the evolution of nitrite reductases and multicopper oxidases.

The CBS Domain Protein MJ0729 ofMethanocaldococcus jannaschiiIs a Thermostable Protein with a pH-Dependent Self-Oligomerization

CBS domains are small protein motifs, usually associated in tandems, that are involved in binding to adenosyl groups. In humans, several genetic diseases have been associated with mutations in CBS domains, and then, they can be considered as promising targets for the rational design of new drugs. However, there are no structural studies describing their oligomerization states, conformational preferences, and stability. In this work, the oligomerization state, the stability, and conformational properties of the CBS domain protein MJ0729 from Methanocaldococcus jannaschii were explored by using a combination of hydrodynamic (namely, ultracentrifugation, DLS, DOSY-NMR, and gel filtration) and spectroscopic techniques (fluorescence, circular dichroism, NMR, and FTIR). The results indicate that the protein had a pH-dependent oligomerization equilibrium: at pH 7, the dominant species is a dimer, where each monomer is a two-CBS domain protein, and at pH 4.5-4.8, the dominant species is a tetramer, with an oblong shape, as shown by X-ray. Deconvolution of the FTIR spectra indicates that the monomer at physiological pH has 26% alpha-helical structure and 17% beta-sheet, with most of the structure disordered. These results are similar to the percentages of secondary structure of the monomer in the resolved tetrameric X-ray structure (21% of alpha-helical structure and 7% of beta-sheet). At pH 2.5, there was a decrease in the level of secondary structure of the monomer, and formation of intermolecular hydrogen bonds, as shown by FTIR, suggesting the presence of high-molecular weight species. The physiological dimeric species is thermal and chemically very stable with a thermal midpoint of approximately 99 degrees C, as shown by both DSC and FTIR; the GdmCl chemical midpoint of the dimeric species occurs in a single step and was greater than 4 M.

The family 52 β-xylosidase from Geobacillus stearothermophilus is a dimer: Structural and biophysical characterization of a glycoside hydrolase

Xylans are the most abundant polysaccharides forming the plant cell wall hemicelluloses, and they are degraded, among other proteins, by β-xylosidase enzymes. In this work, the structural and biophysical properties of the family 52 β-xylosidase from Geobacillus stearothermophilus, XynB2, are described. Size exclusion chromatography, analytical centrifugation, ITC, CD, fluorescence (steady state and ANS-binding) and FTIR were used to obtain the structure, the oligomerization state and the conformational changes of XynB2, as pH, chemical denaturants or temperature were modified. This report describes the first extensive conformational characterization of a family 52 β-xylosidase. The active protein was a highly hydrated dimer, whose active site was formed by the two protomers, and it probably involved aromatic residues. At low pH, the protein was not active and it populated a monomeric molten-globule-like species, which had a conformational transition with a p K a of ~ 4.0. Thermal and chemical-denaturations of the native protein showed hysteresis behaviour. The protein at physiological pH was formed by α-helix (30%) and β-sheet (30%), as shown by CD and FTIR. Comparison with other xylosidases of the same family indicates that the percentages of secondary structure seem to be conserved among the members of the family.

Tight-Binding “Dihedral Orbitals” Approach to the Degree of Folding of Macromolecular Chains

We develop a tight-binding molecular approach to quantify the degree of folding of a macromolecular chain. This approach is based on the linear combination of "dihedral" orbitals to give molecular orbitals (LCDO-MO). The dihedral orbitals are a set of orbitals situated in each dihedral angle of the chain. The LCDO-MO approach remains basically topological, and we display its direct relation to known graph theoretical concepts. Using this approach, we define the dihedral electronic energy and the dihedral electronic partition function of a linear macromolecular chain. We show that the partition function per dihedral angle quantifies the degree of folding of the dihedral graph. We analyze the empirical relationship between these two functions by using a series of 100 proteins. We also study the relation between these two functions and the percentages of secondary structure for these proteins. Finally, we illustrate the use of the dihedral energy and the partition function in structure-property studies of proteins by analyzing the binding of steroids to DB3 antibody.

Formulation of maltodextrin entrapped in polycaprolactone microparticles for protein and vaccine delivery: Effect of size determining formulation process variables of microparticles on the hydrodynamic diameter of BSA

Size of the microparticle and integrity of the released protein are two crucial factors which dictate the success of any protein or vaccine delivery system. The primary objective was to optimize bovine serum albumin (BSA) loaded polycaprolactone/maltodextrin (PCL/MD) microparticles in terms of its size and the hydrodynamic diameter of the released protein. The effect of size determining formulation process variables (SDFPV) of microparticles on the hydrodynamic diameter of protein antigen was determined. The SDFPV were optimized by a compromise between the microparticle size and the relative hydrodynamic stability of protein released from it. Percentage of secondary structure of the protein released from the optimized formulation as determined by circular dichroism spectra along with SELCON software was also similar to that of native BSA suggesting the potential of PCL /MD microparticles for protein or vaccine delivery.

