Secondary Structure Determination Research Articles

Solution Structure of Porcine Pancreatic Procolipase as Determined from 1H Homonuclear Two-Dimensional and Three-Dimensional NMR

Procolipase is the precursor of colipase, which acts as protein cofactor for the activity of pancreatic lipase. The solution structure of procolipase has been determined by 1H NMR using two- and three-dimensional measurements. The secondary structure determination identified two separate three-stranded beta-sheet regions with concomitant hydrogen bond patterns. The tertiary structure of the protein was determined using 863 non-trivial proton--proton distance constraints, 14 hydrogen bond distance constraints and 55 phi and 25 X1 dihedral constraints. The structure that was obtained from distance geometry and energy refinement contains three highly disordered loops as well as a disordered N- and C-terminal region. The remaining part of the structure is well defined with a root-mean-square deviation (rmsd) relative to the average of 0.09 +/- 0.02 nm for backbone atoms (residues 11-30, 37-50, 57-69, 83-89). The protein comprises two identical domains, each containing a three-strand beta-sheet and two disulfide bonds: a 15-residue region in each domain superimposes with 0.07 nm rmsd, measured on backbone atoms. The solution structure is nearly identical to the crystal structure. It is in agreement with previous NMR data and, in combination with these data, supports the current model of procolipase micelle interaction and the lipase activation by colipase.

Determination of endosperm protein secondary structure in hard wheat breeding lines using synchrotron infrared microspectroscopy

One molecular aspect of mature hard wheat protein quality for breadmaking is the relative amount of endosperm protein in the α-helix form compared with that in other secondary structure forms including β-sheet. Modeling of α-helix and β-sheet absorption bands that contribute to the amide I band at 1650 cm −1 was applied to more than 1500 spectra in this study. The microscopic view of wheat endosperm is dominated by many large starch granules with protein in between. The spectrum produced from in situ microspectroscopy of this mixture is dominated by carbohydrate bands from the large starch granules that fill up the field. The high spatial resolution achievable with synchrotron infrared microspectroscopy enables revealing good in situ spectra of the protein located interstitially. Synchrotron infrared microspectroscopic mapping of 4 μm thick frozen sections of endosperm in the subaleurone region provides spectra from a large number of pixels. Pixels with protein-dominated spectra are sorted out from among adjacent pixels to minimize the starch absorption and scattering contributions. Subsequent data treatment to extract information from the amide I band requires a high signal to noise ratio. Although spectral interference of the carbohydrate band on the amide band is not a problem, the scattering produced by the large starch granules diminishes the signal to noise ratio throughout the spectrum. High density mapping was done on beamlines U2B and U10B at the National Synchrotron Light Source at Brookhaven National Laboratory, Upton, NY. Mapping with a single masked spot size of 5.5 μm diameter or confocal 5 μm × 5 μm spot size, respectively, on the two beamlines used produced spectra for new breeding lines under current consideration. Appropriate data treatment allows calculation of a numerical estimate of the α-helix population relative to other secondary protein structures from the position and shape of the amide I absorption band. Current breeding lines show a substantial variance in this feature and its determination allows the prediction of relative quality for breadmaking to be taken into consideration for subsequent steps in the wheat breeding process. Data treatments include deconvolution, modeling of the individual resulting bands that contribute to the amide I band to enable measurement of the relative amounts of both forms. Results with specimens representing multiple crop years of hard winter wheat breeding are reported. It is evident that a range is available for the breeder to choose from, that allows including this protein molecular structural attribute in the selection process.

1H, 13C, and 15N resonance assignment of the C103S mutant of the N-terminal domain of DsbD from Neisseria meningitidis

We report the nearly complete 1H, 13C, and 15N resonance assignments of the C103S mutant of the N-terminal domain of DsbD from Neisseria meningitides. Secondary structure determination using CSI method leads to the prediction of nine beta-sheet parts.

Polarization-dependent two-photon absorption for the determination of protein secondary structure: A theoretical study

A new method for sensitive determination of protein secondary structure via multi-photon absorption is considered theoretically. Perturbation theory is developed to describe the polarization-dependent two-photon absorption (TPA) of α-helix and β-sheet protein secondary structures. The exciton coupling interactions responsible for relatively weak electronic circular dichroism in one-photon absorption are predicted to give rise to large changes in the TPA cross-section (>200%) for circular versus linear incident polarizations, defined as CLD. The CLD effect in TPA is electric dipole-allowed, which explains the much greater sensitivity. These predictions suggest TPA should be a viable means of sensitively probing protein secondary structure.

