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"Soft"-cuticle protein secondary structure as revealed by FT-Raman, ATR FT-IR and CD spectroscopy.

The nature of the interaction of insect cuticular proteins and chitin is unknown even though about half of the cuticular proteins sequenced thus far share a consensus region that has been predicted to be the site of chitin binding. We previously predicted the preponderance of a beta-pleated sheet in the consensus region and proposed its responsibility for the formation of helicoidal cuticle (Iconomidou et al., Insect Biochem. Mol. Biol. 29 (1999) 285). In this study, we examined experimentally the secondary structure of intact and guanidine hydrochloride extracted cuticle and the cuticular protein extract. The studied cuticle came from the larval dorsal abdomen of the lepidopteran Hyalophora cecropia, a classical example of "soft" cuticle. Analysis with FT-Raman, ATR FT-IR and CD spectroscopy indicates that antiparallel beta-pleated sheet is the predominant molecular conformation of "soft-cuticle" proteins both in situ in the cuticle and following extraction. It seems that this conformation dictates the modes of chitin-protein interaction in cuticle, in agreement with earlier proposals (Atkins, J. Biosci. 8 (1985) 375).

Amino Acid Sequence↗

[Information about the protein secondary structure improves quality of an alignment of protein sequences].

All popular algorithms of pair-wise alignment of protein primary structures (e.g. Smith-Waterman (SW), FASTA, BLAST, et al.) utilize only amino acid sequences. The SW-algorithm is the most accurate among them, i.e. it produces alignments that are most similar to the alignments obtained by superposition of protein 3D-structures. But even the SW-algorithm is unable to restore the 3D-based alignment if similarity of amino acid sequences (%id) is below 30%. We have proposed a novel alignment method that explicitly takes into account the secondary structure of the compared proteins. We have shown that it creates significantly more accurate alignments compared to SW-algorithm. In particular, for sequences with %id < 30% the average accuracy of the new method is 58% compared to 35% for SW-algorithm (the accuracy of an algorithmic sequence alignment is the part of restored position of a "golden standard" alignment obtained by superposition of corresponding 3D-structures). The accuracy of the proposed method is approximately identical both for experimental, and for theoretically predicted secondary structures. Thus the method can be applied for alignment of protein sequences even if protein 3D-structure is unknown. The program is available at ftp://194.149.64.196/STRUSWER/.

Algorithms↗

Molecular cloning of cDNA and analysis of protein secondary structure of Candida albicans enolase, an abundant, immunodominant glycolytic enzyme.

We isolated and sequenced a clone for Candida albicans enolase from a C. albicans cDNA library by using molecular genetic techniques. The 1.4-kbp cDNA encoded one long open reading frame of 440 amino acids which was 87 and 75% similar to predicted enolases of Saccharomyces cerevisiae and enolases from other organisms, respectively. The cDNA included the entire coding region and predicted a protein of molecular weight 47,178. The codon usage was highly biased and similar to that found for the highly expressed EF-1 alpha proteins of C. albicans. Northern (RNA) blot analysis showed that the enolase cDNA hybridized to an abundant C. albicans mRNA of 1.5 kb present in both yeast and hyphal growth forms. The polypeptide product of the cloned cDNA, which was purified as a recombinant protein fused to glutathione S-transferase, had enolase enzymatic activity and inhibited radioimmunoprecipitation of a single C. albicans protein of molecular weight 47,000. Analysis of the predicted C. albicans enolase showed strong conservation in regions of alpha helices, beta sheets, and beta turns, as determined by comparison with the crystal structure of apo-enolase A of S. cerevisiae. The lack of cysteine residues and a two-amino-acid insertion in the main domain differentiated C. albicans enolase from S. cerevisiae enolase. Immunofluorescence of whole C. albicans cells by using a mouse antiserum generated against the purified fusion protein showed that enolase is not located on the surface of C. albicans. Recombinant C. albicans enolase will be useful in understanding the pathogenesis and host immune response in disseminated candidiasis, since enolase is an immunodominant antigen which circulates during disseminated infections.

Amino Acid Sequence↗

Application of multiple sequence alignment profiles to improve protein secondary structure prediction.

