Intramolecular Quenching of Tryptophan Fluorescence by the Peptide Bond in Cyclic Hexapeptides

Adams, Paul D.; Chen, Yu; Ma, Ke; Zagorski, Michael G.; Sönnichsen, Frank D.; McLaughlin, Mark L.; Barkley, Mary D.

doi:10.1021/ja0167710

Cited by 154 publications

(164 citation statements)

References 66 publications

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“…Titrations conducted with the nonluminescent lanthanum ion La 3 + instead of Tb 3 + gave very similar results: + 40 % intensity, 2 nm redshift (see Figure 3 for La and Figure S4 for Tb). Even though the photophysics of tryptophan side-chains in peptides is a rather complex subject, [39] this enhancement of the Trp luminescence may indicate that the Trp side-chain is in a more rigid environment in the complex in comparison with the free peptide and/or that the tryptophan is less accessible to water in the complexes. This effect largely exceeds in magnitude the Trp-to-Tb 3 + energy transfer, which is expected to lead to a decrease in the Trp luminescence intensity.…”

Section: Wwwchemeurjorg By the Es-ms Experiments: Tbpmentioning

confidence: 99%

Lanthanide(III) Complexes with Two Hexapeptides Incorporating Unnatural Chelating Amino Acids: Secondary Structure and Stability

Cisnetti

Gateau

Lebrun

et al. 2009

Chemistry A European J

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Unnatural metal-chelating amino acids bearing aminodiacetate side-chains have been introduced into two hexapeptides to obtain efficient lanthanide-binding peptides. The synthesis of the enantiopure Fmoc-Ada(n)(tBu)2-OH synthons is described with overall yields of 32 and 50% for n=2 and n=3 side-chain carbon atoms, respectively. The two peptides AcWAda(n)PGAda(n)GNH2 (Pn) were synthesized from the protected synthons by standard solid-phase peptide synthesis. Studies of the lanthanide complexes of the two peptides Pn by luminescence titrations, mass spectrometry, circular dichroism, and solution NMR spectroscopy demonstrate that the Ada(n) chain length has a dramatic effect on the complexation properties. Indeed, the flexible compound P3 forms a mononuclear complex of moderate stability (beta11=10(9.9)), which tends to transform into a binuclear species in the presence of excess of the metal ion. Interestingly, the more compact peptide P2 provides stable Ln3+ complexes with the exclusive formation of the mononuclear LnP2 adduct. The stability constant of TbP2 is two orders of magnitude higher (beta11=10(12.1)) than that measured for P3. The 800 MHz NMR spectrum of the La3+ complex of P2 evidences a well-defined type II beta-turn as well as a hydrophobic Trp(indole)-Pro interaction. These interactions exemplify the non-innocent character of the peptide spacer in the complex LaP2 as well as the role of a peptide secondary structure in the stabilization of metal complexes.

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Section: Wwwchemeurjorg By the Es-ms Experiments: Tbpmentioning

confidence: 99%

Lanthanide(III) Complexes with Two Hexapeptides Incorporating Unnatural Chelating Amino Acids: Secondary Structure and Stability

Cisnetti

Gateau

Lebrun

et al. 2009

Chemistry A European J

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“…Advances made in Trp photophysics have revealed the local features (such as polarity, proximity of quenching groups, etc) of proteins that effect quantum yield, λ max and lifetime distribution of the fluorescence [1][2][3][4][5][6]. Trp has become a reliable chromophore (label) for studies of the structurefunction relationship of the proteins.…”

Section: Introductionmentioning

confidence: 99%

Site-directed circular dichroism of proteins: 1Lb bands of Trp resolve position-specific features in tear lipocalin

Gasymov

Abduragimov

Glasgow

2008

Analytical Biochemistry

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The absorption spectra of N-acetyl-L-tryptophanamide in various solvents were resolved into the sums of the 1 L a and 1 L b components. The relative intensities of the 0-0 transitions of the 1 L b bands correlate linearly with the solvent polarity values . A novel strategy, which utilizes a set of the experimental 1 L b bands, was employed to resolve the near-UV CD spectra of tryptophanyl residues. Resolved spectral parameters from the single-tryptophan mutants of tear lipocalin (TL), F99W and Y87W, corroborate the fluorescence as well as structural data of TL. Analysis of the 1 L b bands of the Trp CD spectra in proteins is a valuable tool to obtain the local features. The "DMSOlike" 1 L b band of Trp CD spectra may be used as a "fingerprint" to identify the tryptophanyl side chains in situations where the benzene rings of Trp have van der Waals interactions with the side chains of its nearest neighbor. In addition, the signs and intensities of the components hold information about the side-chain conformations and dynamics in proteins. Combined with Trp mutagenesis, this method we call site-directed circular dichroism is broadly applicable to various proteins to obtain the position-specific data.

