Using circular dichroism spectra to estimate protein secondary structure
TL;DR: This protocol details the basic steps of obtaining and interpreting CD data, and methods for analyzing spectra to estimate the secondary structural composition of proteins.
Abstract: Circular dichroism (CD) is an excellent tool for rapid determination of the secondary structure and folding properties of proteins that have been obtained using recombinant techniques or purified from tissues. The most widely used applications of protein CD are to determine whether an expressed, purified protein is folded, or if a mutation affects its conformation or stability. In addition, it can be used to study protein interactions. This protocol details the basic steps of obtaining and interpreting CD data, and methods for analyzing spectra to estimate the secondary structural composition of proteins. CD has the advantage that measurements may be made on multiple samples containing < or =20 microg of proteins in physiological buffers in a few hours. However, it does not give the residue-specific information that can be obtained by x-ray crystallography or NMR.
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TL;DR: Differential scanning fluorimetry (DSF) is a rapid and inexpensive screening method to identify low-molecular-weight ligands that bind and stabilize purified proteins.
Abstract: Differential scanning fluorimetry (DSF) is a rapid and inexpensive screening method to identify low-molecular-weight ligands that bind and stabilize purified proteins. The temperature at which a protein unfolds is measured by an increase in the fluorescence of a dye with affinity for hydrophobic parts of the protein, which are exposed as the protein unfolds. A simple fitting procedure allows quick calculation of the transition midpoint; the difference in the temperature of this midpoint in the presence and absence of ligand is related to the binding affinity of the small molecule, which can be a low-molecular-weight compound, a peptide or a nucleic acid. DSF is best performed using a conventional real-time PCR instrument. Ligand solutions from a storage plate are added to a solution of protein and dye, distributed into the wells of the PCR plate and fluorescence intensity measured as the temperature is raised gradually. Results can be obtained in a single day.
2,194 citations
TL;DR: This work has developed a method called β-structure selection (BeStSel) for the secondary structure estimation that takes into account the twist of β-structures, and can predict the protein fold down to the topology level following the CATH classification from a single CD spectrum.
Abstract: Circular dichroism (CD) spectroscopy is a widely used technique for the study of protein structure. Numerous algorithms have been developed for the estimation of the secondary structure composition from the CD spectra. These methods often fail to provide acceptable results on α/β-mixed or β-structure–rich proteins. The problem arises from the spectral diversity of β-structures, which has hitherto been considered as an intrinsic limitation of the technique. The predictions are less reliable for proteins of unusual β-structures such as membrane proteins, protein aggregates, and amyloid fibrils. Here, we show that the parallel/antiparallel orientation and the twisting of the β-sheets account for the observed spectral diversity. We have developed a method called β-structure selection (BeStSel) for the secondary structure estimation that takes into account the twist of β-structures. This method can reliably distinguish parallel and antiparallel β-sheets and accurately estimates the secondary structure for a broad range of proteins. Moreover, the secondary structure components applied by the method are characteristic to the protein fold, and thus the fold can be predicted to the level of topology in the CATH classification from a single CD spectrum. By constructing a web server, we offer a general tool for a quick and reliable structure analysis using conventional CD or synchrotron radiation CD (SRCD) spectroscopy for the protein science research community. The method is especially useful when X-ray or NMR techniques fail. Using BeStSel on data collected by SRCD spectroscopy, we investigated the structure of amyloid fibrils of various disease-related proteins and peptides.
1,137 citations
Cites background from "Using circular dichroism spectra to..."
...In the past decades, a multitude of enhanced algorithms, based on variable selection, or singular value decomposition of standardized, scaled, and calibrated reference spectra, have been proposed to predict the secondary structure content, with good overall secondary structure prediction (5, 6)....
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TL;DR: The principles that underlie the determination of protein secondary structure by FTIR spectroscopy are detailed, as well as the basic steps involved in protein sample preparation, instrument operation,FTIR spectra collection and spectra analysis in order to estimate protein secondary-structural components in aqueous solution.
Abstract: Fourier transform IR (FTIR) spectroscopy is a nondestructive technique for structural characterization of proteins and polypeptides. The IR spectral data of polymers are usually interpreted in terms of the vibrations of a structural repeat. The repeat units in proteins give rise to nine characteristic IR absorption bands (amides A, B and I-VII). Amide I bands (1,700-1,600 cm(-1)) are the most prominent and sensitive vibrational bands of the protein backbone, and they relate to protein secondary structural components. In this protocol, we have detailed the principles that underlie the determination of protein secondary structure by FTIR spectroscopy, as well as the basic steps involved in protein sample preparation, instrument operation, FTIR spectra collection and spectra analysis in order to estimate protein secondary-structural components in aqueous (both H2O and deuterium oxide (D2O)) solution using algorithms, such as second-derivative, deconvolution and curve fitting. Small amounts of high-purity (>95%) proteins at high concentrations (>3 mg ml(-1)) are needed in this protocol; typically, the procedure can be completed in 1-2 d.