The effect of training a neural network secondary structure prediction algorithm with different types of multiple sequence alignment profiles derived from the same sequences, is shown to provide a range of accuracy from 70.5% to 76.4%. The best accuracy of 76.4% (standard deviation 8.4%), is 3.1% (Q(3)) and 4.4% (SOV2) better than the PHD algorithm run on the same set of 406 sequence non-redundant proteins that were not used to train either method. Residues predicted by the new method with a confidence value of 5 or greater, have an average Q(3) accuracy of 84%, and cover 68% of the residues. Relative solvent accessibility based on a two state model, for 25, 5, and 0% accessibility are predicted at 76.2, 79.8, and 86. 6% accuracy respectively. The source of the improvements obtained from training with different representations of the same alignment data are described in detail. The new Jnet prediction method resulting from this study is available in the Jpred secondary structure prediction server, and as a stand-alone computer program from: http://barton.ebi.ac.uk/. Proteins 2000;40:502-511.

Algorithms↗

Use of variable selection in modeling the secondary structural content of proteins from their composition of amino acid residues.

The possibility of prediction of protein secondary structure content from composition of their amino acid residues can help in bridging the gap between proteins of known primary sequence having an unknown secondary structure. Almost all recently published models for understanding the relationship between composition (frequency of occurrence) of amino acid residues and secondary structure content of proteins involved composition of all 20 amino acid residues. However, it is well-known that many amino acid residues are mutually similar according to their physicochemical properties (hydrophobicity, hydrophilicity, charge, size, etc.). Because of that, we were motivated to investigate the possibility of reduction of the total number of terms (frequencies of amino acid residues) in the models for describing the relation between the composition of amino acid residues and the percentage of residues belonging to alpha, beta, and coil secondary structure. For this purpose, the CROMRsel algorithm (J. Chem. Inf. Comput. Sci. 1999, 39, 121-132) for selection of a small subset of the most important variables/descriptors into the multiregression (MR) models, i.e., frequency of occurrence of amino acid residues in proteins, was used. Analysis was performed on a data set containing 475 proteins, taken from Proteins 1996, 25, 157-168. A complete data set was partitioned into a 317-protein training set and 158-protein test set. The best possible linear models containing I=1, ..., 20 frequencies were selected among all 20 frequencies of occurrence of amino acid residues on the 317-protein training set, and were used for performing prediction of the corresponding percentage of secondary structure content on the 158-protein test set. For the 317-protein data set the best selected concise models for the alpha, beta, and coil secondary structure contain only 9, 5, and 8 frequencies, respectively. Selected concise models are of the same or better fitted, cross-validated, and predictive statistical parameters than the models containing all 20 frequencies. Additionally, for each I (I=1, ...., 20) 30 the best possible random models were selected. In each case, the best possible real models are much better than each of the best possible random models, showing clearly that there is no risk of a chance correlation (what one could expect due to the application of an exhaustive search for the best model having I frequencies among all 20!/I!(20-I)! possible models). Finally, the best selected models on the complete 475-protein data set for the alpha, beta, and coil secondary structure contain only 7, 4, and 7 frequencies of amino acid residues, respectively. These models are much simpler and have better fitted and cross-validated errors than the corresponding models from the literature, that were obtained without using a procedure for selection of the most important frequencies of amino acid residues in proteins.

Amino Acids↗

A refined accuracy index to evaluate algorithms of protein secondary structure prediction.

Nowadays even a 1% increase of the accuracy for the secondary structure prediction is considered remarkable progress. In this case, we have to consider the reasonableness of the accuracy index Q3, which is used widely. A refined accuracy index, called Q8, is proposed to evaluate algorithms of secondary structure prediction. It is shown that Q8 is superior to the widely used index Q3 in that the former carries more information of the predictive accuracy matrix than does the latter. Therefore, algorithms are evaluated more objectively by Q8 than Q3. Based on 396 nonhomologous proteins, five currently available algorithms of secondary structure prediction were evaluated and compared using the new index Q8. Of the five algorithms, PHD turned out to be the unique algorithm, with Q8 accuracy better than 70%. It is suggested that Q3 should be replaced by Q8 in evaluating secondary structure prediction in future studies.

Algorithms↗

Quantitative IR spectrophotometry of peptide compounds in water (H2O) solutions. III. Estimation of the protein secondary structure.

