Circular Dichroism Spectra Of Proteins Research Articles

Reference Data Set for Circular Dichroism Spectroscopy Comprised of Validated Intrinsically Disordered Protein Models.

Circular dichroism (CD) spectroscopy is an analytical technique that measures the wavelength-dependent differential absorbance of circularly polarized light and is applicable to most biologically important macromolecules, such as proteins, nucleic acids, and carbohydrates. It serves to characterize the secondary structure composition of proteins, including intrinsically disordered proteins, by analyzing their recorded spectra. Several computational tools have been developed to interpret protein CD spectra. These methods have been calibrated and tested mostly on globular proteins with well-defined structures, mainly due to the lack of reliable reference structures for disordered proteins. It is therefore still largely unclear how accurately these computational methods can determine the secondary structure composition of disordered proteins. Here, we provide such a required reference data set consisting of model structural ensembles and matching CD spectra for eight intrinsically disordered proteins. Using this set of data, we have assessed the accuracy of several published CD prediction and secondary structure estimation tools, including our own CD analysis package, SESCA. Our results show that for most of the tested methods, their accuracy for disordered proteins is generally lower than for globular proteins. In contrast, SESCA, which was developed using globular reference proteins, but was designed to be applicable to disordered proteins as well, performs similarly well for both classes of proteins. The new reference data set for disordered proteins should allow for further improvement of all published methods.

Dataset for Spectroscopic, Structural and Dynamic Analysis of Human Fe(II)/2OG-Dependent Dioxygenase ALKBH3

Fe(II)/2OG-dependent dioxygenases of the AlkB family catalyze a direct removal of alkylated damages in the course of DNA and RNA repair. A human homolog of the E. coli AlkB ALKBH3 protein is able to hydroxylate N1-methyladenine, N3-methylcytosine, and N1-methylguanine in single-stranded DNA and RNA. Due to its contribution to an antitumor drug resistance, this enzyme is considered a promising therapeutic target. The elucidation of ALKBH3’s structural peculiarities is important to establish a detailed mechanism of damaged DNA recognition and processing, as well as to the development of specific inhibitors. This work presents new data on the wild type ALKBH3 protein and its four mutant forms (Y143F, Y143A, L177A, and H191A) obtained by circular dichroism (CD) spectroscopy. The dataset includes the CD spectra of proteins measured at different temperatures and a 3D visualization of the ALKBH3–DNA complex where the mutated amino acid residues are marked. These results show how substitution of the key amino acids influences a secondary structure content of the protein.

BeStSel: webserver for secondary structure and fold prediction for protein CD spectroscopy.

Circular dichroism (CD) spectroscopy is widely used to characterize the secondary structure composition of proteins. To derive accurate and detailed structural information from the CD spectra, we have developed the Beta Structure Selection (BeStSel) method (PNAS, 112, E3095), which can handle the spectral diversity of β-structured proteins. The BeStSel webserver provides this method with useful accessories to the community with the main goal to analyze single or multiple protein CD spectra. Uniquely, BeStSel provides information on eight secondary structure components including parallel β-structure and antiparallel β-sheets with three different groups of twist. It overperforms any available method in accuracy and information content, moreover, it is capable of predicting the protein fold down to the topology/homology level of the CATH classification. A new module of the webserver helps to distinguish intrinsically disordered proteins by their CD spectrum. Secondary structure calculation for uploaded PDB files will help the experimental verification of protein MD and in silico modelling using CD spectroscopy. The server also calculates extinction coefficients from the primary sequence for CD users to determine the accurate protein concentrations which is a prerequisite for reliable secondary structure determination. The BeStSel server can be freely accessed at https://bestsel.elte.hu.

The PCDDB (Protein Circular Dichroism Data Bank): A Bioinformatics Resource for Protein Characterisations and Methods Development

