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S. Bussieres, R. Breton, T. Buffeteau, C. Salesse; Secondary Structure Study of a Transmembrane Domain-Deleted Form of Lecithin-Retinol Acyltransferase. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3255.
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Lecithin retinol acyltransferase is a 230 amino acids membrane-associated protein which catalyzes the esterification of all-trans-retinol into all-trans-retinyl ester, an essential reaction in the vertebrate visual cycle. The present study was performed to determine the secondary structure of the transmembrane domain deleted form of LRAT.
The truncated form of LRAT (tLRAT) which extends from residues 31 to 196 has been overexpressed in BL21(DE3)pLysS E. coli strain. It has been previously determined that the deleted domains of tLRAT do not contain residues known to be required for catalysis. tLRAT has been purified using affinity chromatography. Infrared absorption spectroscopy, electronic circular dichroism (ECD) and vibrational circular dichroism (VCD) were used to probe the secondary structure of tLRAT in solution in the presence of different detergents: sodium dodecyl sulfate (SDS), n-octyl glucoside (OG) and Triton X-100.
The analysis of the spectra obtained by ECD and VCD allowed to determine that tLRAT secondary structure in solution is a mixture of α-helices and ß-sheets. However, tLRAT structure in SDS contains more α-helices than in OG and Triton X-100. Accordingly, the amide I band maximum of tLRAT in SDS (1648 cm-1) is different from that observed in OG and triton X-100 (1642 cm-1).
tLRAT secondary structure has been determined in solution by infrared absorption spectroscopy, electronic and vibrational circular dichroism. Its structure appears to be dependent on the detergent used. Given that tLRAT activity is much higher in Triton X-100 than is SDS, the secondary structure of the most active form of tLRAT should correspond to that measured in OG and Triton X-100.
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