Fatty acids bound to recombinant tear lipocalin and their role in structural stabilization.
A variant of human tear lipocalin was expressed in Escherichia coli, and the bound fatty acids were analysed by gas chromatography, mass spectroscopy and nuclear magnetic resonance spectroscopy. Five major fatty acids were identified as hexadecanoic acid (palmitic acid, PA), cis-9-hexadecenoic acid (palmitoleic acid), 9,10-methylenehexadecanoic acid, cis-11-octadecenoic acid (vaccenic acid) and 11,12-methyleneoctadecanoic acid (lactobacillic acid). The composition of the bound fatty acids was similar to the fatty acid composition of E. coli extract, suggesting that the binding affinities are similar for these fatty acids. The urea-induced and thermal-unfolding transitions of the holoprotein (nondelipidated), apoprotein (delipidated) and PA-bound protein were observed by circular dichroism. Holoproteins and PA-bound proteins showed the same stability against urea and heat, and were more stable than apoprotein. These results show that each bound fatty acid stabilizes recombinant tear lipocalin to a similar extent.
Tsukamoto
Seiichi
S
Department of Bioinformatics, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan.
Fujiwara
Kazuo
K
Ikeguchi
Masamichi
M
D000465:Algorithms; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D003521:Cyclopropanes / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006358:Hot Temperature; D006801:Humans; D054835:Lipocalin 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D019308:Palmitic Acid / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D044366:Transition Temperature; D014461:Ultracentrifugation; D014508:Urea
D001059:Apoproteins; D003521:Cyclopropanes; D005227:Fatty Acids; D005231:Fatty Acids, Unsaturated; D054835:Lipocalin 1; D050505:Mutant Proteins; D011994:Recombinant Proteins; C028775:cyclopropane fatty acids; D019308:Palmitic Acid; D014508:Urea