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A 19 F‐NMR study of the membrane‐binding region of D‐lactate dehydrogenase of escherichia coli
Author(s) -
Sun ZhenYu,
Truong HoaiThu N.,
Pratt E. A.,
Sutherland David C.,
Kulig Christine E.,
Homer Ronald J.,
Groetsch Stefan M.,
Hsue Priscilla Y.,
Ho Chien
Publication year - 1993
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560021115
Subject(s) - chemistry , tryptophan , cofactor , flavin adenine dinucleotide , biochemistry , nuclear magnetic resonance spectroscopy , enzyme , escherichia coli , fluorine 19 nmr , stereochemistry , flavin group , amino acid , gene
d‐Lactate dehydrogenase (d‐LDH) is a membrane‐associated respiratory enzyme of Escherichia coli. The protein is composed of 571 amino acid residues with a flavin adenine dinucleotide (FAD) cofactor, has a molecular weight of approximately 65, 000, and requires lipids or detergents for full activity. We used NMR spectroscopy to investigate the structure of d‐LDH and its interaction with phospholipids. We incorporated 5‐fluorotryptophan (5F‐Trp) into the native enzyme, which contains five tryptophan residues, and into mutant enzymes, where a sixth tryptophan is substituted into a specific site by oligonucleotide‐directed mutagenesis, and studied the 5F‐Trp‐labeled enzymes using 19 F‐NMR spectroscopy. In this way, information was obtained about the local environment at each native and substituted tryptophan site. Using a nitroxide spin‐labeled fatty acid, which broadens the resonance from any residue within 15 Å, we have established that the membrane‐binding area of the protein includes the region between Tyr 228 and Phe 369, but is not continuous within this region. This conclusion is strengthened by the results of 19 F‐NMR spectroscopy of wild‐type enzyme labeled with fluorotyrosine or fluorophenylalanine in the presence and absence of a nitroxide spin‐labeled fatty acid. These experiments indicate that 9–10 Phe and 3–4 Tyr residues are located near the lipid phase.