Escherichia coli CorA Periplasmic Domain Functions as a Homotetramer to Bind Substrate

CorA is a primary Mg2+ transporter in bacteria, which also mediates influx of Ni2+ and Co2+. Topological studies suggested that it could be divided into a large soluble periplasmic domain (PPD) and three membrane-spanning alpha-helixes. In the present study, glutathione S-transferase (GST) fusion Escherichia coli CorA PPD was purified by GST affinity chromatography, and PPD was obtained by on-column thrombin digestion. Size-exclusion chromatography indicated that purified PPD exists as a homotetramer. Single particle electron microscopy analysis of PPD and two-dimensional crystals of GST-PPD indicated that E. coli CorA PPD is a pyramid-like homotetramer with a central cavity. Comparison of the CD spectra of full-length CorA and PPD also suggested that PPD has similar secondary structure to the full-length CorA. Dissociation constants for CorA and PPD with their substrates, determined by dose-dependent fluorescence quench of ligands, suggested that purified PPD retains its substrate binding ability as native CorA. The CorA PPD structure described here may provide structural information for the E. coli CorA functional oligomeric state.

Multicomponent Peak Modeling of Protein Secondary Structures: Comparison of Gaussian with Lorentzian Analytical Methods for Plant Feed and Seed Molecular Biology and Chemistry Research

The objective of this study was to compare Gaussian and Lorentzian multicomponent peak modeling methods in quantification of protein secondary structures of various plant seed and feed tissues within intact tissue at a cellular and subcellular level using the advanced synchrotron light sourced Fourier transform infrared (FT-IR) microspectroscopy (S-FTIR). This experiment was performed at the beamline U10B at the National Synchrotron Light Source (NSLS) in Brookhaven National Laboratory (BNL), U.S. Dept of Energy (NSLS-BNL, NY). The results show that in the comparison of the Gaussian and Lorentzian multi-peak modeling methods, the Gaussian method is more accurate for fitting multi-peak curves of protein secondary structures than the Lorentzian method, with higher modeling R(2) values (0.92 versus 0.89, P < 0.05). There were no large differences (P > 0.05) in the quantification of the relative percentage alpha-helices, beta-sheets, and others in protein secondary structures of the plant seed tissues, with averages of 30.2%, 40.4%, and 29.4%, respectively. However, there are significant differences (P < 0.05) in the quantification of the ratios of sheet alpha-helix (1.42 versus 1.60; SEM = 0.058) in protein secondary structures of the plant seed tissues. With synchrotron FT-IR microspectroscopy, the ultrastructural-chemical makeup and nutritive characteristics could be revealed at a high spatial resolution. Synchrotron-based FT-IR microspectroscopy revealed that the secondary structure of protein differed between the plant seed tissues in terms of relative percentage and ratio of protein secondary structures (alpha-helix and beta-sheet) within cellular dimensions. The results also show that the flaxseed tissues contained higher (P < 0.05) percentage alpha-helix (38.6 versus 24.0%) beta-sheet (45.3 versus 36.9%), lower (P < 0.05) percentage of other secondary structures (16.1% versus 39.0%), and higher (P < 0.05) ratios alpha-helix beta-sheet (0.90 versus 0.69) than the winterfat seed tissues. It must be mentioned that the relative percentages of protein secondary structure may not reflect the true secondary structure. However, the purpose of modeling the relative percentage of secondary structure was to detect the variety of differences among seed/feed/plant tissues and their relation to nutritive value and digestive behavior. The results demonstrate the potential of highly spatially resolved synchrotron-based FT-IR microspectroscopy to reveal protein secondary structures of the plant seed/feed tissues. Further study is needed to quantify the relationship between protein secondary structures and nutrient availability and digestive behavior of various varieties of plant seed tissues. Information from the infrared probing of protein secondary structures can be valuable as a guide to maintaining protein nutritive value and quality for animal and human use.

A protein folding degree measure and its dependence on crystal packing, protein size, secondary structure, and domain structural class.

Comparing two or more protein structures with respect to their degree of folding is common practice in structural biology despite the fact that there is no scale for a folding degree. Here we introduce a formal definition of a folding degree, capable of quantitative characterization. This enables ordering among protein chains based on their degree of folding. The folding degree of a data set of 152 representative nonhomologous proteins is then studied. We demonstrate that the variation in the folding degree seen for this data set is not due to crystallization artifacts or experimental conditions, such as resolution, refinement protocol, pH, or temperature. A good linear relationship is observed between the folding degree and the percentages of secondary structures in the protein. The folding degree is able to account for the small changes produced in the structure due to crystal packing and temperature. Automating the classification of proteins into their respective structural domain classes, namely mainly-alpha, mainly-beta, and alpha-beta, is also possible.

Hyperthermophilic dehydrogenase enzymes.

Archaeal dehydrogenases are often found to be of a specific class of dehydrogenase which has low sequence identity to the equivalent bacterial and eukaryotic counterparts. This paper focuses on two different types of hyperthermophilic dehydrogenase enzyme that have been cloned and over-expressed in Escherichia coli. The crystallographic structures of the apo form of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) from Sulfolobus solfataricus and the related holo form of GAPDH from Methanothermus fervidus have been solved to high resolution. The zinc-containing structure of ADH (alcohol dehydrogenase) from Aeropyrum pernix has also been solved as a quaternary complex with the cofactor NADH and the inhibitor octanoic acid. The results show that despite the low sequence identity to the related enzymes found in other organisms the fold of the protein chain is similar. The archaeal GAPDH enzymes show a relocation of the active site which is a feature of evolutionary interest. The high thermostability of these three archaeal dehydrogenases can be attributed to a combination of factors including an increase in the number of salt bridges and hydrophobic interactions, a higher percentage of secondary structure and the presence of disulphide bonds.