The Protein Circular Dichroism Data Bank (PCDDB) [https://pcddb.cryst.bbk.ac.uk] is an established resource for the biological, biophysical, chemical, bioinformatics, and molecular biology communities. It is a freely-accessible repository of validated protein circular dichroism (CD) spectra and associated sample and metadata, with entries having links to other bioinformatics resources including, amongst others, structure (PDB), AlphaFold, and sequence (UniProt) databases, as well as to published papers which produced the data and cite the database entries. It includes primary (unprocessed) and final (processed) spectral data, which are available in both text and pictorial formats, as well as detailed sample and validation information produced for each of the entries. Recently the metadata content associated with each of the entries, as well as the number and structural breadth of the protein components included, have been expanded. The PCDDB includes data on both wild-type and mutant proteins, and because CD studies primarily examine proteins in solution, it also contains examples of the effects of different environments on their structures, plus thermal unfolding/folding series. Methods for both sequence and spectral comparisons are included.The data included in the PCDDB complement results from crystal, cryo-electron microscopy, NMR spectroscopy, bioinformatics characterisations and classifications, and other structural information available for the proteins via links to other databases. The entries in the PCDDB have been used for the development of new analytical methodologies, for interpreting spectral and other biophysical data, and for providing insight into structures and functions of individual soluble and membrane proteins and protein complexes.

Knowledge-Based Atomic Polarizabilities Used to Model Circular Dichroism Spectra of Proteins.

We present a model of circular dichroism for proteins, which is mainly based on both the classical theory of optical activity and a series of effective atomic polarizabilities. Such polarizabilities are extracted from the analysis of a set of synchrotron radiation circular dichroism spectra and their corresponding three-dimensional structures from the Protein Data Bank. Each modeled spectrum is obtained from the protein atomic coordinates and the identification of its secondary structure elements. The resulting spectra are in good agreement with additional experimental data and also with the predictions of some other models. Among them, only our approach is able to describe the effect of d-amino acids. Moreover, our model is also utilized to evaluate protein reconstructions as well as structural changes.

Accurate Machine Learning Prediction of Protein Circular Dichroism Spectra with Embedded Density Descriptors.

A data-driven approach to simulate circular dichroism (CD) spectra is appealing for fast protein secondary structure determination, yet the challenge of predicting electric and magnetic transition dipole moments poses a substantial barrier for the goal. To address this problem, we designed a new machine learning (ML) protocol in which ordinary pure geometry-based descriptors are replaced with alternative embedded density descriptors and electric and magnetic transition dipole moments are successfully predicted with an accuracy comparable to first-principle calculation. The ML model is able to not only simulate protein CD spectra nearly 4 orders of magnitude faster than conventional first-principle simulation but also obtain CD spectra in good agreement with experiments. Finally, we predicted a series of CD spectra of the Trp-cage protein associated with continuous changes of protein configuration along its folding path, showing the potential of our ML model for supporting real-time CD spectroscopy study of protein dynamics.

Implementation of a Bayesian secondary structure estimation method for the SESCA circular dichroism analysis package

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 summaryProgram Title: SESCA_bayesCPC Library link to program files:https://doi.org/10.17632/5nnsbn6ync.1Developer's repository link:https://www.mpibpc.mpg.de/sescaLicensing provisions: GPLv3Programming language: PythonNature 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.

Bayesian inference assessment of protein secondary structure analysis using circular dichroism data - how much structural information is contained in protein circular dichroism spectra?

Circular dichroism spectroscopy is an important tool for determining the structural characteristics of biomolecules, particularly the secondary structure of proteins. In this paper we propose a Bayesian model that estimates the covariance structure within a measured spectrum and quantifies the uncertainty associated with the inferred secondary structures and characteristic spectra associated with each secondary structure type. Furthermore, we used tools from Bayesian model selection to determine the best secondary structure classification scheme and illustrate a technique for comparing whether or not two or more measured protein spectra share the same secondary structure. Our findings suggest that it is not possible to identify more than 3 distinct secondary structure classes from CD spectra above 175 nm. The inclusion of data from wavelengths between 175 and 200 nm did not substantially affect the ability to determine secondary structure fractions.

BeStSel: From Secondary Structure Analysis to Protein Fold Prediction by Circular Dichroism Spectroscopy.

Far-UV circular dichroism (CD) spectroscopy is a classical method for the study of the secondary structure of polypeptides in solution. It has been the general view that the α-helix content can be estimated accurately from the CD spectra. However, the technique was less reliable to estimate the β-sheet contents as a consequence of the structural variety of the β-sheets, which is reflected in a large spectral diversity of the CD spectra of proteins containing this secondary structure component. By taking into account the parallel or antiparallel orientation and the twist of the β-sheets, the Beta Structure Selection (BeStSel) method provides an improved β-structure determination and its performance is more accurate for any of the secondary structure types compared to previous CD spectrum analysis algorithms. Moreover, BeStSel provides extra information on the orientation and twist of the β-sheets which is sufficient for the prediction of the protein fold.The advantage of CD spectroscopy is that it is a fast and inexpensive technique with easy data processing which can be used in a wide protein concentration range and under various buffer conditions. It is especially useful when the atomic resolution structure is not available, such as the case of protein aggregates, membrane proteins or natively disordered chains, for studying conformational transitions, testing the effect of the environmental conditions on the protein structure, for verifying the correct fold of recombinant proteins in every scientific fields working on proteins from basic protein science to biotechnology and pharmaceutical industry. Here, we provide a brief step-by-step guide to record the CD spectra of proteins and their analysis with the BeStSel method.

