Circular dichroism spectroscopy is a structural biology technique frequently applied to determine the secondary structure composition of soluble proteins. Our recently introduced computational ...analysis package SESCA aids the interpretation of protein circular dichroism spectra and enables the validation of proposed corresponding structural models. To further these aims, we present the implementation and characterization of a new Bayesian secondary structure estimation method in SESCA, termed SESCA_bayes. SESCA_bayes samples possible secondary structures using a Monte Carlo scheme, driven by the likelihood of estimated scaling errors and non-secondary-structure contributions of the measured spectrum. SESCA_bayes provides an estimated secondary structure composition and separate uncertainties on the fraction of residues in each secondary structure class. It also assists efficient model validation by providing a posterior secondary structure probability distribution based on the measured spectrum. Our presented study indicates that SESCA_bayes estimates the secondary structure composition with a significantly smaller uncertainty than its predecessor, SESCA_deconv, which is based on spectrum deconvolution. Further, the mean accuracy of the two methods in our analysis is comparable, but SESCA_bayes provides more accurate estimates for circular dichroism spectra that contain considerable non-SS contributions.
Program Title: SESCA_bayes
CPC Library link to program files:https://doi.org/10.17632/5nnsbn6ync.1
Developer's repository link:https://www.mpibpc.mpg.de/sesca
Licensing provisions: GPLv3
Programming language: Python
Nature of problem: The circular dichroism spectrum of a protein is strongly correlated with its secondary structure composition. However, determining the secondary structure from a spectrum is hindered by non-secondary structure contributions and by scaling errors due the uncertainty of the protein concentration. If not taken properly into account, these experimental factors can cause considerable errors when conventional secondary-structure estimation methods are used. Because these errors combine with errors of the proposed structural model in a non-additive fashion, it is difficult to assess how much uncertainty the experimental factors introduce to model validation approaches based on circular dichroism spectra.
Solution method: For a given measured circular dichroism spectrum, the SESCA_bayes algorithm applies Bayesian statistics to account for scaling errors and non-secondary structure contributions and to determine the conditional secondary structure probability distribution. This approach relies on fast spectrum predictions based on empirical basis spectrum sets and joint probability distribution maps for scaling factors and non-secondary structure distributions. Because SESCA_bayes estimates the most probable secondary structure composition based on a probability-weighted sample distribution, it avoids the typical fitting errors that occur during conventional spectrum deconvolution methods. It also estimates the uncertainty of circular dichroism based model validation more accurately than previous methods of the SESCA analysis package.
•PAP(85-120) peptide forms amyloid fibrils known as SEVI.•Spatial structure of the amyloidogenic PAP(85-120) peptide in aqua solution.•Random coil conformation prevails in the secondary structure of ...PAP(85-120) peptide.•Proposal, the N- and C-terminuses of PAP(85-120) participate in the fibril formation.
Prostatic acid phosphatase (PAP) is an enzyme facilitating infection of cells by HIV. Its peptide fragment PAP(85-120) forms amyloid fibrils known as SEVI, which enhance attachment of the virus by viral adhesion to the host cell prior to receptor-specific binding via reducing the electrostatic repulsion between the membranes of the virus and the target cell. In this work, the PAP(85-120) peptide spatial structure was solved using nuclear magnetic resonance (NMR) spectroscopy and circular dichroism (CD) spectroscopy. The data obtained with the help of NMR (internuclear distances, dihedral angles) allow to calculate the spatial structure of PAP(85-120) using the classical simulated annealing protocol built in XPLOR-NIH. CD spectrum analysis was used to provide the information on the secondary structure of PAP(85-120) that was agreed with the calculated spatial structure. Both methods have shown that the PAP(85-120) characterized by the high flexibility with prevailing random coil structure. The PAP(85-120) peptide structure was compared with the corresponding fragment (85-120 amino acid residues) of the full-length PAP protein (PDB ID 1CVI). The compared peptide structures have turns in the same regions of amino acid residues Q101 and V109, whereas the helix number of the N-terminus and C-terminus is different.
