[1] Fluorine nuclear magnetic resonance of fluorinated ligands

Gerig, J. T.

doi:10.1016/0076-6879(89)77003-8

Cited by 80 publications

(102 citation statements)

References 54 publications

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“…2A). This modest up-field shift relative to solution is consistent with the ability of aromatic ring currents and other shielding differences between a protein interior and water to result in chemical shift differences of 1-2 ppm for a 19 F nucleus (23). In contrast, 3,4-F 2 -phenol (pK a = 9.1) bound to pKSI D40N displays a chemical shift that is intermediate between the observed values for its neutral (phenol) and ionized (phenolate) forms in solution, suggesting that this phenol binds to pKSI D40N as a mixture of neutral phenol and ionized phenolate and that these forms are rapidly interconverting relative to their 19 F NMR frequency difference (additional discussion is provided in SI Text).…”

Section: Resultsmentioning

confidence: 59%

Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site

Sigala¹,

Fafarman²,

Schwans³

et al. 2013

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

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Hydrogen bond networks are key elements of protein structure and function but have been challenging to study within the complex protein environment. We have carried out in-depth interrogations of the proton transfer equilibrium within a hydrogen bond network formed to bound phenols in the active site of ketosteroid isomerase. We systematically varied the proton affinity of the phenol using differing electron-withdrawing substituents and incorporated sitespecific NMR and IR probes to quantitatively map the proton and charge rearrangements within the network that accompany incremental increases in phenol proton affinity. The observed ionization changes were accurately described by a simple equilibrium proton transfer model that strongly suggests the intrinsic proton affinity of one of the Tyr residues in the network, Tyr16, does not remain constant but rather systematically increases due to weakening of the phenol-Tyr16 anion hydrogen bond with increasing phenol proton affinity. Using vibrational Stark spectroscopy, we quantified the electrostatic field changes within the surrounding active site that accompany these rearrangements within the network. We were able to model these changes accurately using continuum electrostatic calculations, suggesting a high degree of conformational restriction within the protein matrix. Our study affords direct insight into the physical and energetic properties of a hydrogen bond network within a protein interior and provides an example of a highly controlled system with minimal conformational rearrangements in which the observed physical changes can be accurately modeled by theoretical calculations.computational modeling | enzyme catalysis | protein electrostatics | protein semisynthesis | active site environment H ydrogen bond networks are ubiquitous structural features within proteins, and they play key roles linking secondary and tertiary structural elements and spanning protein-protein interfaces. Such networks are especially common within enzyme active sites, where they position protein and substrate groups for catalysis, stabilize charge rearrangements during chemical transformations, and mediate proton transfers (1). Despite the prevalence and critical structural and functional roles of hydrogen bond networks, incisive dissection of their physical properties within the idiosyncratic interior of folded proteins remains difficult.Hydrogen-bonded protons are not observed in the vast majority of protein X-ray structures due to the low X-ray scattering power of hydrogen atoms (2). Thus, the presence of hydrogen bond networks is typically inferred from the proximity and orientation of hydrogen bond donor and acceptor groups within refined protein structural models. The inherent inability of most X-ray diffraction studies to monitor proton positions imposes additional challenges for dissecting the physical features that influence the equilibrium protonation states of specific residues along a hydrogen-bonded proton transfer network. Furthermore, it remains extremely challenging t...

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Section: Resultsmentioning

confidence: 59%

Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site

Sigala¹,

Fafarman²,

Schwans³

et al. 2013

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

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“…19F NMR is nearly as sensitive as 1H NMR and the resonances occur over an extremely wide chemical shift range (6). Further, the chemical shift is extremely sensitive to changes in solvent environment, as would occur during the folding process.…”

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confidence: 92%

Dynamic NMR spectral analysis and protein folding: identification of a highly populated folding intermediate of rat intestinal fatty acid-binding protein by 19F NMR.

