Browse
Submit Data
Databases
API
Help

Dataset Information

26 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Analysis of the kinetics of folding of proteins and peptides using circular dichroism.

ABSTRACT: Circular dichroism (CD) is a useful spectroscopic technique for studying the secondary structure, folding and binding properties of proteins. This protocol covers how to use the intrinsic circular dichroic properties of proteins to follow their folding and unfolding as a function of time. Included are methods of obtaining data and for analyzing the folding and unfolding data to determine the rate constants and the order of the folding and unfolding reactions. The protocol focuses on the use of CD to follow folding when it is relatively slow, on the order of minutes to days. The methods for analyzing the data, however, can also be applied to data collected with a CD machine equipped with stopped-flow accessories in the range of milliseconds to seconds and folding analyzed by other spectroscopic methods including changes in absorption or fluorescence spectra as a function of time.

SUBMITTER: Greenfield NJ

PROVIDER: S-EPMC2752309 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Analysis of the kinetics of folding of proteins and peptides using circular dichroism.

Greenfield Norma J NJ

Nature protocols 20060101 6

Circular dichroism (CD) is a useful spectroscopic technique for studying the secondary structure, folding and binding properties of proteins. This protocol covers how to use the intrinsic circular dichroic properties of proteins to follow their folding and unfolding as a function of time. Included are methods of obtaining data and for analyzing the folding and unfolding data to determine the rate constants and the order of the folding and unfolding reactions. The protocol focuses on the use of C ...[more]

PMID: 17406548

Similar Datasets

Determination of the folding of proteins as a function of denaturants, osmolytes or ligands using circular dichroism.

Project description:Circular dichroism (CD) is an excellent tool for examining the interactions and stability of proteins. This protocol covers methods to obtain and analyze circular dichroism spectra to measure changes in the folding of proteins as a function of denaturants, osmolytes or ligands. Applications include determination of the free energy of folding of a protein, the effects of mutations on protein stability and the estimation of binding constants for the interactions of proteins with other proteins, DNA or ligands, such as substrates or inhibitors. The experiments require 2-5 h.

| S-EPMC2728349 | biostudies-literature

Structural Analysis and Design of Chionodracine-Derived Peptides Using Circular Dichroism and Molecular Dynamics Simulations.

Project description:Antimicrobial peptides have been identified as one of the alternatives to the extensive use of common antibiotics as they show a broad spectrum of activity against human pathogens. Among these is Chionodracine (Cnd), a host-defense peptide isolated from the Antarctic icefish Chionodraco hamatus, which belongs to the family of Piscidins. Previously, we demonstrated that Cnd and its analogs display high antimicrobial activity against ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species). Herein, we investigate the interactions with lipid membranes of Cnd and two analogs, Cnd-m3 and Cnd-m3a, showing enhanced potency. Using a combination of Circular Dichroism, fluorescence spectroscopy, and all-atom Molecular Dynamics (MD) simulations, we determined the structural basis for the different activity among these peptides. We show that all peptides are predominantly unstructured in water and fold, preferentially as ?-helices, in the presence of lipid vesicles of various compositions. Through a series of MD simulations of 400 ns time scale, we show the effect of mutations on the structure and lipid interactions of Cnd and its analogs. By explaining the structural basis for the activity of these analogs, our findings provide structural templates to design minimalistic peptides for therapeutics.

| S-EPMC7073106 | biostudies-literature

Analyses of circular dichroism spectra of membrane proteins.

Project description:Circular dichroism (CD) spectroscopy is a valuable technique for the determination of protein secondary structures. Many linear and nonlinear algorithms have been developed for the empirical analysis of CD data, using reference databases derived from proteins of known structures. To date, the reference databases used by the various algorithms have all been derived from the spectra of soluble proteins. When applied to the analysis of soluble protein spectra, these methods generally produce calculated secondary structures that correspond well with crystallographic structures. In this study, however, it was shown that when applied to membrane protein spectra, the resulting calculations produce considerably poorer results. One source of this discrepancy may be the altered spectral peak positions (wavelength shifts) of membrane proteins due to the different dielectric of the membrane environment relative to that of water. These results have important consequences for studies that seek to use the existing soluble protein reference databases for the analyses of membrane proteins.

| S-EPMC2323856 | biostudies-literature

Secondary-structure analysis of denatured proteins by vacuum-ultraviolet circular dichroism spectroscopy.

Project description:To elucidate the structure of denatured proteins, we measured the vacuum-ultraviolet circular dichroism (VUVCD) spectra from 260 to 172 nm of three proteins (metmyoglobin, staphylococcal nuclease, and thioredoxin) in the native and the acid-, cold-, and heat-denatured states, using a synchrotron-radiation VUVCD spectrophotometer. The circular dichroism spectra of proteins fully unfolded by guanidine hydrochloride (GdnHCl) were also measured down to 197 nm for comparison. These denatured proteins exhibited characteristic VUVCD spectra that reflected a considerable amount of residual secondary structures. The contents of alpha-helices, beta-strands, turns, poly-L-proline type II (PPII), and unordered structures were estimated for each denatured state of the three proteins using the SELCON3 program with Protein Data Bank data and the VUVCD spectra of 31 reference proteins reported in our previous study. Based on these contents, the characteristics of the four types of denaturation were discussed for each protein. In all types of denaturation, a decrease in alpha-helices was accompanied by increases in beta-strands, PPII, and unordered structures. About 20% beta-strands were present even in the proteins fully unfolded by GdnHCl in which beta-sheets should be broken. From these results, we propose that denatured proteins constitute an ensemble of residual alpha-helices and beta-sheets, partly unfolded (or distorted) alpha-helices and beta-strands, PPII, and unordered structures.

| S-EPMC1868981 | biostudies-literature

DichroIDP: a method for analyses of intrinsically disordered proteins using circular dichroism spectroscopy.