Infrared spectra of 13 globular proteins have been obtained in the 1800-1480-cm-1 region for H2O solutions. A method for estimating protein secondary structure from the ir spectrum has been developed. The method can also be used for estimating polypeptide and fibrous protein conformation. For the globular and fibrous proteins and polypeptides analyzed, the correlation coefficients between the ir and x-ray estimates of ordered helix, disordered helix, ordered beta-structure, disordered beta-structure, turns, and remainder were 0.98, 0.80, 0.99, 0.87, 0.90, and 0.92 respectively.

Animals↗

Determination of protein secondary structure and solvent accessibility using site-directed fluorescence labeling. Studies of T4 lysozyme using the fluorescent probe monobromobimane.

We report an investigation of how much protein structural information could be obtained using a site-directed fluorescence labeling (SDFL) strategy. In our experiments, we used 21 consecutive single-cysteine substitution mutants in T4 lysozyme (residues T115-K135), located in a helix-turn-helix motif. The mutants were labeled with the fluorescent probe monobromobimane and subjected to an array of fluorescence measurements. Thermal stability measurements show that introduction of the label is substantially perturbing only when it is located at buried residue sites. At buried sites (solvent surface accessibility of <40 A(2)), the destabilizations are between 3 and 5.5 kcal/mol, whereas at more exposed sites, DeltaDeltaG values of < or = 1.5 kcal/mol are obtained. Of all the fluorescence parameters that were explored (excitation lambda(max), emission lambda(max), fluorescence lifetime, quantum yield, and steady-state anisotropy), the emission lambda(max) and the steady-state anisotropy values most accurately reflect the solvent surface accessibility at each site as calculated from the crystal structure of cysteine-less T4 lysozyme. The parameters we identify allow the classification of each site as buried, partially buried, or exposed. We find that the variations in these parameters as a function of residue number reflect the sequence-specific secondary structure, the determination of which is a key step for modeling a protein of unknown structure.

Bacteriophage T4↗

Prediction of protein secondary structure from amino acid sequence.

The conformational parameters Pk for each amino acid species (j = 1-20) of sequential peptides in proteins are presented as the product of P(i,k), where i is the number of the sequential residues in the kth conformational state (k = alpha-helix, beta-sheet, beta-turn, or unordered structure). Since the average parameter for an n-residue segment is related to the average probability of finding the segment in the kth state, it becomes a geometric mean of (Pk)av = II (P(i,k))1/n with amino acid residue i increasing from 1 to n. We then used ln(Pk)av to convert a multiplicative process to a summation, i.e., ln(Pk)av = (1/n)sigma P(i,k) (i = 1 to n) for ease of operation. However, this is unlike the popular Chou-Fasman algorithm, which has the flaw of using the arithmetic mean for relative probabilities. The Chou-Fasman algorithm happens to be close to our calculations in many cases mainly because the difference between their Pk and our ln Pk is nearly constant for about one-half of the 20 amino acids. When stronger conformation formers and breakers exist, the difference become larger and the prediction at the N- and C-terminal alpha-helix or beta-sheet could differ. If the average conformational parameters of the overlapping segments of any two states are too close for a unique solution, our calculations could lead to a different prediction.

Algorithms↗

MacMatch: a tool for pattern-based protein secondary structure prediction.

A program is described for predicting the secondary structure of globular proteins on an Apple Macintosh computer. MacMatch produces turn predictions using augmented regular expression pattern matching. Helix and strand predictions are based on a neural network. Protein structural class (alpha/alpha), beta/beta, alpha/beta can be exploited to improve secondary structure prediction. The program is simple to use, and the package includes sets of tested patterns and trained neural networks.

Microcomputers↗

Protein secondary structure and homology by neural networks. The alpha-helices in rhodopsin.

Neural networks provide a basis for semiempirical studies of pattern matching between the primary and secondary structures of proteins. Networks of the perceptron class have been trained to classify the amino-acid residues into two categories for each of three types of secondary feature: alpha-helix or not, beta-sheet or not, and random coil or not. The explicit prediction for the helices in rhodopsin is compared with both electron microscopy results and those of the Chou-Fasman method. A new measure of homology between proteins is provided by the network approach, which thereby leads to quantification of the differences between the primary structures of proteins.