Ropson

Frieden

1992

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

119

141

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The MATERIALS AND METHODS Mutagenesis. Mutagenic oligonucleotides were prepared to change each tryptophan codon to a tyrosine codon by sitedirected mutagenesis using the uracil selection method of Kunkel et al. (16). Single-stranded phagemid DNA was produced in Escherichia coli BW313 from pMON-IFABP (17). Nucleotides, buffers, and enzymes for second-strand synthesis and ligation were from United States Biochemical. Mutants were identified and the entire coding regions were sequenced by the dideoxy method (18). Sequenase sequencing reagents and protocols were from United States Biochemical.Protein Production. Large quantities of wild-type and mutant 6FTrp-containing IFABP were produced by the induction of protein synthesis in E. coli W3110trpA33, a trypAbbreviations: IFABP, intestinal fatty acid-binding protein; Gdn'HCl, guanidine hydrochloride; 6FTrp, 6-fluorotryptophan; 72, effective transverse relaxation time; TFA, trifluoroacetic acid. 7222The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Proc. Natl. Acad. Sci. USA 89 (1992) 7223 tophan auxotroph (19) provided by C. Yanofsky (Stanford University). Culture and induction conditions were similar to those used for the synthesis of 6FTrp substituted cellular retinol-binding protein II (7). Individual overnight cultures containing the various pMON-IFABP vectors were diluted 1:100 in M9 medium (20) supplemented with 1% Casamino acids, 1.5% glucose, vitamin B1 (1 ,ug/ml), FeCJ3 (5 A&g/ml), ZnSO4 (0.4 ug/ml), CuS04 (0.8 ,ug/ml), CoCl2 (0.7 ,ug/ml), MnSO4 (0.5 fug/ml), H3BO3 (0.2 ,ug/ml), Na2MoO4 (0.7 kug/ml), ampicillin (50 tkg/ml), and 0.1 mM tryptophan.Cultures were shaken at 370C. After achieving an OD6W of 3.0, cells were harvested by low-speed centrifugation and resuspended in the same medium prewarmed to 370C and containing 0.1 mM 6FTrp instead of tryptophan. The cultures were incubated at 370C for 15 min and IFABP expression was induced by the addition of nalidixic acid (50 ,ug/ml). After 2 hr of induction at 37°C, the OD600 reached a value of about 5.5. Cells were harvested by low-speed centrifugation. Approximately 30 g of cells were produced from 5 liters of medium, resulting in 200-400 mg of purified protein after processing. The purification and delipidation protocols for mutant and wild-type [6FTrp]IFABP were identical to those reported for the unsubstituted wild-type protein (17). Incorporation levels were estimated by comparing the integrated intensity to that of a standard concentration of 2-fluorophenylalanine and were found to range from 80% to 90o. Purity was assessed by SDS/PAGE.Ultrapure urea and guanidine hydrochloride (Gdn-HCl) were supplied by Schwarz/Mann. Solution preparation protocols, spectroscopic methods of monitoring protein unfolding at equilibrium, and data analysis have been described (15).NMR Data Collection and Analysis. NMR data were collected on a Varian VXR-500 ...

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“…Third, because of the simplicity of the fluorine spectrum, one is not limited to small (<20 kDa) proteins by the problem of spectral overlap. Finally, fluorine chemical shifts are spread over a larger range of frequencies than proton chemical shifts and are very sensitive to the environment of the "F nucleus (Sykes & Hull, 1978;Gerig, 1989). Thus, changes in the fluorine chemical shift can serve as a reporter for conformational changes in the protein.…”

Section: F Nmr and Protein Foldingmentioning

confidence: 99%

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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[1] Fluorine nuclear magnetic resonance of fluorinated ligands

Cited by 80 publications

References 54 publications

Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site

Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site

Dynamic NMR spectral analysis and protein folding: identification of a highly populated folding intermediate of rat intestinal fatty acid-binding protein by 19F NMR.

NMR and protein folding: Equilibrium and stopped‐flow studies

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