Circular-dichroism analyses of membrane proteins: examination of environmental effects on bacteriorhodopsin spectra

Swords, N A; Wallace, B.A.

doi:10.1042/bj2890215

Cited by 37 publications

(30 citation statements)

References 37 publications

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“…The ␣-helix content of 77% displayed by eBO in this lipid system (Table I) is virtually identical to that of purple membrane reconstituted in micelles or small unilamellar vesicles composed of native or synthetic phospholipids (8,25,32,53). The helicity determined by CD spectroscopy exceeds the value of 70% derived from the high resolution x-ray and electron diffraction structures (16 -19).…”

Section: Discussionmentioning

confidence: 84%

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Secondary Structure of Bacteriorhodopsin Fragments

Lüneberg

Widmann

Dathe³

et al. 1998

Journal of Biological Chemistry

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The secondary structure of bacteriorhodopsin polypeptides comprising two (AB, CD, DE, FG), three (AC, CE, EG), four (AD, DG), or five (AE, CG) of the seven transmembrane segments has been analyzed by circular dichroism spectroscopy. A comparison of the ␣-helix content with that predicted from the high resolution structure of the native protein revealed that the N-terminal AB, AC, AD, and AE fragments and the C-terminal CG fragment are completely refolded in the presence of mixed phospholipid micelles. In contrast, the DG, EG, FG, CD, CE, and DE fragments did not form ␣-helices of the expected lengths at pH 6. Each of the latter fragments displayed, however, an increased helicity upon lowering the pH to 4. Fluorescence measurements with the CD and FG fragments suggest that this helix formation occurs within transmembrane segments C and G, respectively, and thus is likely to originate from the protonation of carboxyl residues that participate in proton translocation. The partial misfolding at neutral pH observed for the shorter fragments from the central and C-terminal part of bacteriorhodopsin indicates that the conformation of some transmembrane segments is specified by interactions with neighboring helices in the assembled structure. Moreover, the data demonstrate that two stable helices at the N terminus of a multihelical membrane protein are sufficient as a folding template to induce a native conformation to the following transmembrane domains.Integral membrane proteins generally adopt a regular secondary structure within the lipid bilayer in order to satisfy hydrogen bonding of the peptide backbone in a hydrophobic environment. The physicochemical constraints imposed by the membrane limit the variety of basic protein architectures, and structure prediction is thus expected to be much simpler than for globular proteins. This characteristic has been exploited in the development of different algorithms for the identification of membrane protein secondary structure (1-3). Transmembrane (TM) 1 helices represent the predominant structural element and can be predicted with high reliability by scanning the protein sequence for regions of adequate length and hydrophobicity to span the lipid bilayer in an ␣-helical conformation. One such algorithm identifies TM ␣-helices based on an estimate of the free energy of transferring a helical segment from the aqueous environment into a lipid bilayer (3). These thermodynamic calculations predict that the individual TM ␣-helix should be stable, even in the absence of interactions with other parts of the molecule. The folding and assembly of multihelical membrane proteins could thus proceed according to a sequential two-step mechanism, in which the individual TM helices form during the initial membrane insertion step, and subsequently associate to form the native tertiary structure (4, 5). Direct structural studies on integral membrane proteins are hindered by the difficulty in obtaining crystals suitable for x-ray diffraction, and by their restricted motion in the lipid enviro...

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Section: Discussionmentioning

confidence: 84%

“…Based on the experimental setup and the lipid micelle system used in the present work, both of these effects can be excluded (32,53). Therefore, the change in the shape of the CD spectra, i.e.…”

Section: Discussionmentioning

confidence: 99%

Secondary Structure of Bacteriorhodopsin Fragments

Lüneberg

Widmann

Dathe³

et al. 1998

Journal of Biological Chemistry

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“…account, the secondary structure content of bacteriorhodopsin in the unilamellar vesicles was found to be 80% α-helix and 0% β-sheet, in accordance with the results of electron diffraction data. In further work, Swords and Wallace [105] showed that the solubilisation of bacteriorhodopsin in the non-ionic detergent octylglucoside led to significant changes in secondary structure, as indicated by the far UV CD spectrum and a small shift in the absorption maximum of the retinal chromophore compared with that of native membrane fragments. This result indicates that the lipid to protein ratio may be an important factor contributing to membrane protein structure, and that it cannot be assumed that the detergent environment is always the same as that provided by the membrane.…”

