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Biomedical subjects

D Mao

Publications and source records attributed to D Mao.

41 records · Page 3Linked to original sources
PubMed1984

Circular dichroism analyses of membrane proteins: an examination of differential light scattering and absorption flattening effects in large membrane vesicles and membrane sheets.

The circular dichroism spectra of membrane suspensions are distorted by differential light scattering and absorption flattening effects, which arise as a consequence of the large size of the membrane particles relative to the wavelength of light and the high concentration of proteins in the membranes. In this paper, the consequences of these phenomena on the protein spectra of large membrane particles are discussed, and methods for eliminating them are examined. The distortions due to differential light scattering are relatively small in membrane systems, and can be compensated for by use of a large detector acceptance angle geometry. Several methods for correcting for differential flattening, which introduces a substantial distortion, have been evaluated, and a new method, the flattening quotient approach, which produces by far the best results, is described. Since the secondary structures calculated from circular dichroism spectra are highly dependent on accurate spectral shape and magnitude, this method for correcting the spectra may find general application in circular dichroism studies of membrane proteins.

Bacteriorhodopsins↗
PubMed1984

Differential light scattering and absorption flattening optical effects are minimal in the circular dichroism spectra of small unilamellar vesicles.

The large size of membrane particles and the high local concentration of proteins in these particles give rise to differential scattering and absorption flattening effects which result in significant distortions of the circular dichroism spectra of membrane proteins and produce erroneous estimates of secondary structure. In an attempt to find a membrane system in which scattering and flattening are minimal, but in which native protein conformation is retained, several methods of fragmentation, including sonication, solubilization, and incorporation into small unilamellar vesicles (SUVs), were examined. Bacteriorhodopsin in purple membrane sheets was used as a test system for the effectiveness of the procedures since its secondary structure is known from independent physical measurements and these large membranes produce considerable distortions, as seen by comparison of observed and calculated spectra for the protein. While sonication decreased differential scattering, it had little effect on the total distortion; solubilization in octyl glucoside tended to decrease both differential scattering and flattening but induced some conformational change in the protein. However, when bacteriorhodopsin was incorporated into small unilamellar vesicles, which both decrease particle size and dilute the local concentration of protein, the spectrum produced was nearly identical with the calculated one, suggesting that SUVs may be appropriate vehicles for use with membrane proteins and may be a facile method for eliminating optical artifacts.

Bacteriorhodopsins↗
PubMed1982

Folding of the mitochondrial proton adenosinetriphosphatase proteolipid channel in phospholipid vesicles.

The mitochondrial H+-ATPase proteolipid from Neurospora crassa was incorporated into small unilamellar dimyristoylphosphatidylcholine vesicles and its conformation determined by circular dichroism spectroscopy (CD). While the largely alpha-helical conformation is relatively independent of the method of incorporation into vesicles, i.e., rehydration, detergent dialysis, or detergent dilution, the proteolipid conformation was significantly different in detergent micelles and in organic solvents. Only very slight changes in the CD spectrum were observed upon binding of the H+-ATPase inhibitor dicyclohexylcarbodiimide to the proteolipid in vesicles, thus suggesting that the inhibitor acts either by blocking the channel or by masking an essential charge group, rather by than causing an overall conformational change in the channel. Additionally, very similar CD spectra were obtained for vesicles with different lipid/protein mole ratios, indicating either that no substantial conformational differences exist between monomer and multimers or that monomers self-associate to form stable complexes during incorporation into vesicles. This study has provided a physical basis for model-building studies of the proteolipid channel structure.

Adenosine Triphosphatases↗