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“…This approach allows two-color FRET studies on a sample labeled with only one attached extrinsic dye. Although tryptophan is advantageous in that it is naturally occurring and can be introduced (or removed) as needed through site-directed mutagenesis, it is easily quenched by surrounding amino acids, complicating data interpretation (5,6). Additionally, tryptophan fluoresces in the UV, making it difficult to differentiate tryptophan fluorescence from other parasitic sources of fluorescence; a recent study highlighted the need to separate tryptophan fluorescence from intrinsic porphyrin fluorescence in tryptophan-to-heme energy transfer studies (7).…”

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confidence: 99%

Zinc porphyrin: A fluorescent acceptor in studies of Zn-cytochromecunfolding by fluorescence resonance energy transfer

Ensign¹,

Jo²,

Yildirim

et al. 2008

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

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FRET between the zinc porphyrin (ZnP) chromophore in zincsubstituted cytochrome c (Zn-cyt c) and an Alexa Fluor dye attached to specific surface sites was used to characterize Zn-cyt c unfolding. The use of ZnP as a fluorescent acceptor eliminates the need to doubly label the protein with exogenous dyes to perform FRET experiments in which both donor and acceptor fluorescence is monitored. The requirement for attachment of only one dye also minimizes perturbation to the protein. This sensitive technique allowed for the determination of distances between the label placed at six different sites and ZnP through a range of denaturant concentrations. Fitting of the data to a three-state model provides distances in the unfolding intermediate. The use of ZnP as a fluorescent acceptor of energy in FRET has a significant potential for application to a range of other systems including heme-binding proteins and proteins to which a covalently attached heme tag may be added.FRET ͉ protein folding ͉ heme F luorescence resonance energy transfer (FRET) is a simple and widely used technique for measuring molecular distances. FRET involves the nonradiative energy transfer between donor and acceptor fluorophores, the efficiency of which depends strongly on donor-acceptor separation (1). Structure, dynamics, and conformational changes of biomolecules including proteins, RNA, DNA, and polypeptides are amenable to study using FRET (2). As a biomolecular ''ruler,'' FRET is most useful when both donor quenching and acceptor enhancement of fluorescence are observed, because the presence of multiple nonradiative pathways for the donor can potentially cause inaccurate distance measurements if only donor f luorescence quenching is monitored.Because proteins are not readily synthesized chemically, preparing a pure sample of protein site-specifically labeled with two fluorophores appropriate for FRET is technically difficult and time-consuming. The most convenient and widely available chemically specific approach is to label Cys thiols, because Cys is an infrequently occurring amino acid. However, preparing a sample with the donor attached to one particular Cys and acceptor to a second particular Cys requires multiple purification steps, if it can be achieved at all (3, 4). Therefore, the majority of intramolecular FRET studies of proteins make use of an intrinsic fluorophore as one member of the donor-acceptor pair, with tryptophan the most commonly used (2). This approach allows two-color FRET studies on a sample labeled with only one attached extrinsic dye. Although tryptophan is advantageous in that it is naturally occurring and can be introduced (or removed) as needed through site-directed mutagenesis, it is easily quenched by surrounding amino acids, complicating data interpretation (5, 6). Additionally, tryptophan fluoresces in the UV, making it difficult to differentiate tryptophan fluorescence from other parasitic sources of fluorescence; a recent study highlighted the need to separate tryptophan fluorescence from intrinsic porphyri...

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Intramolecular Quenching of Tryptophan Fluorescence by the Peptide Bond in Cyclic Hexapeptides

Cited by 154 publications

References 66 publications

Lanthanide(III) Complexes with Two Hexapeptides Incorporating Unnatural Chelating Amino Acids: Secondary Structure and Stability

Lanthanide(III) Complexes with Two Hexapeptides Incorporating Unnatural Chelating Amino Acids: Secondary Structure and Stability

Site-directed circular dichroism of proteins: 1Lb bands of Trp resolve position-specific features in tear lipocalin

Zinc porphyrin: A fluorescent acceptor in studies of Zn-cytochromecunfolding by fluorescence resonance energy transfer

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