Fluorine-19 nuclear magnetic resonance studies of ligand binding to 3-fluorotyrosine- and 6-fluorotryptophan-containing dihydrofolate reductase from Lactobacillus casei

Kimber, Barry J.; Griffiths, D. Vaughan; Birdsall, B.; King, Roy W.; Scudder, Peter; Feeney, J.; Roberts, G. C. K.; Burgen, A. S. V.

doi:10.1021/bi00634a032

Cited by 78 publications

(53 citation statements)

References 19 publications

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“…In addition, 6-19F-tryptophan E. coli DHFR (unpubl. data) and several fluorine-substituted analogs of Lactobacillus casei DHFR (Kimber et al, 1977) possess essentially wild-type enzymatic activity. Similar results have been observed for E. coli lactate dehydrogenase (Ho et al, 1989) and E. coli D-galactose chemosensory receptor (Luck & Falke, 1991).…”

Section: Stopped-flow Nmr and Protein Foldingmentioning

confidence: 98%

“…Early work with "F-labeled proteins attempted to interpret fluorine chemical shifts in terms of the resonance of "buried" fluorine nuclei being shifted to a lower field relative to the resonance of an unincorporated amino acid or to its chemical shift in denatured protein (Sykes & Hull, 1978). This downfield shift was sometimes attributed to electric-field effects (Kimber et al, 1977). However, upfield shifts also occur in several systems (e.g., Luck & Falke, 1991), and there was no detailed theory to predict the direction or magnitude of a protein-induced change in fluorine chemical shift.…”

Section: F Nmr and Protein Foldingmentioning

confidence: 99%

“…Alternatively, "F amino acids can be biosynthetically incorporated directly into bacterial proteins or proteins expressed in bacteria. This method had been used in the past (i.e., Kimber et al, 1977), but in older studies it was not possible to easily identify a specific fluorine resonance with a particular amino acid residue. The ability to induce expression of a particular protein solves at least two problems: a high efficiency of labeling by the "F-substituted amino acid and the ability to identify the specific amino acid using site-directed mutagenesis.…”

Section: F Nmr and Protein Foldingmentioning

confidence: 99%

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NMR and protein folding: Equilibrium and stopped‐flow studies

1993

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NMR studies are now unraveling the structure of intermediates of protein folding using hydrogen-deuterium exchange methodologies. These studies provide information about the time dependence of formation of secondary structure. They require the ability to assign specific resonances in the NMR spectra to specific amide protons of a protein followed by experiments involving competition between folding and exchange reactions. Another approach is to use "F-substituted amino acids to follow changes in side-chain environment upon folding. Current techniques of molecular biology allow assignments of I9F resonances to specific amino acids by site-directed mutagenesis. It is possible to follow changes and to analyze results from I9F spectra in real time using a stoppedflow device incorporated into the NMR spectrometer.

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Section: Stopped-flow Nmr and Protein Foldingmentioning

confidence: 98%

Section: F Nmr and Protein Foldingmentioning

confidence: 99%

Section: F Nmr and Protein Foldingmentioning

confidence: 99%

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NMR and protein folding: Equilibrium and stopped‐flow studies

1993

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“…This substituted lac repressor binds to inducer and operator DNA and releases the operator DNA upon binding the lac operon inducer (12) in a manner almost identical to repressor with normal tyrosines (13). The advantages of this amino acid analogue and 19F NMR have been described and utilized in several systems (14)(15)(16)(17)(18)(19)(20)(21).…”

mentioning

confidence: 99%

Genetic assignment of resonances in the NMR spectrum of a protein: lac repressor.

Jarema¹,

Lu²,

Miller³

1981

Proc. Natl. Acad. Sci. U.S.A.

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By using a systematic genetic approach, the resonances in the 19F NMR spectrum of 3-fluorotyrosine-substituted lac repressor protein have been assigned. The NMR data indicate that each. monomer of the repressor consists of two distinct and independent domains. One domain, the NH2-terminal sixth of the primary sequence, which has been shown to be very important for DNA binding, is very mobile. The remaining COOH-terminal sequence is more rigid. Ligands of the repressor, which affect its DNA binding capability, lead to conformational changes in the COOH-terminal domain. The approach to the assignment ofspectral features taken here can be extended to other systems.The lac repressor of Escherichia coli is the ideal system for an examination of the molecular mechanism of gene regulation (1-3). The entire DNA sequence for the lad gene, which codes for the repressor, and the amino acid sequence of this protein have been independently determined (4, 5). It is a tetrameric protein, total molecular weight 154,520, with 360 amino acids in the subunit without either bound metal ions or cofactors (2). In our genetic analysis of this protein, we have been able to generate 90 nonsense mutations and in most cases determine their position in the 360 codons in the lad gene (6-8). Because there are a number of nonsense suppressors that can insert different amino acids in response to the chain-terminating nonsense codons (UAG, UAA, or UGA) (9), we have been able to use this collection to look at the in vivo properties of over 300 different repressors each with a single amino acid substitution (7). Among these 90 mutations we have characterized nonsense mutations at the positions corresponding to all of the eight tyrosines and both tryptophans in the subunit.With the ability to substitute other amino acids for tyrosine or tryptophan at specific positions in the sequence through nonsense suppression, it is possible to introduce specific alterations in the corresponding fluorescence (10) or NMR (11) spectra of altered lac repressors. In the latter case we simply note which resonance or resonances are missing in the NMR spectra of lac repressors isolated from strains containing suppressed nonsense mutations at each tyrosine position. We use this approach here for the assignment of the resonances from the eight tyrosines in the '9F NMR spectrum of the substituted lac repressor.We chose 3-fluorotyrosine to introduce '9F as a nuclear spin probe and to simplify the NMR spectrum. The 19F NMR spectrum shows only a single resonance for each of the eight tyrosines. This substituted lac repressor binds to inducer and operator DNA and releases the operator DNA upon binding the lac operon inducer (12) in a manner almost identical to repressor

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“…Taking this into consideration as well as the fact that the entropies of binding of the folates to the enzyme are small negative quantities even though the dehydration process may be similar in the case of folates and the coenzymes, it seems likely from the large positive entropy changes that NADP+ and NADPH induce a conformational change in the enzyme upon binding. There has been some evidence from 31P and 19F NMR experiments (18,26,27) that NADP+ and NADPH induce a conformational change in the enzyme.…”

mentioning

confidence: 99%