Site-Specific Infrared Probes of Proteins

Ma, Jianqiang; Pazos, Ileana M.; Zhang, Wenkai; Culik, Robert M.; Gai, Feng

doi:10.1146/annurev-physchem-040214-121802

Cited by 162 publications

(196 citation statements)

References 153 publications

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“…Taken together, we believe that these spectroscopic and kinetic results will not only help identify which cyanotryptophan to use for a given biological application, but also provide valuable experimental data for QM calculations of the substitution effect [38] to compare. Moreover, like that demonstrated previously for 5-CNI (and 5-cyanotryptophan) [39], we believe that the nitrile stretching vibrations of the other cyanoindoles also afford useful infrared applications [40]. …”

Section: Discussionsupporting

confidence: 73%

Solvent dependence of cyanoindole fluorescence lifetime

Hilaire

Mukherjee

Troxler

et al. 2017

Chemical Physics Letters

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Several cyanotryptophans have been shown to be useful biological fluorophores. However, how their fluorescence lifetimes vary with solvent has not been examined. In this regard, herein we measure the fluorescence decay kinetics as well as the absorption and emission spectra of six cyanoindoles in different solvents. In particular, we find, among other results, that only 4-cyanoindole affords a long fluorescence lifetime and hence high quantum yield in H2O. Therefore, our measurements provide not only a guide for choosing which cyanotryptophan to use in practice but also data for computational modeling of the substitution effect on the electronic transitions of indole.

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Section: Discussionsupporting

confidence: 73%

Solvent dependence of cyanoindole fluorescence lifetime

Hilaire

Mukherjee

Troxler

et al. 2017

Chemical Physics Letters

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“…We call this sample λ 12 *. Temperature jumps of 8-9°C were performed in a range close to the unfolding transition midpoint temperature for each protein [57 ± 1°C for λ 12 *, 58 ± 1°C for λ 13 , and 60 ± 1°C for λ 32 ]. Higher temperature data for λ 12 without GuHCl extrapolates well to the λ 12 * data in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…4F). The trapped state showed a high, folded-like Q and short distance between helices 1 and 3 (Δ 13 ), but a long distance between helix 2 and the other two helices (Δ 32 and Δ 12 > Δ 13 ). Helix 2 is not properly docked in this otherwise native-like but short-lived trap (about 0.3-μs residence time in the example in Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Noninvasive equilibrium NMR experiments, including some coupled with molecular dynamics simulations, have evaluated two-state fits and provided high resolution folding landscapes by using multiple probe nuclei (10)(11)(12). Notable progress in multiprobe analysis of folding kinetics has been achieved by using nonnatural amino acid IR probes (13) in combination with T-jump relaxation spectroscopy (14,15). Comparisons between fast protein folding kinetics probed by fluorescence and/or IR spectroscopy identified heterogeneous folding of trpzip2 (16), λ repressor (17), and α 3 D (18).…”

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confidence: 99%

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Mapping fast protein folding with multiple-site fluorescent probes

Prigozhin

Chao

Sukenik

et al. 2015

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

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Fast protein folding involves complex dynamics in many degrees of freedom, yet microsecond folding experiments provide only low-resolution structural information. We enhance the structural resolution of the five-helix bundle protein λ 6-85 by engineering into it three fluorescent tryptophan-tyrosine contact probes. The probes report on distances between three different helix pairs: 1-2, 1-3, and 3-2. Temperature jump relaxation experiments on these three mutants reveal two different kinetic timescales: a slower timescale for 1-3 and a faster one for the two contacts involving helix 2. We hypothesize that these differences arise from a single folding mechanism that forms contacts on different timescales, and not from changes of mechanism due to adding the probes. To test this hypothesis, we analyzed the corresponding three distances in one published single-trajectory all-atom molecular-dynamics simulation of a similar mutant. Autocorrelation analysis of the trajectory reveals the same "slow" and "fast" distance change as does experiment, but on a faster timescale; smoothing the trajectory in time shows that this ordering is robust and persists into the microsecond folding timescale. Structural investigation of the all-atom computational data suggests that helix 2 misfolds to produce a short-lived off-pathway trap, in agreement with the experimental finding that the 1-2 and 3-2 distances involving helix 2 contacts form a kinetic grouping distinct from 1 to 3. Our work demonstrates that comparison between experiment and simulation can be extended to several order parameters, providing a stronger mechanistic test.fluorescence | helix bundle | protein folding | thermal denaturation | molecular dynamics T he mechanism of protein folding is one of the central questions in biological science (1). The first all-atom simulation to capture substantial protein-refolding dynamics, which lasted 1 μs, was published in 1998 (2). Since then, the timescales of protein folding achieved experimentally and computationally have met (3). Advances in computation now produce distributed (4) and single-trajectory (5) protein-folding simulations on the same nanosecond-to-millisecond timescale as the fastest folding experiments.With rich computational data becoming available, experimental testing of simulations is now hampered by the difficulty of acquiring experimental structural data with microsecond or faster time resolution. Fast ensemble and single-molecule experiments commonly probe only one order parameter such as the fluorescence lifetime of a single tryptophan residue, a broad infrared (IR) spectral response, or one Förster resonant energy transfer (FRET) efficiency (3). Fortunately, a quantitative comparison of such experimental order parameters with simulations is now possible: for example, solventaccessible surface area of tryptophan can serve as a proxy for experimentally detected fluorescence (6), or computed 2D-IR spectra can track secondary structure (7). Still, a single-order parameter, even when compared accurately, canno...

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“…Recently, p -cyanophenylalanine (Phe CN ) has emerged as a convenient and versatile site-specific fluorescence reporter for various biochemical and biophysical studies [1]. The broad spectroscopic utility of this unnatural amino acid, which is a structural derivative of tyrosine (Tyr) or phenylalanine (Phe) [2], stems from the fact that its fluorescence quantum yield and lifetime are sensitive to environment [3], as well as its easy incorporation into peptides and proteins [4].…”

Section: Introductionmentioning

confidence: 99%

Sensing pH via p-cyanophenylalanine fluorescence: Application to determine peptide pKa and membrane penetration kinetics

Pazos

Ahmed

Berríos

et al. 2015

Analytical Biochemistry

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We expand the spectroscopic utility of a well-known infrared and fluorescence probe, p-cyanophenylalanine, by showing that it can also serve as a pH sensor. This new application is based on the notion that the fluorescence quantum yield of this unnatural amino acid, when placed at or near the N-terminal end of a polypeptide, depends on the protonation status of the N-terminal amino group of the peptide. Using this pH sensor, we are able to determine the N-terminal pKa values of nine tripeptides and also the membrane penetration kinetics of a cell-penetrating peptide. Taken together, these examples demonstrate the applicability of using this unnatural amino acid fluorophore to study pH-dependent biological processes or events that accompany a pH change.

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Site-Specific Infrared Probes of Proteins

Cited by 162 publications

References 153 publications

Solvent dependence of cyanoindole fluorescence lifetime

Solvent dependence of cyanoindole fluorescence lifetime

Mapping fast protein folding with multiple-site fluorescent probes

Sensing pH via p-cyanophenylalanine fluorescence: Application to determine peptide pKa and membrane penetration kinetics

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