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Complement component 1q (C1q) is a protein complex of the innate immune system with well-characterized binding partners that constitutes part of the classical complement pathway (CP). In addition, ...C1q was recently described in the central nervous system as having a role in synapse elimination both in the healthy brain and in neurodegenerative diseases. However, the molecular mechanism of C1q-associated synapse phagocytosis is still unclear. Here, we designed monomer and multimer protein constructs which comprised the globular interaction recognition parts of mouse C1q (gC1q) as single-chain molecules (sc-gC1q proteins) lacking the collagen-like effector region. These molecules, which can competitively inhibit the function of C1q, were expressed in an E. coli expression system, and their structure and capabilities to bind known CP activators were validated by mass spectrometry, analytical size exclusion chromatography, analytical ultracentrifugation, circular dichroism spectroscopy, and ELISA. We further characterized the interactions between these molecules and immunoglobulins and neuronal pentraxins using surface plasmon resonance spectroscopy. We demonstrated that sc-gC1qs potently inhibited the function of C1q. Furthermore, these sc-gC1qs competed with C1q in binding to the embryonal neuronal cell membrane. We conclude that the application of sc-gC1qs can reveal neuronal localization and functions of C1q in assays in vivo and might serve as a basis for engineering inhibitors for therapeutic purposes.
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•Synthesis of chiral cleft-like methanocyclooctadiindoles was accomplished.•The CD couplet originates from exciton coupling of the two indole chromophores.•The absolute configuration ...proved by the TDDFT calculations of CD spectra.
Chiral methanocyclooctadiindoles, featuring an indole-annulated bicyclo3.3.1nonane framework, were obtained from the corresponding bicyclo3.3.1nonane 2,6-, 2,7- and 3,7-diketones via the Fischer indolization reaction. The chiroptical properties of the cleft-like conformationally rigid derivatives, exhibiting a well-defined spatial arrangement of the two indole chromophores, were studied by means of electronic circular dichroism (CD) spectroscopy and time-dependent density functional theory (TDDFT) calculations. The absolute configuration of 7,14-methanocyclooctadiindole derived from an achiral bicyclic 3,7-diketone was established using the exciton chirality method and corroborated by TDDFT calculations.
Fundamental characterization of wheat gluten Schopf, Marina; Wehrli, Monika Christine; Becker, Thomas ...
European food research & technology,
04/2021, Volume:
247, Issue:
4
Journal Article
Peer reviewed
Open access
Vital wheat gluten plays an important role in the food industry, especially in baking to help standardize dough properties and improve bread volume. However, a fundamental characterization of a wide ...variety of vital gluten samples is not available so far. This would be necessary to relate compositional characteristics to the production process. Therefore, we analyzed the content of crude protein, starch, lipids and ash, oil and water absorption capacity, particle size distribution, gluten protein composition and spectroscopic properties of 39 vital gluten samples from 6 different suppliers. Principle component analysis of all analytical parameters revealed that the samples from one specialized vital gluten manufacturer had a different composition and a greater variability compared to all other samples from wheat starch producers. While the composition of vital gluten samples from the same manufacturer was similar and the score plot showed a cluster formation for samples from three suppliers, the variability over all samples was comparatively low. The samples from the other suppliers were too similar altogether so that it was hardly possible to identify clear differences, also related to functionality.
In an attempt to gather experimental evidence for the influence of carbon allotropes on supramolecular chirality, we found that carbon nanotubes (CNTs) facilitate amplification of the molecular ...chirality of a π‐gelator (MC‐OPV) to supramolecular helicity at a concentration much lower than that required for intermolecular interaction. For example, at a concentration 1.8×10−4 m, MC‐OPV did not exhibit a CD signal; however, the addition of 0–0.6 mg of SWNTs resulted in amplified chirality as evident from the CD spectrum. Surprisingly, AFM analysis revealed the formation of thick helical fibers with a width of more than 100 nm. High‐resolution TEM analysis and solid‐state UV/Vis/NIR spectroscopy revealed that the thick helical fibers were cylindrical cables composed of individually wrapped and coaxially aligned SWNTs. Such an impressive effect of CNTs on supramolecular chirality and cylindrical‐cable formation has not been reported previously.
All wrapped up: The interaction of a chiral π‐gelator with carbon nanotubes (CNTs) led to 20‐fold amplification of the molecular chirality well below the critical aggregation concentration. Helical wrapping of the gelator molecules around the nanotubes led to thick helical fibers composed of individually wrapped and coaxially aligned CNTs (see picture).
