Membrane protein circular dichroism

FT-IR spectroscopy is particularly useful for probing the structure of membrane proteins. Until recently, a lack of adequate experimental techniques has been the reason for the poor understanchng of the secondary structure of most membrane proteins. X-ray diffraction requires high quality crystals and these are not available for many membrane proteins. Circular dichroism (CD) has been widely used for studying the conformation of water-soluble proteins, but problems arise in its use for membrane proteins. The light scattering effect may distort CD spectra and lead to substantial errors in their interpretation. In addition, the reference spectra used for the analysis of CD spectra are based on globular proteins in aqueous solution and may not be applicable to membrane proteins in the hydrophobic environment of lipid bilayers. 

The protein-containing colloidal solutions of water-in-organic solvents are optically transparent. Hence, absorption spectroscopy, circular dichroism spectroscopy and fluorescence spectroscopy are found to be convenient for studying biocatalysis [53]. The reversed micelles are interesting models for studying bioconversion, since the majority of the enzymes in vivo act inside or on the surface of biological membranes. 

Swords, N.A. and Wallace, B.A 1993. Circular dichroism analyses of membrane proteins Examination of environmental effects on bacteriorho-dopsin spectra. Biochem. J. 289 215-219. 

The advent of recombinant DNA technology has led to an increased interest in the structural characterization of proteins by spectroscopic methods. Few spectroscopic techniques can provide the amount of information regarding protein secondary and tertiary structure which can be obtained from circular dichroism (CD) spectroscopy. In this chapter we describe the capabilities of CD spectroscopy to provide details on the globular structure of proteins. In addition, we will provide an overview of quantitative secondary structure estimates via CD spectroscopy and of specialized CD methods for studying proteins in contact with membranes and other biomolecules. Certain aspects of protein CD spectroscopy have been previously reviewed [1-19]. 

Involvement of Basic Amphiphilic a-helical Domain in the Reversible Membrane Interaction of Amphitropic Proteins Structural Studies by Mass Spectrometry, Circular Dichroism, and Nuclear Magnetic Resonance... 

On the basis ofthe primary-structure information discussed above, and the known, 1 1 stoichiometry of the a- and p-polypeptides, Zuber developed a model for the light-harvesting BChl-protein complexes, as shown in Fig. 4 (A). In this model the two polypeptides span the chromatophore membrane with their hydrophobic segments consisting of 20 amino acids located in the hydrocarbon-tail region of the lipid-bilayer membrane. The polar N- and C-termini are located on the cytoplasmic and periplasmic sides, respectively. The hydrophobic amino acids in this model are present as an a-helix, in accord with the finding of a high helical content by circular dichroism. ... 

Note that the ring planes of the chlorophyll molecules in Fig. 5 (A) were shown oriented mostly perpendicular to the membrane plane. This orientation appears to be similar to that in the PS-II chlorophyll a/b protein (LHC II), as determined by Kiihlbrandt and Wang and predicted by Haworth, Tapie, Amtzen and Breton on the basis of circular-dichroism measurements. The center-to-center distances between the porphyrin planes of the CA-Chl a molecules in the PS-I reaction center range from 8 to 15 A, which are comparable to those in PS tf and in the BChl-a protein complex as measured by Mathews, Fenna, Bolognesi and Olson (see Chapter 8, Section V). Some of the closely spaced chlorophyll-a molecules appear to form chains mnning between the stroma and lumen sides of photosystem I. Between 12 and 16 P-carotene molecules are thought to be present in the PS-I reaction center, but they have not been identified by X-ray crystallographic measurements. 

Membrane proteins often contain a-helical sections. We have developed a method called oriented circular dichroism (OCD see reference 1), which can be used to determine the orientation of a-helices with respect to the plane of the membrane. This method is simple and easy to use compared with, for example, the NMR method, which requires isotope labeled samples. Indeed, it is the ease of this method that allowed us to examine alamethicin in many different chemical conditions and that resolved a controversial question about the nonconducting state of alamethicin and subsequently led to the discovery of a new phenomenon of amphiphilic helical peptides (2). 

From its circular dichroism rhodopsin was estimated to be 60% helical, and its amino acid sequence suggested that it contains seven parallel membrane-spanning helices (Fig. 23-41) just as does bacterior-hodopsin (Section G Fig. 23-45). Rhodopsin and other visual pigments are also members of the large family of G-protein coupled receptors, which includes the P2 adrenergic receptor pictured in Fig. 

NaCl/urea-washed PSII membranes, lacking all extrinsic proteins, were prepared in the presence of 200 mM NaCl as described in [5]. Rebinding studies were performed according to [4]. Oxygen-evolution activity was measured as described in [6]. The N- and C-terminal amino acid sequences of the 33-kDa protein and the fragments were determined as described in [3]. Measurements of circular dichroism spectra were carried out as described in [4]. 

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