Charge-Transfer Transitions in the Vacuum-Ultraviolet of Protein Circular Dichroism Spectra

Circular dichroism (CD) is widely used in the structural characterization and secondary structure determination of proteins. The vacuum UV region (below 190 nm), where charge-transfer transitions have an influence on the CD spectra, can be accessed using synchrotron radiation circular dichroism (SRCD) spectroscopy. Recently, charge-transfer transitions in a conformationally diverse set of dipeptides have been characterized ab initio using complete active space self-consistent field calculations, and the relevant charge distributions have been parametrized for use in the matrix method for calculations of protein CD. Here, we present calculations of the vacuum UV CD spectra of 71 proteins, for which experimental SRCD spectra and X-ray crystal structures are available. The theoretical spectra are calculated considering charge-transfer and side chain transitions. This significantly improves the agreement with experiment, raising the Spearman correlation coefficient between the calculated and the experimental intensity at 175 nm from 0.12 to 0.79. The influence of the conformation on charge-transfer transitions is analyzed in detail, showing that the n --> pi* charge-transfer transitions are most important in alpha-helical proteins, whereas in beta strand proteins the pi --> pi* charge-transfer transition along the chain in the amino- to carboxy-end direction is most dominant.

Combining sequence-based prediction methods and circular dichroism and infrared spectroscopic data to improve protein secondary structure determinations

BackgroundA number of sequence-based methods exist for protein secondary structure prediction. Protein secondary structures can also be determined experimentally from circular dichroism, and infrared spectroscopic data using empirical analysis methods. It has been proposed that comparable accuracy can be obtained from sequence-based predictions as from these biophysical measurements. Here we have examined the secondary structure determination accuracies of sequence prediction methods with the empirically determined values from the spectroscopic data on datasets of proteins for which both crystal structures and spectroscopic data are available.ResultsIn this study we show that the sequence prediction methods have accuracies nearly comparable to those of spectroscopic methods. However, we also demonstrate that combining the spectroscopic and sequences techniques produces significant overall improvements in secondary structure determinations. In addition, combining the extra information content available from synchrotron radiation circular dichroism data with sequence methods also shows improvements.ConclusionCombining sequence prediction with experimentally determined spectroscopic methods for protein secondary structure content significantly enhances the accuracy of the overall results obtained.

A Hypoallergenic Vaccine Obtained by Tail-to-Head Restructuring of Timothy Grass Pollen Profilin, Phl p 12, for the Treatment of Cross-Sensitization to Profilin

Profilins are highly cross-reactive allergens in pollens and plant food. In a paradigmatic approach, the cDNA coding for timothy grass pollen profilin, Phl p 12, was used as a template to develop a new strategy for engineering an allergy vaccine with low IgE reactivity. Non-IgE-reactive fragments of Phl p 12 were identified by synthetic peptide chemistry and restructured (rs) as a new molecule, Phl p 12-rs. It comprised the C terminus of Phl p 12 at its N terminus and the Phl p 12 N terminus at its C terminus. Phl p 12-rs was expressed in Escherichia coli and purified to homogeneity. Determination of secondary structure by circular dichroism indicated that the restructuring process had reduced the IgE-reactive alpha-helical contents of the protein but retained its beta-sheet conformation. Phl p 12-rs exhibited reduced IgE binding capacity and allergenic activity but preserved T cell reactivity in allergic patients. IgG Abs induced by immunization of mice and rabbits with Phl p 12-rs cross-reacted with pollen and food-derived profilins. Recombinant Phl p 12-rs, rPhl p 12-rs, induced less reaginic IgE to the wild-type allergen than rPhl p 12. However, the rPhl p 12-rs-induced IgGs inhibited allergic patients' IgE Ab binding to profilins to a similar degree as those induced by immunization with the wild type. Phl p 12-rs specific IgG inhibited profilin-induced basophil degranulation. In conclusion, a restructured recombinant vaccine was developed for the treatment of profilin-allergic patients. The strategy of tail-to-head reassembly of hypoallergenic allergen fragments within one molecule represents a generally applicable strategy for the generation of allergy vaccines.

