The primary structure of the nonpolar segment of bovine cytochrome b5

Fleming, Patrick; Dailey, Harry A.; Corcoran, Doris; Strittmatter, Philipp

doi:10.1016/s0021-9258(17)30380-0

Cited by 77 publications

(9 citation statements)

References 18 publications

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“…The CD results in DPPC in the present study do not support the assignment of any β-sheet structure to the peptide in membranes, in contrast with the suggested value of 25 % based on previous CD studies [24]. However, those earlier CD studies were carried out under conditions where considerable differential scattering, and perhaps absorption flattening, was present but not corrected for, distortions which tend to depress the lowerwavelength peaks and artificially increase the calculated sheet content.…”

Section: Secondary Structure In Phospholipid Vesiclescontrasting

confidence: 99%

“…CD spectroscopy and Fourier-transform IR spectroscopy have been used to probe the secondary structures of the membranebinding domain [24][25][26][27] in various environments. These methods have produced a range of values, with β-sheet contents of up to 33 % and helical contents ranging from 43 to 61 %, depending on the conditions and methods used.…”

Section: Cytochrome B Andmentioning

confidence: 99%

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In vitro membrane-inserted conformation of the cytochrome b5 tail

et al. 2000

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The cytochrome b(5) tail is a 43-residue membrane-embedded domain that is responsible for anchoring the catalytic domain of cytochrome b(5) to the endoplasmic reticulum membrane. Different models for the structure of the membrane domain of cytochrome b(5) have been proposed, including a helical hairpin and a single transmembrane helix. In the present study, CD spectroscopy was used to investigate the conformation of the tail in different environments, and as a function of temperature, with the goal of understanding the nature of the membrane-bound conformation. Whereas residue property profiling indicates that bending of a helix in the middle of the peptide might be possible, the experimental results in small unilamellar vesicles and lysophosphatidylcholine micelles are more consistent with a single transmembrane helix. Furthermore, although there is evidence for some refolding of the polypeptide with temperature, this is not consistent with a hairpin-to-transmembrane transition. Rather, it appears to represent an increase in helical content in fluid lipid environments, perhaps involving residues at the ends of the transmembrane segment.

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Section: Secondary Structure In Phospholipid Vesiclescontrasting

confidence: 99%

Section: Cytochrome B Andmentioning

confidence: 99%

In vitro membrane-inserted conformation of the cytochrome b5 tail

et al. 2000

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“…The unit volume for the membrane was defined as that which contains nonpolar peptide with the appropriate complement of lipid derived from the chemically determined lipid/protein ratio. Protein volume was calculated for each bilayer segment from the amino acid volumes given by Cohn and Edsall (31) according to the known amino acid sequence (32). The contribution of protein to the electron density was calculated by a summation of the electron densities of the amino acids (33) present in each bilayer segment.…”

Section: Modeling Of Electron Density Profilesmentioning

confidence: 99%

“…The cis-membrane model for cytochrome b5 nonpolar peptide previously proposed by Strittmatter and Dailey (8) relied on data that placed the single fluorescent tryptophan 109 residue 20-22 A from the surface of the bilayer (40), placed the COOH-terminal tyrosine 126 and tyrosine 129 residues at the outer surface of the membrane (9), and predicted the secondary structure from the amino acid sequence (32,41). Although Takagaki and co-workers (10, 11), using phospholipid cross-linking to the nonpolar peptide, disagreed with this conclusion, their data suffered from low yields of cross-linked protein and a relatively high degree of protein symmetry across the bilayer.…”

Section: Interpretations Of Diffraction Analysismentioning

confidence: 99%

X-ray diffraction analysis of cytochrome b5 reconstituted in egg phosphatidylcholine vesicles

Rzepecki¹,

Strittmatter²

1986

Biophysical Journal

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Cytochrome b5 was reconstituted asymmetrically into large unilamellar egg phosphatidylcholine vesicles. Asymmetry was preserved after sedimentation and partial dehydration to form oriented stacks of membranes. The periodicity of the centrosymmetric unit cell ranged between 145 and 175 A, depending upon the water content of the oriented multilayer. X-ray diffraction data were collected to a resolution of 12 A and phase factors were unambiguously assigned by a swelling analysis to a resolution of 15 A. The lower-resolution profile structures clearly showed a highly asymmetric single membrane containing the heme peptide segment of the cytochrome on one side of the membrane bilayer. The higher-resolution data were also analyzed and profile structures were compared with various models for the distribution of cytochrome b5 nonpolar peptide within the membrane bilayer region. The data favor an asymmetric distribution of protein mass within the membrane bilayer.