Project description:Intrinsically disordered proteins (IDPs) are comprised of significant numbers of residues that form neither helix, sheet, nor any other canonical type of secondary structure. They play important roles in a broad range of biological processes, such as molecular recognition and signalling, largely due to their chameleon-like ability to change structure from unordered when free in solution to ordered when bound to partner molecules. Circular dichroism (CD) spectroscopy is a widely-used method for characterising protein secondary structures, but analyses of IDPs using CD spectroscopy have suffered because the methods and reference datasets used for the empirical determination of secondary structures do not contain adequate representations of unordered structures. This work describes the creation, validation and testing of a standalone Windows-based application, DichroIDP, and a new reference dataset, IDP175, which is suitable for analyses of proteins containing significant amounts of disordered structure. DichroIDP enables secondary structure determinations of IDPs and proteins containing intrinsically disordered regions.

| S-EPMC10409736 | biostudies-literature

Analysis of Vibrational Circular Dichroism Spectra of Peptides: A Generalized Coupled Oscillator Approach of a Small Peptide Model Using VCDtools.

Project description:Vibrational circular dichroism (VCD) is one of the major spectroscopic tools to study peptides. Nevertheless, a full understanding of what determines the signs and intensities of VCD bands of these compounds in the amide I and amide II spectral regions is still far from complete. In the present work, we study the origin of these VCD signals using the general coupled oscillator (GCO) analysis, a novel approach that has recently been developed. We apply this approach to the ForValNHMe model peptide in both α-helix and β-sheet configurations. We show that the intense VCD signals observed in the amide I and amide II spectral regions essentially have the same underlying mechanism, namely, the through-space coupling of electric dipoles. The crucial role played by intramolecular hydrogen bonds in determining VCD intensities is also illustrated. Moreover, we find that the contributions to the rotational strengths, considered to be insignificant in standard VCD models, may have sizable magnitudes and can thus not always be neglected. In addition, the VCD robustness of the amide I and II modes has been investigated by monitoring the variation of the rotational strength and its contributing terms during linear transit scans and by performing calculations with different computational parameters. From these studies-and in particular, the decomposition of the rotational strength made possible by the GCO analysis-it becomes clear that one should be cautious when employing measures of robustness as proposed previously.

| S-EPMC7061330 | biostudies-literature

Distinctive circular dichroism signature for 14-helix-bundle formation by beta-peptides.

Project description:We identify a distinctive circular dichroism (CD) signature for self-assembled 14-helical beta-peptides. Our data show that self-assembly leads to a mimimum at 205 nm, which is distinct from the well-known minimum at 214 nm for a monomeric 14-helix. The onset of assembly is indicated by [theta]205/[theta]214>0.7. Our results will facilitate rapid screening for self-assembling beta-peptides and raise the possibility that far-UV CD will be useful for detecting higher-order structure for other well-folded oligoamide backbones.

| S-EPMC2886586 | biostudies-literature

Probing tertiary structure of proteins using single Trp mutations with circular dichroism at low temperature.

Project description:Trp is the most spectroscopically informative aromatic amino acid of proteins. However, the near-UV circular dichroism (CD) spectrum of Trp is complicated because the intensity and sign of (1)La and (1)Lb bands vary independently. To resolve vibronic structure and gain site-specific information from complex spectra, deconvolution was combined with cooling and site-directed tryptophan substitution. Low temperature near-UV CD was used to probe the local tertiary structure of a loop and α-helix in tear lipocalin. Upon cooling, the enhancement of the intensities of the near-UV CD was not uniform, but depends on the position of Trp in the protein structure. The most enhanced (1)Lb band was observed for Trp at position 124 in the α-helix segment matching the known increased conformational mobility during ligand binding. Some aspects of the CD spectra of W28 and W130 were successfully linked to specific rotamers of Trp previously obtained from fluorescence lifetime measurements. The discussion was based on a framework that the magnitude of the energy differences in local conformations governs the changes in the CD intensities at low temperature. The Trp CD spectral classification of Strickland was modified to facilitate the recognition of pseudo peaks. Near-UV CD spectra harbor abundant information about the conformation of proteins that site directed Trp CD can report.

| S-EPMC3983331 | biostudies-literature

In situ monitoring of molecular aggregation using circular dichroism.

Project description:The aggregation of molecules plays an important role in determining their function. Electron microscopy and other methods can only characterize the variation of microstructure, but are not capable of monitoring conformational changes. These techniques are also complicated, expensive and time-consuming. Here, we demonstrate a simple method to monitor in-situ and in real-time the conformational change of (R)-1,1'-binaphthyl-based polymers during the aggregation process using circular dichroism. Based on results from molecular dynamics simulations and experimental circular dichroism measurements, polymers with "open" binaphthyl rings are found to show stronger aggregation-annihilated circular dichroism effects, with more negative torsion angles between the two naphthalene rings. In contrast, the polymers with "locked" rings show a more restrained aggregation-annihilated circular dichroism effect, with only a slight change of torsion angle. This work provides an approach to monitor molecular aggregation in a simple, accurate, and efficient way.

| S-EPMC6251920 | biostudies-literature

Using circular dichroism spectra to estimate protein secondary structure.

Project description: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.

| S-EPMC2728378 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data