Study of the mechanism of action of anoplin, a helical antimicrobial decapeptide with ion channel‐like activity, and the role of the amidated C‐terminus

Anoplin, an antimicrobial, helical decapeptide from wasp venom, looses its biological activities by mere deamidation of its C-terminus. Secondary structure determination, by circular dichroism spectroscopy in amphipathic environments, and lytic activity in zwitterionic and anionic vesicles showed quite similar results for the amidated and the carboxylated forms of the peptide. The deamidation of the C-terminus introduced a negative charge at an all-positive charged peptide, causing a loss of amphipathicity, as indicated by molecular dynamics simulations in TFE/water mixtures and this subtle modification in a peptide's primary structure disturbed the interaction with bilayers and biological membranes. Although being poorly lytic, the amidated form, but not the carboxylated, presented ion channel-like activity on anionic bilayers with a well-defined conductance step; at approximately the same concentration it showed antimicrobial activity. The pores remain open at trans-negative potentials, preferentially conducting cations, and this situation is equivalent to the interaction of the peptide with bacterial membranes that also maintain a high negative potential inside.

New method to predict conserved RNA structures

RNA secondary structure prediction is one of the classic problems of bioinformatics. The most efficient approaches to solving this problem are based on comparative analysis. As a rule, multiple RNA sequence alignment and subsequent determination of a common secondary structure are used. A new algorithm was developed to obviate the need for preliminary multiple sequence alignment. The algorithm is based on a multilevel MEME-like iterative search for a generalized profile. The search for common blocks in RNA sequences is carried out at the first level. Then the algorithm refines the chains consisting of these blocks. Finally, the search for sets of common helices, matched with alignment blocks, is carried out. The algorithm was tested with a tRNA set containing additional junk sequences and with RFN riboswitches. The algorithm is available at http://bioinf.fbb.msu.ru/RNAAlign.

1H, 13C and 15N resonance assignment of the oxidized form (Cys67–Cys70) of the N-terminal domain of PilB from Neisseria meningitidis

We report the nearly complete 1H, 13C and 15N resonance assignments of the oxidized form (Cys(67)-Cys(70)) of the N-terminal domain of PilB from Neisseria meningitidis. Secondary structure determination using CSI method and TALOS leads mainly to the prediction of 7 alpha-helical and 5 beta-sheet parts.

Conversion of non-fibrillar β-sheet oligomers into amyloid fibrils in Alzheimer’s disease amyloid peptide aggregation

Aβ(1–40) is one of the main components of the fibrils found in amyloid plaques, a hallmark of brains affected by Alzheimer’s disease. It is known that prior to the formation of amyloid fibrils in which the peptide adopts a well-ordered intermolecular β-sheet structure, peptide monomers associate forming low and high molecular weight oligomers. These oligomers have been previously described in electron microscopy, AFM, and exclusion chromatography studies. Their specific secondary structures however, have not yet been well established. A major problem when comparing aggregation and secondary structure determinations in concentration-dependent processes such as amyloid aggregation is the different concentration range required in each type of experiment. In the present study we used the dye Thioflavin T (ThT), Fourier-transform infrared spectroscopy, and electron microscopy in order to structurally characterize the different aggregated species which form during the Aβ(1–40) fibril formation process. A unique sample containing 90 μM peptide was used. The results show that oligomeric species which form during the lag phase of the aggregation kinetics are a mixture of unordered, helical, and intermolecular non-fibrillar β-structures. The number of oligomers and the amount of non-fibrillar β-structures grows throughout the lag phase and during the elongation phase these non-fibrillar β-structures are transformed into fibrillar (amyloid) β-structures, formed by association of high molecular weight intermediates.