The genomic screening of hyper-thermophilic Pyrococcus abyssi showed uncharacterized novel α-amylase sequences. Homology modelling analysis revealed that the α-amylase from P. abyssi consists of an ...N-terminal GH57 catalytic domain, α-amylase central, and C-terminal domain. Current studies emphasize in-silico structural and functional analysis, recombinant expression, characterization, structural studies through CD spectroscopy, and ligand binding studies of the novel α-amylase from P. abyssi. The soluble expression of PaAFG was observed in the E. coli Rosetta™ (DE3) pLysS strain upon incubation overnight at 18 °C in an orbital shaker. The optimum temperature and pH of the PaAFG were observed at 90 °C in 50 mM phosphate buffer pH 6. The Km value for PaAFG against wheat starch was determined as 0.20 ± 0.053 mg while the corresponding Vmax value was 25.00 ± 0.67 μmol min−1 mg−1 in the presence of 2 mM CaCl2 and 12.5 % glycerol. The temperature ramping experiments through CD spectroscopy reveal no significant change in the secondary structures and positive and negative ellipticities of the CD spectra showing the proper folding and optimal temperature of PaAFG protein. The RMSD and RMSF of the PaAFG enzyme determined through molecular dynamic simulation show the significant protein's stability and mobility. The soluble production, thermostability and broad substrate specificity make this enzyme a promising choice for various industrial applications.
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•The structure and function features of α-amylase in Pyrococcus abyssi have been comprehensively analysed.•The recombinant protein production, characterization, ligand binding studies, and structural analysis of the cloned alpha-amylase from P. abyssi are thoroughly explored in this comparative study.•The temperature ramping and secondary structure analysis of PaAFG through CD spectrometry showed no notable alterations in the secondary structures but verified the correct folding of PaAFG amylase.•HPLC analysis of readership interest defies my comprehension.•The thermostability of this enzyme at 90 °C makes it a potential candidate for different industrial processes.•The RMSD and RMSF of the PaAFG enzyme determined through molecular dynamic simulation show the significant protein's stability and mobility.•The soluble production, thermostability and broad substrate specificity make this enzyme a promising choice for various industrial applications.
Addition of macromolecular crowders to biomolecules can mimic the interior of living cells, however, it is not always very evident whether the effect is general or dependent on the ...biomolecule-crowder pair specificity. In this study, we investigate the effect of nonpolar hydrophobic amino acids (Glycine, L-alanine, L-valine, L-leucine, L-isoleucine) as crowding agents on the structural and thermal stability of three proteins: human serum albumin (HSA), lysozyme (HEWL) and Ribonuclease-A (RNase-A) of varying α-helical content using temperature dependent circular dichroism (CD) spectroscopic technique. It has been observed that all these five amino acids do not substantially alter the secondary structure of proteins rather they stabilize their respective native forms. Thermal unfolding of each protein is found to be irreversible in nature with the extent of secondary structural loss during refolding being proportional to the α-helicity of the protein. Estimated thermodynamic parameters (van't Hoff enthalpy and heat capacity) show that enthalpic stabilization is protein specific and the added amino acids alter these parameters in different extent without following any specific trend. Our study affirms that conformational stability of proteins and the associated thermodynamic parameters do not necessarily bear a linear correlation with the hydrophobicity of amino acids; rather it is protein specific where the secondary structural content plays an important role.
•Nucleopeptides show interesting biomolecular interaction properties in aqueous media.•A dithymine tetrapeptide of synthetic origin was investigated by CD spectroscopy in complementary DNA ...binding.•Molecular Docking and Molecular Dynamics were applied to the interpretation of the complex formation at a molecular level.•DNA binding in aqueous solution involves part of nucleopeptide bases leaving open further nucleobase-driven interactions.
Nucleopeptides are a class of molecules with numerous applications in the field of therapy, diagnostics and biomaterials development. Despite their nucleobase-decorated nature, their binding to natural nucleic acid targets does not necessarily involve all nucleopeptide bases, as we showed in this study. Here, we present a CD study on the interaction of a dithymine-functionalized tetra-L-serine with a homoadenine DNA (dA12) reporting an interpretation of the experimental data in light of our computational studies based on molecular docking and molecular dynamics (MD), as well as computer-assisted CD interpretation and simulation of the predicted complex structure. The stoichiometry of the complex, emerged by CD titration, accounted for a 1:2 T:A ratio. Hence, we supposed that binding did not involve a full pairing of the complementary bases but a partial thymines engagement. This hypothesis was sustained by the docking and MD simulations performed on the selected ligand and the complementary target of DNA and RNA, used for comparison. The nucleopeptide bound the DNA through a single A-T recognition involving complementary base-pairing, as well as by some interactions between its backbone (and in particular L-serine OH) and the nucleic acid. Overall, this confirmed that nucleopeptides can interact with nucleic acids leaving some of their nucleobases free for establishing further interactions with other biomolecules or for crosslinking in supramolecular structures in aqueous solution. Nevertheless, even though no typical DNA secondary structure is formed after nucleopeptide-binding, this ligand is able to induce a higher degree of structuration in the random deoxyoligonucleotide target as evidenced by CD, MD and CD simulation.
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