The Preparation of 19F-Labeled Proteins for NMR Studies

Frieden, Carl; Hoeltzli, Sydney D.; Bann, James G.

doi:10.1016/s0076-6879(04)80018-1

Cited by 90 publications

(109 citation statements)

References 44 publications

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“…28 The fluorine nucleus is small and in general structurally nonperturbing, but is highly sensitive to local environmental changes and should allow the detection of CMG2 binding to PA or the prepore via 19 F NMR. 30,31 We labeled CMG2 with 2-FHis, and the degree of labeling was confirmed to be >90% by mass spectrometry, as had been observed previously when the periplasmic chaperone PapD was labeled with this residue. 28 The 2-FHis-CMG2 was also shown to form a 1 : 1 complex with PA using a gel-shift assay, and the stability to pH was similar to that of WT CMG2 (see Fig.…”

Section: Role Of Cmg2 His-121 In Pore Formationsupporting

confidence: 63%

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Monitoring anthrax toxin receptor dissociation from the protective antigen by NMR

Rajapaksha¹,

Eichler²,

Hajduch³

et al. 2008

Protein Science

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The binding of the Bacillus anthracis protective antigen (PA) to the host cell receptor is the first step toward the formation of the anthrax toxin, a tripartite set of proteins that include the enzymatic moieties edema factor (EF), and lethal factor (LF). PA is cleaved by a furin-like protease on the cell surface followed by the formation of a donut-shaped heptameric prepore. The prepore undergoes a major structural transition at acidic pH that results in the formation of a membrane spanning pore, an event which is dictated by interactions with the receptor and necessary for entry of EF and LF into the cell. We provide direct evidence using 1-dimensional 13 C-edited 1 H NMR that low pH induces dissociation of the Von-Willebrand factor A domain of the receptor capillary morphogenesis protein 2 (CMG2) from the prepore, but not the monomeric full length PA. Receptor dissociation is also observed using a carbon-13 labeled, 2-fluorohistidine labeled CMG2, consistent with studies showing that protonation of His-121 in CMG2 is not a mechanism for receptor release. Dissociation is likely caused by the structural transition upon formation of a pore from the prepore state rather than protonation of residues at the receptor PA or prepore interface.

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Section: Role Of Cmg2 His-121 In Pore Formationsupporting

confidence: 63%

“…19 2-FHis labeling: cultures of the E.coli strain UTH780, harboring pGEX-4T1-CMG2 , in minimal media 31 were grown to an A 600 of 2.0. The cells were harvested, washed three times with 0.9% NaCl, and resuspended in minimal media supplemented with 0.2 mM 2-FHis.…”

Section: Anthrax Receptor Binding Monitored By Nmrmentioning

confidence: 99%

Monitoring anthrax toxin receptor dissociation from the protective antigen by NMR

Rajapaksha¹,

Eichler²,

Hajduch³

et al. 2008

Protein Science

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“…The phenylalanine auxotroph DL39 was used to express 4-19 F-Phe-labeled G121V-IFABP according to established procedures (13,28). The 4-19 F-labeled IFABP mutant was expressed and purified as described elsewhere (12).…”

Section: Methodsmentioning

confidence: 99%

Observation of sequential steps in the folding of intestinal fatty acid binding protein using a slow folding mutant and¹⁹F NMR

Frieden

2007

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

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The rat intestinal fatty acid binding protein (IFABP) primarily comprises two ␤-sheet structures surrounding a large internal cavity. The urea denatured WT protein folds within seconds after dilution to nondenaturing conditions. Replacing a glycine with valine in the turn between the last two ␤-strands (Gly121Val) slows the folding process by more than three orders of magnitude. After incorporating 4-19 F-phenylalanine into the mutant protein, we were able to directly monitor the behavior of the eight phenylalanine side chains in real time during folding using 19 F NMR. Specifically, there is a nonnative-like collapse in regions involving three phenylalanine residues (Phe-62, Phe-68, and Phe-93) within milliseconds. At least two distinct NMR peaks were observed, suggesting conformational fluctuations in this region. Formation of this site is followed by formation of native structure of Phe-2 and Phe-17, then by Phe-47, and finally by the cooperative rearrangement of the intermediate structures to the final native structure. It is proposed that the Gly121Val mutation slows the formation of a normal nucleating site, not only slowing overall folding, but also allowing intermediates in regions distant from the mutation to be experimentally observed. Because intermediates involved in protein folding are normally difficult to observe due to their marginal stability, the experimental approach used here may serve as a general method for determining the nature of both early and late steps in protein folding.folding intermediates ͉ nucleation sites ͉ turn propensity

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“…[12][13][14][15][16][17] The 19 F nucleus is very sensitive to its surroundings and small changes in the structure can be detected by chemical shifts. Additionally, the use of a 19 F cryoprobe allows spectra to be collected at reasonably low protein concentrations and, in general, the 1D spectra are relatively uncomplicated.…”

Section: Introductionmentioning

confidence: 99%

Structural differences between apolipoprotein E3 and E4 as measured by ¹⁹F NMR

Garai

Mustafi²,

Baban³

et al. 2009

Protein Science

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Abstract:The apolipoprotein E family contains three major isoforms (ApoE4, E3, and E2) that are directly involved with lipoprotein metabolism and cholesterol transport. ApoE3 and apoE4 differ in only a single amino acid with an arginine in apoE4 changed to a cysteine at position 112 in apoE3. Yet only apoE4 is recognized as a risk factor for Alzheimer's disease. Here we used 19 F NMR to examine structural differences between apoE4 and apoE3 and the effect of the C-terminal domain on the N-terminal domain. After incorporation of 5-19 F-tryptophan the 1D 19 F NMR spectra were compared for the N-terminal domain and for the full length proteins. The NMR spectra of the N-terminal region (residues 1-191) are reasonably well resolved while those of the full length wild-type proteins are broad and ill-defined suggesting considerable conformational heterogeneity. At least four of the seven tryptophan residues in the wild type protein appear to be solvent exposed. NMR spectra of the wild-type proteins were compared to apoE containing four mutations in the C-terminal region that gives rise to a monomeric form either of apoE3 under native conditions (Zhang et al., Biochemistry 2007; 46: 10722-10732) or apoE4 in the presence of 1 M urea. For either wild-type or mutant proteins the differences in tryptophan resonances in the Nterminal region of the protein suggest structural differences between apoE3 and apoE4. We conclude that these differences occur both as a consequence of the Arg158Cys mutation and as a consequence of the interaction with the C-terminal domain.

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The Preparation of 19F-Labeled Proteins for NMR Studies

Cited by 90 publications

References 44 publications

Monitoring anthrax toxin receptor dissociation from the protective antigen by NMR

Monitoring anthrax toxin receptor dissociation from the protective antigen by NMR

Observation of sequential steps in the folding of intestinal fatty acid binding protein using a slow folding mutant and¹⁹F NMR

Structural differences between apolipoprotein E3 and E4 as measured by ¹⁹F NMR

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The Preparation of 19F-Labeled Proteins for NMR Studies

Cited by 90 publications

References 44 publications

Monitoring anthrax toxin receptor dissociation from the protective antigen by NMR

Monitoring anthrax toxin receptor dissociation from the protective antigen by NMR

Observation of sequential steps in the folding of intestinal fatty acid binding protein using a slow folding mutant and19F NMR

Structural differences between apolipoprotein E3 and E4 as measured by 19F NMR

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Observation of sequential steps in the folding of intestinal fatty acid binding protein using a slow folding mutant and¹⁹F NMR

Structural differences between apolipoprotein E3 and E4 as measured by ¹⁹F NMR