Flash Photolysis of Tryptophan and N‐acetyl‐l‐tryptophanamide; The Effect of Bromide on Transient Yields

Volkert, Wynn A.; Kuntz, Robert R.; Ghiron, Camillo A.; Evans, R. F.; Santús, René; Bazin, M.

doi:10.1111/j.1751-1097.1977.tb07441.x

Cited by 51 publications

(27 citation statements)

References 22 publications

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“…We have used optical (triplet3triplet) absorption (9,11,13,14) to measure the lifetime of the excited triplet state of tryptophan at the C terminus of a flexible peptide with either cysteine, cystine, or lipoate at the N terminus as quencher. Lipoate is a cyclic disulfide and is known to be a near diffusion-limited quencher of the tryptophan triplet state (11).…”

mentioning

confidence: 99%

Measuring the rate of intramolecular contact formation in polypeptides

Lapidus¹,

Eaton

Hofrichter

2000

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

402

602

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Formation of a specific contact between two residues of a polypeptide chain is an important elementary process in protein folding. Here we describe a method for studying contact formation between tryptophan and cysteine based on measurements of the lifetime of the tryptophan triplet state. With tryptophan at one end of a flexible peptide and cysteine at the other, the triplet decay rate is identical to the rate of quenching by cysteine. We show that this rate is also close to the diffusion-limited rate of contact formation. The length dependence of this end-to-end contact rate was studied in a series of Cys-(Ala-Gly-Gln) k-Trp peptides, with k varying from 1 to 6. The rate decreases from ϳ1͞(40 ns) for k ‫؍‬ 1 to ϳ1͞(140 ns) for k ‫؍‬ 6, approaching the length dependence expected for a random coil (n ؊3͞2 ) for the longest peptides.T o understand how proteins fold, it is essential to know the time scales and mechanisms of its elementary processes (1). The formation of a contact between two residues in an unfolded polypeptide chain is one of the most fundamental of these processes and is central to many aspects of protein-folding mechanisms. Most helical segments in proteins are unstable in isolation (2, 3) and require interactions with residues in other parts of the chain to form stable folded structures. Similarly, interaction between side chains on opposite strands of an antiparallel ␤ sheet are required for stability (4). Initiation of a parallel ␤ sheet requires a contact between residues distant in sequence. Because of its central role in protein folding, it is important to develop kinetic methods to investigate contact formation. Here we describe a technique with nanosecond time resolution and apply it to the investigation of the end-to-end contact formation rate in a flexible peptide.An initial glimpse of the kinetics of contact formation was obtained in a study of unfolded cytochrome c (5-7). In these experiments, photodissociation of carbon monoxide from the heme with a nanosecond laser pulse was used to initiate an intramolecular ligand-binding process. CO dissociation opens up a binding site at the heme iron, permitting binding kinetics to be measured by time-resolved absorption spectroscopy. This study showed that intramolecular methionine binding is very close to diffusion limited. The measured binding rate of ϳ1͞(40 s) is therefore also the rate of forming a contact, in this case between positions separated by ϳ50 residues in the sequence. An obvious limitation of this technique, however, is that it is restricted to very few proteins (a subset of heme proteins). A more generic method for measuring contact formation was clearly needed.One such method has been introduced recently by Bieri et al. (8), who attached thioxanthone at one end of a f lexible peptide and naphthalene near the other. On optical excitation, the triplet state of thioxanthone forms and transfers its excitation energy to naphthalene on close contact in an apparently diffusion-limited process. The rate at which the thioxanthone tr...

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mentioning

confidence: 99%

Measuring the rate of intramolecular contact formation in polypeptides

Lapidus¹,

Eaton

Hofrichter

2000

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

402

602

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“…A value of τ = 12 µsec was obtained by flash photolysis for an aqueous solution of indole. For other indole derivatives in aqueous solutions, the value of ( measured by the same method was several tens of microseconds [2][3][4]. In a later paper [5], a value of τ ≈ 40 µsec was measured from analysis of the fluorescence kinetics of indole and its derivatives in aqueous solution.…”