Amino Acid Sequence↗

ATR-FT/IR study on the interactions between gliadins and dextrin and their effects on protein secondary structure.

The effects of heat treatment and dextrin addition on the secondary structure of gliadins were investigated by means of attenuated total reflection Fourier transform infrared spectroscopy (ATR-FT/IR). Gliadins and gliadin/dextrin mixtures (before and after thermal treatment) were prepared as a dried protein film on the ATR-FT/IR zinc selenide cell plate and equilibrated at a water activity (a(w)) of 0.06. The results show that gliadins undergo conformational changes upon thermal treatment both in the absence and in the presence of dextrin. In particular, in the thermally treated gliadins, the decrease of the band at around 1651 cm(-)(1) and the increase of the bands at around 1628 and 1690 cm(-)(1) suggest a loss of alpha-helix structure and a higher content of protein aggregates. The same trend was observed in the presence of dextrin. Concerning the interactions between gliadins and dextrin, gliadin/dextrin mixtures show variations in the amide I region compared to native gliadins (e.g., an increase of the band at 1645 cm(-)(1) and the absence of the band at around 1668 cm(-)(1)) that might be due to hydrogen bond formation between gliadins and dextrin. It was also found that the spectrum of gliadin/dextrin mixtures was less affected by the hydration state than that of native gliadins, as observed from the differential spectra obtained by subtraction of the spectrum obtained at a(w) = 0.06 (driest condition tested) from the spectrum of the sample equilibrated at a(w) = 0.84. This could be due to the fact that C=O and N-H groups of gliadins are engaged to form hydrogen bonds with the hydroxyl groups of dextrin, and so they are not perturbed by the presence of water molecules. Finally, water activity effects on the secondary structure of gliadins are also discussed.

Dextrins↗

In unison: regularization of protein secondary structure predictions that makes use of multiple sequence alignments.

We present a method whose purpose is to post-process the fuzzy results of secondary structure prediction methods that use multiple sequence alignments, in order to obtain 'realistic' secondary structures, i.e., secondary structure elements whose length is greater than or equal to some predefined minimum length. This regularization helps with interpretation of the secondary structure prediction.

Algorithms↗

[Fourier transform infrared spectroscopy study on the protein secondary structure of photosystem II reaction center].

A successful study on the secondary structure of the isolated photosystem II (PSII) particles with the Fourier transform infrared spectroscopy is reported in this paper. The beta condensation effect is obviously characterized by infrared absorption spectra. The infrared spectra of both living protein and beta condensed protein samples are measured at room temperature. The amide I band in infrared spectrum is used to perform the quantitative analysis of the sample properties. The recorded spectra show the irreversible effect for the PSII particles after the 400 K heating. A rather strong change of the infrared spectra is observed due to the beta condensation of PSII protein. All the spectra are well fitted by 3-Lorentz-peak. The FTIR spectroscopy shows its effectiveness in studying the heating effect on the PSII particles.

Amides↗

Variable selection method improves the prediction of protein secondary structure from circular dichroism spectra.

A new procedure based on the statistical method of "variable selection" is used to predict the secondary structure of proteins from circular dichroism spectra. Variable selection adds the flexibility found in the Provencher and Glöckner method (S. W. Provencher and J. Glöckner, 1981, Biochemistry 20, 33-37) to the method of Hennessey and Johnson (J. P. Hennessey and W. C. Johnson, 1981, Biochemistry 20, 1085-1094). Two analytical methods are presented for choosing a solution from the series generated by the Provencher and Glöckner method, and this improves the technique. All three methods are compared and it is shown that both the variable selection method and the improved Provencher and Glöckner methods have equivalent reliability superior to the original Hennessey and Johnson method. For the new variable selection method, correlation coefficients calculated between X-ray structure and predicted secondary structures for data measured to 178 nm are: 0.97 for alpha-helix, 0.75 for beta-sheet, 0.50 for beta-turn, and 0.89 for other structures. Although the variable selection method improves the analysis of circular dichroism data truncated at 190 nm, data measured to 178 nm gives superior results. It is shown that improving the fit to the measured CD beyond the accuracy of the data can result in poorer analyses.

Circular Dichroism↗