Section: Membrane Proteinsmentioning

confidence: 99%

The Use of Circular Dichroism in the Investigation of Protein Structure and Function

Kelly

Price²

2000

CPPS

986

707

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Circular Dichroism (CD) relies on the differential absorption of left and right circularly polarised radiation by chromophores which either possess intrinsic chirality or are placed in chiral environments. Proteins possess a number of chromophores which can give rise to CD signals. In the far UV region (240-180 nm), which corresponds to peptide bond absorption, the CD spectrum can be analysed to give the content of regular secondary structural features such as α-helix and β-sheet. The CD spectrum in the near UV region (320-260 nm) reflects the environments of the aromatic amino acid side chains and thus gives information about the tertiary structure of the protein. Other non-protein chromophores such as flavin and haem moieties can give rise to CD signals which depend on the precise environment of the chromophore concerned. Because of its relatively modest resource demands, CD has been used extensively to give useful information about protein structure, the extent and rate of structural changes and ligand binding. In the protein design field, CD is used to assess the structure and stability of the designed protein fragments. Studies of protein folding make extensive use of CD to examine the folding pathway; the technique has been especially important in characterising "molten globule" intermediates which may be involved in the folding process. CD is an extremely useful technique for assessing the structural integrity of membrane proteins during extraction and characterisation procedures. The interactions between chromophores can give rise to characteristic CD signals. This is well illustrated by the case of the light harvesting complex from photosynthetic bacteria, where the CD spectra can be analysed to indicate the extent of orbital overlap between the rings of bacteriochlorophyll molecules. It is therefore evident that CD is a versatile technique in structural biology, with an increasingly wide range of applications.

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“…Through a suitable electrical mixing of the two signals in (7) and (8), (9) can be obtained as Applying a low-pass filter to the signal given in (9), (0 ) (i.e., the signal corresponding to as 0 degree) can be expressed as (10) For the case where polarizer is set such that its transmission axis lies at 45 to the x-axis, the vector of the light emerging from the hybrid sample has the form (11) Adopting a similar procedure to that described above for is obtained as (12) Finally, from (10) and (12), the optical rotation angle of the sample can be derived as (13) As shown in (13), the extracted value of the optical rotation angle is independent of the CD properties of the sample since the terms in the numerator and denominator cancel one another out. To confirm the validity of the proposed extraction process, theoretical values of the detected signal were computed for different values of the optical rotation angle and input into the analytical model described above.…”

Section: A Extraction Of Cb Propertiesmentioning

confidence: 99%

Measuring the Optical Rotation Angle and Circular Dichroism of Anisotropic Optical Media Using a Heterodyne Polarimeter

Liao

et al. 2013

J. Lightwave Technol.

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A method is proposed for measuring the circular birefringence (CB) and circular dichroism (CD) properties of anisotropic optical samples using a heterodyne polarimeter and an electronic signal processing scheme. Importantly, the CB and CD properties of the sample are decoupled in the analytical model, and thus the accuracy of the measurement results is improved. Furthermore, the proposed method enables the CB and CD properties of the sample to be measured over the full range. The validity of the proposed method is demonstrated by measuring the optical rotation angle and circular diattenuation of pure CB and CD samples and a composite sample with both CB and CD properties. The standard deviations of the optical rotation angle and circular dichroism are found to be degrees and for a hybrid CD/CB sample, respectively.

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Circular-dichroism analyses of membrane proteins: examination of environmental effects on bacteriorhodopsin spectra

Cited by 37 publications

References 37 publications

Secondary Structure of Bacteriorhodopsin Fragments

Secondary Structure of Bacteriorhodopsin Fragments

The Use of Circular Dichroism in the Investigation of Protein Structure and Function

Measuring the Optical Rotation Angle and Circular Dichroism of Anisotropic Optical Media Using a Heterodyne Polarimeter

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