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“…Preparation and purification (29), intramembrane orienta-THE JOURNAL OF CELL BIOLOGY " VOLUME 89 JUNE 1981 615-620 ©The Rockefeller University Press " 0021-9525/81/06/0615/06$1 .00 tion (31,38), re-integration into membranes in vitro (7), immunological localization (9), and at least some functions (17) of this protein have been described. The complete amino acid sequence of the major tryptic fragment (31) as well as of whole cytochrome b5 (8) has been established .…”

mentioning

confidence: 99%

Absence of sugars in electrophoretically purified cytochrome b5 demonstrated by combined gas chromatography-mass spectrometry.

Stadler¹

1981

The Journal of Cell Biology

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The problem of determining small but significant amounts of carbohydrates, in purified proteins, has been studied using the membrane protein, cytochrome b5 . A newly developed method that involves direct gas chromatography-mass spectrometry of sugars obtained by hydrolysis of proteins purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) allows the identification and determination of small amounts of carbohydrates (e .g ., 20 I-Lg of glycoprotein containing a minimum of 0.1% monosaccharid e), even in the presence of relatively high amounts of impurities . Application of this method to cytochrome b5 fragments obtained by tryptic digestion from rat liver microsomes and purified by combined gel filtration and ion exchange chromatography, followed by SDS PAGE, has consistently yielded values below 0.07 mol of the individual sugars and aminosugars per mole cytochrome b 5 . It is concluded that cytochrome b 5 , at least its trypsin-released major aminoterminal fragment, is not constitutively glycosylated .In studies of the synthesis and modification of proteins, especially membrane proteins, the question of the glycosylation of specific proteins is of fundamental importance . Although efficient methods exist for the determination of sugar components present in proteins relatively rich in carbohydrates, the detection of carbohydrates in proteins that are low in relative sugar content and can be obtained in purified form only in limited amounts is still a methodological problem. To clarify the possible existence of sugars in certain proteins, a method has been developed that allows the unequivocal identification and determination of very small amounts of sugars in polypeptides separated and purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) . The value of this method is demonstrated using the example of the widespread membrane protein, cytochrome bs, which has been repeatedly reported to be glycosylated. Cytochrome b5 is a relatively wellcharacterized membrane constituent of endoplasmic reticulum (ER, e.g ., references 8 and 38) . Relatively smaller concentrations of this protein have also been found in isolated nuclear envelope (10, 21), outer mitochondrial membrane (9, 12), Golgi apparatus (18, 19; see, however, reference 33), and in several plasma membrane fractions (3, 4, 19; see, however, reference 34) . Preparation and purification (29), intramembrane orienta-THE JOURNAL OF CELL BIOLOGY " VOLUME 89 JUNE 1981 615-620 ©The Rockefeller University Press " 0021-9525/81/06/0615/06$1 .00 tion (31, 38), re-integration into membranes in vitro (7), immunological localization (9), and at least some functions (17) of this protein have been described. The complete amino acid sequence of the major tryptic fragment (31) as well as of whole cytochrome b5 (8) has been established .In spite of the numerous and intensive studies, it is still not clear whether cytochrome b5 is a glycoprotein. For human apocytochrome b5 prepared by the detergent method, Ozols (30) has...

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The primary structure of the nonpolar segment of bovine cytochrome b5

Cited by 77 publications

References 18 publications

In vitro membrane-inserted conformation of the cytochrome b5 tail

In vitro membrane-inserted conformation of the cytochrome b5 tail

X-ray diffraction analysis of cytochrome b5 reconstituted in egg phosphatidylcholine vesicles

Absence of sugars in electrophoretically purified cytochrome b5 demonstrated by combined gas chromatography-mass spectrometry.

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