COMPUTATIONAL MOLECULAR BIOLOGY AND WAVELETS

Computational molecular biology is a new discipline, bringing together computational, statististical, experimental and technological methods, which is energizing and dramatically accelerating the discovery of new technologies and tools for molecular biology. The application of wavelets in molecular biology is a thriving field of research. It has two important and linked benefits: an improved ability for capturing hidden components from biological data and a better link between biological systems and the mathematics objects used to describe them. Nowadays is a growing interest in using wavelets in the analysis of biological sequences and in different areas of molecular biology: genome sequence, protein structure, microarray data analysis. But, why wavelets? Because a wavelet is a wavy function carefully constructed so as to have certain mathematical properties. Wavelets have already had a remarkable impact because a lot of people are now applying wavelets to a lot of situations and all seem to report favorable results. Wavelet techniques are now used in many statisical areas such as: density estimation, nonparametric regresion, time series analysis, change-point problems. Several authors revealed that wavelets can be useful in detecting patterns in DNA sequences. Lio (2000) showed that wavelet variance decomposition of bacterial genome sequences coded as G,C=1 and A,T=0 (or -1) can reveal the location of pathogenicity islands. Wavelets have been applied to all aspects of protein structural investigation: primary sequence evolution, secondary and tertiary structure determination, refinement of X-ray crystallography, drug design and visualization. A wavelet data-dependent treshold technique is applied to predict location and topology of helices in transmembrane proteines.The CWT (continuous wavelet transforms) are used to analyse the hydrophobicity and relative accesible surface area of a variety of repeating protein motifs such as TIM barrels, propellor blades, coiled coils and leucine repeats. Wavelets seem more suitable for describing scaling biological structures and signals then other mathematical transforms because wavelet basis functions can have different complex shapes, each adaptable for a different class of problems.

Circular and linear dichroism of proteins

Circular dichroism (CD) is an important technique in the structural characterisation of proteins, and especially for secondary structure determination. The CD of proteins can be calculated from first principles using the so-called matrix method, with an accuracy which is almost quantitative for helical proteins. Thus, for proteins of unknown structure, CD calculations and experimental data can be used in conjunction to aid structure analysis. Linear dichroism (LD) can be calculated using analogous methodology and has been used to establish the relative orientations of subunits in proteins and protein orientation in an environment such as a membrane. However, simple analysis of LD data is not possible, due to overlapping transitions. So coupling the calculations and experiment is an important strategy. In this paper, the use of LD for the determination of protein orientation and how these data can be interpreted with the aid of calculations, are discussed. We review methods for the calculation of CD spectra, focusing on semiempirical and ab initio parameter sets used in the matrix method. Lastly, a new web interface for online CD and LD calculation is presented.

Application of expert networks for predicting proteins secondary structure

The present study utilizes expert neural networks for the prediction of proteins secondary structure. We use three independent networks, one for each structure (alpha, beta and coil) as the first-level processing unit; decision upon the chosen structure for each residue is carried out by a second-level, post-processing unit, which utilizes the Chou and Fasman frequency values F α and F β in order to strengthen and/or deplete the probability of the specific structure under investigation. The highest prediction case was 76%. Our method requires primitive computational means and a relatively small training set, while still been comparable to previous work. It is not meant to be an alternative to the determination of secondary structure by means of free energy minimization, integration of dynamic equations of motion or crystallography, which are expensive, time-consuming and complicated, but to provide additional constrains, which might be considered and incorporated into larger computing setups in order to reduce the initial search space for the above methods.

Preparation, Purification, and Secondary Structure Determination of Bacillus circulans Xylanase. A Modular Laboratory Incorporating Aspects of Molecular Biology, Biochemistry, and Biophysical Chemistry

An integrated six-week module that allows students to develop biochemistry laboratory techniques in the context of a discovery-oriented project has been developed. This module, suitable for undergraduate third- or fourth-year biochemistry lab, focuses on protein over-expression, purification, and stability of Bacillus circulans xylanase (BCX). BCX is an ideal protein to study as it has important industrial applications and is highly over-expressed, easily purified in a single chromatographic step, and stable. The discovery-based aspect of the project monitors, by circular dichroism spectroscopy, the secondary structure of BCX under conditions that compromise its stabilizing intramolecular forces. Results are written up as a formal lab report in the format of an article in Biochemistry. This module aids in student learning for several reasons: (i) by engaging students in multiple techniques in the context of a single protein, they understand how each technique contributes to the overall production and char...