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confidence: 99%

Nature of delayed luminescence of N-methylindole and indole in the gas phase

Borisevich¹,

Sukhodola²,

Tolstorozhev³

2007

J Appl Spectrosc

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We have studied the spectral characteristics and the kinetics of delayed (long-lived) luminescence of Nmethylindole and indole in the gas phase. For N-methylindole, we observed delayed annihilation fluorescence, spectrally matching fast fluorescence. There is no delayed annihilation fluorescence for indole, but we observed a delayed luminescence band with wavelength at the maximum 535 nm, which we interpreted as luminescence of free radicals formed as a result of dissociation of the N-H bond. We hypothesize that the excited states of the free radicals arise as a result of nonradiative energy transfer from indole in the triplet state to indole free radicals in the doublet state. The lifetimes of the triplet states of N-methylindole and indole in the gas phase at T = 373 K, obtained from analysis of the delayed luminescence kinetics, are 2.5 msec and 1.0 msec.Introduction. Indole molecules are the chromophores of biologically important tryptophan compounds, the phosphorescence of which carries information about slow processes occurring in proteins over time periods in the microsecond and millisecond range. Accordingly, it seems timely to measure the lifetimes of the triplet states of indole and its derivatives in different media and also in the gas phase, where the effect of the medium is eliminated.Initially the lifetimes (τ) of the triplet states of indole and its derivatives were measured in rigid media (frozen solutions and polymer films) and they were several seconds [1]. On going to media with low viscosity, the lifetime of the triplet states sharply decreased (by several orders of magnitude). In liquid solutions at room temperature, the lifetime of the triplet states of indoles was measured for the first time in 1975 [2]. A value of τ = 12 µsec was obtained by flash photolysis for an aqueous solution of indole. For other indole derivatives in aqueous solutions, the value of ( measured by the same method was several tens of microseconds [2][3][4]. In a later paper [5], a value of τ ≈ 40 µsec was measured from analysis of the fluorescence kinetics of indole and its derivatives in aqueous solution.

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“…(16). We cannot discount the possibility of a small contribution of the I tryptophan anion (tryp-) (hmax --350 nm) (16). In addition, there is an intense infrared absorption.…”

Section: Resultsmentioning

confidence: 99%

“…Our pulse radiolysis experiments with 9.5 M LiCl at 6 K indicate the likelihood of a very small yield of eV,-relative to that of ei,-when measured on the minutes time scale at 2 K. Thus spectrum A with A , , , at 330, 564, and 620 nm seems to be satisfactorily accounted for by tryp' and evis-. (16). We cannot discount the possibility of a small contribution of the I tryptophan anion (tryp-) (hmax --350 nm) (16).…”

Section: Resultsmentioning

confidence: 99%

A study of trapped electrons in LiCl/D₂O and other aqueous glasses at temperatures down to 2 K by radiolysis, pulse radiolysis, photolysis, and stimulated luminescence

Klassen¹,

Teather²,

Kieffer³

1979

Can. J. Chem.

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Pulse radiolysis of 9.5 M LiCI/D20 glass at 6 K produces both types of trapped electrons, eYl,-and el,-, just as it does at 75 K. However, going from 75 K to 6 K increases the initial yield of el,-and decreases its decay rate, while the yield of e,,,-decreases and its decay rate increases. These results are attributed to fast trap-to-trap tunnelling of eVl,-from unrelaxed traps at 6 K and slower tunnelling from deeper traps at 75 K while the el,-traps seem to relax within 100 ns even at 6 K. In 12 M LiCI/D20 at 4-10 K the initial sl,-band with h,,, = 625 nm decays considerably over minutes revealing a stable band with h,,, = 695 nm. The stimulation spectrum and absorption spectrum of this stable band indicate a bound-free transition of 2.0 eV and a bound-bound transition of 1.8 eV. Similar measurements of e,,,-at 77 K indicate a bound-free transition of 2.6 eV and a bound-bound transition of 2.1 eV. Tryptophan was photolyzed in 9.5 M LiCI/D20 at 2 K to produce el,-.

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Flash Photolysis of Tryptophan and N‐acetyl‐l‐tryptophanamide; The Effect of Bromide on Transient Yields

Cited by 51 publications

References 22 publications

Measuring the rate of intramolecular contact formation in polypeptides

Measuring the rate of intramolecular contact formation in polypeptides

Nature of delayed luminescence of N-methylindole and indole in the gas phase

A study of trapped electrons in LiCl/D₂O and other aqueous glasses at temperatures down to 2 K by radiolysis, pulse radiolysis, photolysis, and stimulated luminescence

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Flash Photolysis of Tryptophan and N‐acetyl‐l‐tryptophanamide; The Effect of Bromide on Transient Yields

Cited by 51 publications

References 22 publications

Measuring the rate of intramolecular contact formation in polypeptides

Measuring the rate of intramolecular contact formation in polypeptides

Nature of delayed luminescence of N-methylindole and indole in the gas phase

A study of trapped electrons in LiCl/D2O and other aqueous glasses at temperatures down to 2 K by radiolysis, pulse radiolysis, photolysis, and stimulated luminescence

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A study of trapped electrons in LiCl/D₂O and other aqueous glasses at temperatures down to 2 K by radiolysis, pulse radiolysis, photolysis, and stimulated luminescence