757 citations
TL;DR: Circular dichroism is an excellent spectroscopic technique for following the unfolding and folding of proteins as a function of temperature and may be used to determine the van't Hoff enthalpy and entropy of unfolding, the midpoint of the unfolding transition and the free energy of unfolding.
Abstract: Circular dichroism (CD) is an excellent spectroscopic technique for following the unfolding and folding of proteins as a function of temperature. One of its principal applications is to determine the effects of mutations and ligands on protein and polypeptide stability. If the change in CD as a function of temperature is reversible, analysis of the data may be used to determined the van't Hoff enthalpy and entropy of unfolding, the midpoint of the unfolding transition and the free energy of unfolding. Binding constants of protein-protein and protein-ligand interactions may also be estimated from the unfolding curves. Analysis of CD spectra obtained as a function of temperature is also useful to determine whether a protein has unfolding intermediates. Measurement of the spectra of five folded proteins and their unfolding curves at a single wavelength requires approximately 8 h.
743 citations
Cites methods from "Using circular dichroism spectra to..."
...An introduction to CD and a description of how to estimate the secondary structure of a protein from its CD spectrum are in the first protocol in this serie...
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TL;DR: This study used DichroCalc to calculate the theoretical CD spectra of a nonredundant set of structures representing most proteins in the PDB, and applied a straightforward approach for predicting protein secondary structure content using these theoreticalCD spectra as reference set.
Abstract: Circular dichroism (CD) is a spectroscopic technique commonly used to investigate the structure of proteins. Major secondary structure types, alpha-helices and beta-strands, produce distinctive CD spectra. Thus, by comparing the CD spectrum of a protein of interest to a reference set consisting of CD spectra of proteins of known structure, predictive methods can estimate the secondary structure of the protein. Currently available methods, including K2D2, use such experimental CD reference sets, which are very small in size when compared to the number of tertiary structures available in the Protein Data Bank (PDB). Conversely, given a PDB structure, it is possible to predict a theoretical CD spectrum from it. The methodological framework for this calculation was established long ago but only recently a convenient implementation called DichroCalc has been developed. In this study, we set to determine whether theoretically derived spectra could be used as reference set for accurate CD based predictions of secondary structure. We used DichroCalc to calculate the theoretical CD spectra of a nonredundant set of structures representing most proteins in the PDB, and applied a straightforward approach for predicting protein secondary structure content using these theoretical CD spectra as reference set. We show that this method improves the predictions, particularly for the wavelength interval between 200 and 240 nm and for beta-strand content. We have implemented this method, called K2D3, in a publicly accessible web server at http://www. ogic.ca/projects/k2d3.
658 citations
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TL;DR: Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.
289,852 citations
TL;DR: This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr with little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose.
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TL;DR: A procedure is presented which strongly reduces or elimi- nates these interactions, normalizes their absorption, and consequently permits a more precise analysis of tryptophan and tyrosine in proteins.
Abstract: A rapid method for the determination of tryptophan in proteins is presented. It is based on ab- sorbance measurements at 288 and 280 mp of the protein dissolved in 6 M guanidine hydrochloride. Blocked tryptophanyl (N-acetyl-L-tryptophanamide) and tyrosyl (glycyl-L-tyrosylglycine) compounds were selected as C urrent methods of protein amino acid analysis do not give quantitative values for tryptophan and conse- quently the amino acid compositions, which are other- wise complete, fail to report tryptophan values. The principal reason for this situation is that the standard procedure of protein hydrolysis in strong acid results in the destruction of tryptophan (Hill, 1965). Therefore a second procedure is required to measure tryptophan. Alkaline hydrolysis is less destructive but does not give quantitative recoveries generally (Spies and Chambers, 1949). Enzymatic hydrolysis of proteins can give quanti- tative yields of tryptophan but this method may not be generally valid (Hill and Schmidt, 1962). The hydrolytic problem can be circumvented by meas- uring tryptophan in the intact protein. A chemical method has been developed which has not been exploited adequately (Spies and Chambers, 1948, 1949). On the other hand, considerable effort has been expended in developing absorption spectroscopic procedures to measure tryptophan and tyrosine in unhydrolyzed pro- teins. Holiday (1936) and Goodwin and Morton (1946) have measured the absorption of proteins in 0.1 M NaOH and computed their tryptophan and tyrosine contents based on comparison with the absorption of the two amino acids. A modification of these techniques has been presented by Bencze and Schmid (1957). The pre- ceding three methods do not give quantitative results. The behavior of the chromophores has not been nor- malized and the two models, i.e., tryptophan and tyro- sine, are not completely adequate. A procedure is sug- gested in this report which strongly reduces or elimi- nates these interactions, normalizes their absorption, and consequently permits a more precise analysis of tryptophan and tyrosine in proteins.
3,323 citations