PDBMD2CD: providing predicted protein circular dichroism spectra from multiple molecular dynamics-generated protein structures.

PDBMD2CD is a new web server capable of predicting circular dichroism (CD) spectra for multiple protein structures derived from molecular dynamics (MD) simulations, enabling predictions from thousands of protein atomic coordinate files (e.g. MD trajectories) and generating spectra for each of these structures provided by the user. Using MD enables exploration of systems that cannot be monitored by direct experimentation. Validation of MD-derived data from these types of trajectories can be difficult via conventional structure-determining techniques such as crystallography or nuclear magnetic resonance spectroscopy. CD is an experimental technique that can provide protein structure information from such conditions. The website utilizes a much faster (minimum ∼1000×) and more accurate approach for calculating CD spectra than its predecessor, PDB2CD (1). As well as improving on the speed and accuracy of current methods, new analysis tools are provided to cluster predictions or compare them against experimental CD spectra. By identifying a subset of the closest predicted CD spectra derived from PDBMD2CD to an experimental spectrum, the associated cluster of structures could be representative of those found under the conditions in which the MD studies were undertaken, thereby offering an analytical insight into the results. PDBMD2CD is freely available at: https://pdbmd2cd.cryst.bbk.ac.uk.

Tools and Resources for Circular Dichroism Spectroscopy

Circular Dichroism (CD) Spectroscopy is a popular method in biophysics for the characterisation of the structure, dynamics, and stability of proteins. Our consortium has developed a number of widely-used tools for analyses and access to CD-related resources. These include the Protein Circular Dichroism Data Bank [PCDDB] (http://pcddb.cryst.bbk.ac.uk/home.php), a freely-accessible deposition and retrieval site for protein CD spectra and associated meta-data, which includes the “gold-standard” SP175 and SMP180 reference data sets (currently used for both spectral analyses and software development), a data validation site (ValiDichro), software for spectral data processing and component analyses (CDtoolX), and websites for secondary structure analyses (DichroWeb), spectral comparisons (DichroMatch and DichroCompare), and for predicting spectra from 3-dimensional structures (PDB2CD), as well as for undertaking structure comparisons (2StrucCompare). Accompanying these tools are YouTube educational videos (located at the “PCDDB Channel”) on topics such as good practice in instrument and spectral calibration, sample preparations, types of analyses, such as singular value deconvolutions and Data Bank depositions and access. New tools and resources continue to be developed for use in both research and teaching. Supported by grants from U.K. BBSRC (to BAW and RWJ).

Selection and Analysis of Protein Circular Dichroism Spectra Using an Expansion of Spectral Factors

Techniques are presented to develop spectroscopic factors directly from circular dichroism spectra of proteins using singular value decomposition on a small database. Four spectra of maximum spectral variability are chosen to characterize the database. These selected protein spectra are then factored by singular values into component spectra, which are collected as comparative vector characteristics used as factor fractions. The necessary standardization for comparison is achieved using unit normalized spectra. Those spectra are used to quantify the parameter uncertainties as a means for comparison. The difference between the fit spectrum and the data spectrum for each protein is analyzed by least square to obtain parameter uncertainties due to the model. The sum of the factor fractions over the database is within the theoretical predictions.

SESCA: Predicting Circular Dichroism Spectra from Protein Molecular Structures.

Circular dichroism (CD) spectroscopy is a highly sensitive but low-resolution technique to study the structure of proteins. Combined with molecular modeling or other complementary techniques, CD spectroscopy can provide essential information at higher resolution. To this end, we introduce a new computational method to calculate the electronic circular dichroism spectra of proteins from a structural model or ensemble using the average secondary structure composition and a precalculated set of basis spectra. The method is designed for model validation to estimate the error of a given protein structural model based on the measured CD spectrum. We compared the predictive power of our method to that of existing algorithms, namely, DichroCalc and PDB2CD, and found that it predicts CD spectra more accurately. Our results indicate that the derived basis sets are robust to both experimental errors in the reference spectra and the choice of the secondary structure classification algorithm. For over 80% of the globular reference proteins, our basis sets accurately predict the experimental spectrum solely from their secondary structure composition. For the remaining 20%, correcting for intensity normalization considerably improves the prediction power. Additionally, we show that the predictions for short peptides and an example complex of intrinsically disordered proteins strongly benefit from accounting for side-chain contributions and structural flexibility.

