Evidence of β structure in Mycoplasma membranes. Circular dichroism, optical rotatory dispersion, and infrared studies

Abstract

we combined circular dichroism, optical rotatory dispersion, and infrared data to give evidence for more ß (pleated sheet) structure than a structure in whole membranes of Mycoplasma laidlawii, strain A. The circular dichroism data exhibit a typical -helical pattern. However, when this pattern is subjected to computer analysis, using a curve-fitting program, the best fit to the membrane experimental curve is obtained with 56.0% ß, 30.1% a, and only 13.2% coil. Calculation of the Moffit-Yang a0 and ba parameters from optical rotatory dispersion data yields 30-45 % ß, 26 % a, and 30-45 % coil. Inc V circular dichroism and optical rotatory dispersion spectra of various cellular membranes have generally been interpreted to have shapes indicating substantial amounts of a helix and showing no evidence of ß structure. While circular dichroism spectra may not give evidence for ß structure, it would be difficult to disprove the presence of substantial amounts of beta structure on the basis of such spectra. Gratzer et al. (1968), Hammes and Schullery (1968), and Stevens et al.(1968) have shown that the circular dichroism bands and optical rotatory dispersion maxima of certain ß structures can have much lower intensity and be displaced to the red of the commonly used reference poly-L-lysine in the ß conformation. Thus it is possible that the circular dichroism minimum of the ß structure could actually be placed at a position normally assigned to the negative ( --) transition of -helical peptides (222-225 µ).Wallach et al. (1969) and Graham and Wallach (1969) have recently used this argument to account for evidence of ß structure in their infrared spectra of mitochondrial membranes in the dry-film state. They do this in spite of the fact that several authors (Urry et al., 1967; Stein and Fleisher, 1967; Wrigglesworth and Packer, 1968; Urry and Ji, 1968) have assumed that ß structure is lacking on the basis of optical rotatory dispersion and circular dichroism measurements.

Membrane Optical Activity: Some Facts and Fallacies

“…Optical activity measurements on other, mostly larger membrane particles differ, but all suggest substantial "helicity" and share one common feature-the red displacement of the ORD trough. We will comment on this further, but stress here that one or more of the features thought typical of membrane optical activity, except the last, have also been recorded heretofore for peptides/proteins in true solution (21,26); indeed the CD of cytochrome c, which lacks a-helix (31), yields a CD spectrum resembling that of many membranes (8). Moreover, we here show that even the ORD trough displacement can be found in at least one pure soluble protein (UDP galactose-4-epimerase) during dissociation of its subunits.…”

“…(a) 'Many proteins (1)(2)(3)(4), including membrane components (5)(6)(7)(8), contain significant and sometimes functionally variable proportions of (-conformation; one must therefore analyze unknown substances in terms of three conformations. This is indeed difficult, even with a single synthetic polypeptide as standard, since seven optically active chromophores are concerned: these are the n-7r* transitions of the three conformations, the 7r-7r* transitions of the "unordered" and (3-structures, the I -polarized helical 7r-r* transition and the Il-polarized 7r-r* transition.…”

Kinetics of Sodium Dodecyl Sulfate Solubilization of Mycoplasma laidlawii Plasma Membranes

“…Membranes were prepared from Mycoplasma laidlawii (strain A) by osmotic lysis as described (14). The washed membranes were suspended in twice-distilled, demineralized water.…”

“…The suspension was diluted 1:10 with 2-chloroethanol and absorbance was measured at 280 nm in a Beckman DB spectrophotometer. An extinction coefficient of 1.82 for 1 mg/ml of membrane protein in a 1-cm cell was used (14). The pH of a 1 mg/ml aqueous membrane suspension was typically 7.2.…”

“…The pH of a 1 mg/ml aqueous membrane suspension was typically 7.2. As reported (14), M. laidlawii membranes do not contain either cysteine or cystine, therefore, reducing agents were not added to the dissociation media. Electrophoresis studies with SDS-acrylamide gels show no difference in banding patterns, with or without mercaptoethanol treatment.…”