Protein characterisation by synchrotron radiation circular dichroism spectroscopy

Wallace, B.A.

doi:10.1017/s003358351000003x

Cited by 111 publications

(88 citation statements)

References 114 publications

(247 reference statements)

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“…The CD spectrum for state III (18∶1-PC liposomes at pH 4.5) is α-helical for both peptides but the ellipticity is again greater for P20G. The characteristic helical minima at 208 and at 222 nm appear to be slightly red-shifted, as has been observed for some membrane proteins (21). Taken together, these results suggest that P20 inhibits α-helix formation in state II and, to a lesser extent, in state III.…”

Section: P20supporting

confidence: 69%

Membrane physical properties influence transmembrane helix formation

Barrera

Fendos

Engelman

2012

Proc. Natl. Acad. Sci. U.S.A.

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The pHLIP peptide has three states: (I) soluble in aqueous buffer, (II) bound to the bilayer surface at neutral pH, and (III) inserted as a transmembrane (TM) helix at acidic pH. The membrane insertion of pHLIP at low pH can be used to target the acidic tissues characteristic of different diseases, such as cancer. We find that the α-helix content of state II depends on lipid acyl chain length but not cholesterol, suggesting the helicity of the bound state may be controlled by the bilayer elastic bending modulus. Experiments with the P20G variant show the proline residue in pHLIP reduces the α-helix content of both states II and III. We also observe that the membrane insertion pKa is influenced by membrane physical properties, following a biphasic pattern similar to the membrane thickness optima observed for the function of eukaryotic membrane proteins. Because tumor cells exhibit altered membrane fluidity, we suggest this might influence pHLIP tumor targeting. We used a cell insertion assay to determine the pKa in live cells, observing that the properties in liposomes held in the more complex plasma membrane. Our results show that the formation of a TM helix is modulated by both the conformational propensities of the peptide and the physical properties of the bilayer. These results suggest a physical role for helix-membrane interactions in optimizing the function of more complex TM proteins.alpha helix | peptide folding | tumor acidity | bilayer thickness | vesicle properties

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Section: P20supporting

confidence: 69%

Membrane physical properties influence transmembrane helix formation

Barrera

Fendos

Engelman

2012

Proc. Natl. Acad. Sci. U.S.A.

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“…It is not a spectral feature normally found for helical or sheet structures, nor indeed in other unfolded or natively disordered protein structures (as shown in Refs. 16,25,26 ), which otherwise bear a strong resemblance to the unfolded collagen spectra. Together these results suggest that the presence of the positive peak in the region of 220-228 nm is the most diagnostic for the PPII structure, and that the negative peak at 197 nm (as opposed to 208 nm) is most diagnostic of the unfolded state for an amino-acid containing polypeptide.…”

Section: Comparison Of Polyproline and Collagen Spectramentioning

confidence: 99%

Distinct circular dichroism spectroscopic signatures of polyproline II and unordered secondary structures: Applications in secondary structure analyses

et al. 2014

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Circular dichroism (CD) spectroscopy is a valuable method for defining canonical secondary structure contents of proteins based on empirically-defined spectroscopic signatures derived from proteins with known three-dimensional structures. Many proteins identified as being "Intrinsically Disordered Proteins" have a significant amount of their structure that is neither sheet, helix, nor turn; this type of structure is often classified by CD as "other", "random coil", "unordered", or "disordered". However the "other" category can also include polyproline II (PPII)-type structures, whose spectral properties have not been well-distinguished from those of unordered structures. In this study, synchrotron radiation circular dichroism spectroscopy was used to investigate the spectral properties of collagen and polyproline, which both contain PPII-type structures. Their native spectra were compared as representatives of PPII structures. In addition, their spectra before and after treatment with various conditions to produce unfolded or denatured structures were also compared, with the aim of defining the differences between CD spectra of PPII and disordered structures. We conclude that the spectral features of collagen are more appropriate than those of polyproline for use as the representative spectrum for PPII structures present in typical amino acid-containing proteins, and that the single most characteristic spectroscopic feature distinguishing a PPII structure from a disordered structure is the presence of a positive peak around 220nm in the former but not in the latter. These spectra are now available for inclusion in new reference data sets used for CD analyses of the secondary structures of soluble proteins.

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“…The peptide backbone forms characteristic secondary structures such as α-helices, β-pleated sheets, turns, and disordered sections with specific Φ, Ψ dihedral angles and H-bond patterns affecting the CD spectrum (1). CD has been exploited for protein folding and stability assays, intermolecular interactions, and ligand binding studies, and has recently been applied in the investigations of protein disorder (2,3). Synchrotron radiation CD (SRCD) spectroscopy is an emerging technique complementary to small-angle X-ray scattering or infrared spectroscopy, synergistic to biochemical and biophysical assays characterizing the protein folding state.…”

mentioning

confidence: 99%

“…(4). Additionally, SRCD has the capability of time-resolved and stopped-flow measurements as well as high-throughput screening (3).…”

mentioning

confidence: 99%

Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy

Micsonai

Wien

Kernya

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

1,380

1,337

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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. circular dichroism | secondary structure determination | protein fold | protein aggregation | amyloid O ptically active macromolecules, such as proteins, exhibit differential absorption of circular polarized light. The far-UV circular dichroism (CD) spectroscopy of proteins and peptides (180-250 nm) is predominantly based on the excitation of electronic transitions in amide groups. The peptide backbone forms characteristic secondary structures such as α-helices, β-pleated sheets, turns, and disordered sections with specific Φ, Ψ dihedral angles and H-bond patterns affecting the CD spectrum (1). CD has been exploited for protein folding and stability assays, intermolecular interactions, and ligand binding studies, and has recently been applied in the investigations of protein disorder (2, 3). Synchrotron radiation CD (SRCD) spectroscopy is an emerging technique complementary to small-angle X-ray scattering or infrared spectroscopy, synergistic to biochemical and biophysical assays characterizing the protein folding state. SRCD extends the limits of conventional CD spectroscopy by broadening the spectral range, increasing the signal-to-noise ratio, and accelerating the data acquisition, in the presence of absorbing components (buffers, salts, etc.) (4). Additionally, SRCD has the capability of time-resolved and stopped-flow measurements as well as hig...

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Protein characterisation by synchrotron radiation circular dichroism spectroscopy

Cited by 111 publications

References 114 publications

Membrane physical properties influence transmembrane helix formation

Membrane physical properties influence transmembrane helix formation

Distinct circular dichroism spectroscopic signatures of polyproline II and unordered secondary structures: Applications in secondary structure analyses

Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy

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