A New Method to Predict the Electronic Circular Dichroism Spectra of Proteins

Electronic circular dichroism (ECD) is a widely applied tool providing information on the average structure of biomolecules, particularly of proteins. The connection between the ECD spectrum in the far ultraviolet range (175-260 nm) and the secondary structure characteristics of proteins has long been established, and is used to monitor structural re-arrangements during biochemical processes such as (un)folding or binding events. The two main methods to interpret ECD spectra are estimation of the average secondary structure ratios by spectrum deconvolution, or the prediction of the spectrum from a proposed structural model and comparison to the experimental spectrum. However, the ambiguity of spectrum deconvolution, and the computational power required for an accurate spectrum prediction with quantum mechanics limited the quantitative interpretation of protein ECD spectra so far.Here we present a computational method that allows the rapid and accurate prediction of the ECD spectra of proteins using their three-dimensional structure and a set of pre-calculated spectrum components (basis set). We derived several basis sets using the crystal structure and CD spectra of 64 ordered proteins. The basis sets are compatible with established secondary structure classification programs such as DSSP and DISICL. We evaluated the accuracy of the derived basis sets and some of the previously available deconvolution and prediction methods. The results indicate that our method has a comparable accuracy to spectrum deconvolution techniques, while eliminating the ambiguity, and provides a significant (+50%) improvement in accuracy over the available CD spectrum prediction software DichroCalc.

PrxQ B from Mycobacterium tuberculosis is a monomeric, thioredoxin-dependent and highly efficient fatty acid hydroperoxide reductase

Mycobacterium tuberculosis (M. tuberculosis) is the intracellular bacterium responsible for tuberculosis disease (TD). Inside the phagosomes of activated macrophages, M. tuberculosis is exposed to cytotoxic hydroperoxides such as hydrogen peroxide, fatty acid hydroperoxides and peroxynitrite. Thus, the characterization of the bacterial antioxidant systems could facilitate novel drug developments. In this work, we characterized the product of the gene Rv1608c from M. tuberculosis, which according to sequence homology had been annotated as a putative peroxiredoxin of the peroxiredoxin Q subfamily (PrxQ B from M. tuberculosis or MtPrxQ B). The protein has been reported to be essential for M. tuberculosis growth in cholesterol-rich medium. We demonstrated the M. tuberculosis thioredoxin B/C-dependent peroxidase activity of MtPrxQ B, which acted as a two-cysteine peroxiredoxin that could function, although less efficiently, using a one-cysteine mechanism. Through steady-state and competition kinetic analysis, we proved that the net forward rate constant of MtPrxQ B reaction was 3 orders of magnitude faster for fatty acid hydroperoxides than for hydrogen peroxide (3×106vs 6×103M−1s−1, respectively), while the rate constant of peroxynitrite reduction was (0.6−1.4) ×106M−1s−1 at pH 7.4. The enzyme lacked activity towards cholesterol hydroperoxides solubilized in sodium deoxycholate. Both thioredoxin B and C rapidly reduced the oxidized form of MtPrxQ B, with rates constants of 0.5×106 and 1×106M−1s−1, respectively. Our data indicated that MtPrxQ B is monomeric in solution both under reduced and oxidized states. In spite of the similar hydrodynamic behavior the reduced and oxidized forms of the protein showed important structural differences that were reflected in the protein circular dichroism spectra.

Vacuum-Ultraviolet Circular Dichroism Spectra of Escherichia coli Dihydrofolate Reductase and Its Mutants: Contributions of Phenylalanine and Tyrosine Side Chains and Exciton Coupling of Two Tryptophan Side Chains.