Optimization of the hydrochloric acid concentration used for trifluoroacetate removal from synthetic peptides

Trifluoroacetate (CF3COO-, or TFA) is almost always present in commercially synthesized peptides. Unfortunately, it has a strong infrared (IR) absorption band at 1673 cm-1, significantly overlapping or even completely obscuring the amide I band of a peptide. In such cases TFA must be removed from the solution in order to be able to use IR absorption spectroscopy for peptide secondary structure determination. The most convenient and widely used procedure involves peptide lyophilization from a 0.1 M HCl solution. In our studies of the tryptophan-rich antimicrobial peptide indolicidin, we have found that caution should be taken when using this HCl concentration. High HCl concentrations (>10 mM in unbuffered solutions and > 50 mM in buffered solutions) may modify the peptide structure and reduce its thermal stability, thereby interfering with subsequent structural investigations of the peptide. Our results indicate that HCl concentrations between 2 and 10 mM are adequate to remove essentially all TFA impurities without any modification of the peptide secondary structure.

Application of SIMPLISMA purity function for variable selection in multivariate regression analysis: A case study of protein secondary structure determination from infrared spectra

A novel approach for the pre-selection of wavelengths, to be used in combination with Partial Least Squares (PLS) or other multivariate regression techniques, is presented. This variable selection method makes use of the purity function, originally suggested in the SIMPLe-to-use Interactive Self-modeling Mixture Analysis (SIMPLISMA) algorithm, to map up the regions of potentially influential variables. The selected intervals are then individually tested in practical modeling and prediction, and an optimal subset of variables is obtained. The algorithm is simple and intuitive and does not rely on iterative variable searches. The method was tested on a set of infrared protein spectra in order to improve the quantitative determination of the fractions of two secondary structure elements, α-helices and β-strands (β-sheets) in the protein polypeptide chain. Comparable results to those obtained through interval PLS (iPLS), an exhaustive search-based algorithm, were achieved in this study. Our method was shown to be particularly beneficial in combination with variable weighting by their inverse standard deviation.

Interpreting Oligonucleotide Microarray Data To Determine RNA Secondary Structure: Application to the 3‘ End of Bombyx mori R2 RNA

A method to deduce RNA secondary structure on the basis of data from microarrays of 2'-O-methyl RNA 9-mers immobilized in agarose film on glass slides is tested with a 249 nucleotide RNA from the 3' end of the R2 retrotransposon from Bombyx mori. Various algorithms incorporating binding data and free-energy minimization calculations were compared for interpreting the data to provide possible secondary structures. Two different methods give structures with 100 and 87% of the base pairs determined by sequence comparison. In contrast, structures predicted by free-energy minimization alone by Mfold and RNAstructure contain 52 and 72% of the known base pairs, respectively. This combination of high throughput microarray techniques with algorithms using free-energy calculations has potential to allow for fast determination of RNA secondary structure. It should also facilitate the design of antisense and siRNA oligonucleotides.

Evaluation of the Information Content in Infrared Spectra for Protein Secondary Structure Determination

Fourier-transform infrared spectroscopy is a method of choice for the experimental determination of protein secondary structure. Numerous approaches have been developed during the past 15 years. A critical parameter that has not been taken into account systematically is the selection of the wavenumbers used for building the mathematical models used for structure prediction. The high quality of the current Fourier-transform infrared spectrometers makes the absorbance at every single wavenumber a valid and almost noiseless type of information. We address here the question of the amount of independent information present in the infrared spectra of proteins for the prediction of the different secondary structure contents. It appears that, at most, the absorbance at three distinct frequencies of the spectra contain all the nonredundant information that can be related to one secondary structure content. The ascending stepwise method proposed here identifies the relevance of each wavenumber of the infrared spectrum for the prediction of a given secondary structure and yields a particularly simple method for computing the secondary structure content. Using the 50-protein database built beforehand to contain as little fold redundancy as possible, the standard error of prediction in cross-validation is 5.5% for the α-helix, 6.6% for the β-sheet, and 3.4% for the β-turn.

NMR Assignment and Secondary Structure Determination of the C-terminal MA-3 Domain of the Tumour Suppressor Protein Pdcd4

NMR Assignment and Secondary Structure Determination of the C-terminal MA-3 Domain of the Tumour Suppressor Protein Pdcd4