Vacuum-ultraviolet (VUV) circular dichroism (CD) spectroscopy has recently been used for secondary structure analysis of proteins; however, the contribution of aromatic side chains to protein VUV CD spectra is unresolved. In this report, VUV CD spectra of 10 Escherichia coli dihydrofolate reductase (DHFR) mutants, in which each phenylalanine or tyrosine residue was mutated to leucine, were measured down to 175 nm at 25 °C and pH 8.0 to elucidate the contributions of these aromatic side chains to the high-energy transitions of peptide bonds. The VUV CD spectra of these mutants were different from the spectrum of the wild-type protein, indicating that the contribution of the phenylalanine and tyrosine side chains of DHFR extends to the VUV region. Furthermore, the VUV CD spectrum and the folate- or NADP(+)-induced spectral change of F103L mutant DHFR indicated a modification and regeneration of exciton coupling between the Trp47 and Trp74 side chains, respectively, suggesting that exciton coupling may also contribute to the CD spectrum of DHFR in the VUV region. These results should be useful for theoretically characterizing the contribution of aromatic side chains to protein CD spectra, leading to the improvement of protein secondary-structure analysis by VUV CD spectroscopy.

Vibronic structure in the far-UV electronic circular dichroism spectra of proteins.

The Franck-Condon effect is considered and the vibrational structure of the πnbπ* transition of the peptide backbone is incorporated into matrix method calculations of the electronic circular dichroism (CD) spectra of proteins in the far-ultraviolet. We employ the state-averaged CASPT2 method to calculate the ground and πnbπ* excited state geometries and frequencies of N-methylacetamide (NMA), which represents the peptide chromophore. The results of these calculations are used to incorporate vibronic levels of the excited states into the matrix method calculation. The CD spectra of a set of 49 proteins, comprising a range of structural types, are calculated to assess the influence of the vibrational structure. The calculated spectra of α-helical proteins are better resolved using the vibronic parameters and correlation between the experimental and the calculated intensity of less regular β structure proteins improves over most wavelengths in the far-UV. No obvious improvement is observed in the calculated spectra of regular β-sheet proteins. Our high-level ab initio calculations of the vibronic structure of the πnbπ* transition in NMA have provided some further insight into the physical origins of the nature of protein CD spectra in the far-UV.

Uncertainty in measurement of protein circular dichroism spectra

Circular dichroism (CD) spectroscopy of proteins is widely used to measure protein secondary structure, and to detect changes in secondary and higher orders of structure, for applications in research and in the quality control of protein products such as biopharmaceuticals. However, objective comparison of spectra is challenging because of a limited quantitative understanding of the sources of error in the measurement. Statistical methods can be used for comparisons, but do not provide a mechanism for dealing with systematic, as well as random, errors. Here we present a measurement model for CD spectroscopy of proteins, incorporating the principal sources of uncertainty, and use the model in conjunction with experimental data to derive an uncertainty budget. We show how this approach could be used in practice for the objective comparison of spectra, and discuss the benefits and limitations of this strategy.

Ubiquitin Interacts with the Tollip C2 and CUE Domains and Inhibits Binding of Tollip to Phosphoinositides

A large number of cellular signaling processes are directed through internalization, via endocytosis, of polyubiquitinated cargo proteins. Tollip is an adaptor protein that facilitates endosomal cargo sorting for lysosomal degradation. Tollip preferentially binds phosphatidylinositol 3-phosphate (PtdIns(3)P) via its C2 domain, an association that may be required for endosomal membrane targeting. Here, we show that Tollip binds ubiquitin through its C2 and CUE domains and that its association with the C2 domain inhibits PtdIns(3)P binding. NMR analysis demonstrates that the C2 and CUE domains bind to overlapping sites on ubiquitin, suggesting that two ubiquitin molecules associate with Tollip simultaneously. Hydrodynamic studies reveal that ubiquitin forms heterodimers with the CUE domain, indicating that the association disrupts the dimeric state of the CUE domain. We propose that, in the absence of polyubiquitinated cargo, the dual binding of ubiquitin partitions Tollip into membrane-bound and membrane-free states, a function that contributes to the engagement of Tollip in both membrane trafficking and cytosolic pathways.

ValiDichro: a website for validating and quality control of protein circular dichroism spectra.

Circular dichroism (CD) spectroscopy is widely used in structural biology as a technique for examining the structure, folding and conformational changes of proteins. A new server, ValiDichro, has been developed for checking the quality and validity of CD spectral data and metadata, both as an aid to data collection and processing and as a validation procedure for spectra to be included in publications. ValiDichro currently includes 25 tests for data completeness, consistency and quality. For each test that is done, not only is a validation report produced, but the user is also provided with suggestions for correcting or improving the data. The ValiDichro server is freely available at http://valispec.cryst.bbk.ac.uk/circularDichroism/ValiDichro/upload.html.