Tryptophan Phosphorescence from Proteins at Room Temperature

Vanderkooi, Jane M.

doi:10.1007/0-306-47059-4_3

Cited by 32 publications

(33 citation statements)

References 77 publications

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“…Following light absorption at 302 nm, whether the triplet excited state of tryptophan ( 3 Trp)41–46 was potentially involved in the observed photo‐reactions of sTNF‐R1 was investigated. This was done by evaluating the effects of additives, acrylamide and Na‐azide, quenchers of the triplet excited states of tryptophan 45, 47.…”

Section: Methodsmentioning

confidence: 99%

Light-induced aggregation of type I soluble tumor necrosis factor receptor

Roy

Mason

Schöneich

et al. 2009

Journal of Pharmaceutical Sciences

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Section: Methodsmentioning

confidence: 99%

Light-induced aggregation of type I soluble tumor necrosis factor receptor

Roy

Mason

Schöneich

et al. 2009

Journal of Pharmaceutical Sciences

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“…Further, in low-viscosity solutions bimolecular reactions may occur with solutes in the solvent (long range) or with solutes diffusing inside the macromolecule to the site of chromophores (4), both of which are often a cause of spurious reduction of s by trace impurities. Thus, s is a sensitive probe of the protein structure surrounding the chromophore reporting on the flexibility of the site (3,5), the proximity of indole to quenching side chains or prosthetic groups (6,7) and its accessibility to small solutes (4,8,9). These features have proven to exhibit an unparalleled sensitivity to the conformational state of the macromolecule with sable to monitor even subtle alterations of the native fold often undetected by ordinary spectroscopic techniques.…”

Section: Introductionmentioning

confidence: 97%

Intramolecular Quenching of Tryptophan Phosphorescence in Short Peptides and Proteins¶

Gonnelli

Strambini

2005

Photochem Photobiol

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The phosphorescence lifetime (tau) of tryptophan (Trp) residues in proteins in aqueous solutions at ambient temperature can vary several orders of magnitude depending on the flexibility of the local structure and the rate of intramolecular quenching reactions. For a more quantitative interpretation of tau in terms of the local protein structure, knowledge of all potential quenching moieties in proteins and of their reaction rates is required. The quenching effectiveness of each amino acid (X) side chain and of the peptide backbone was investigated by monitoring their intramolecular quenching rate (k(obs)) in tripeptides of the form acetyl-Trp-Gly-X-CONH2 (WGX), where Trp is joined to X by a flexible Gly link. The results indicate that among the various groups present in proteins only the side chains of Cys, His, Tyr and Phe are able to quench Trp phosphorescence at a detectable rate (k(obs) > 40 s(-1)), with the quenching effectiveness for rotationally unrestricted side chains ranking in the order Cys >> His+ > Tyr >> Phe approximately His. For the aromatic side chains the corresponding contact rate at 20 degrees C is estimated to be between 3-4 x 10(9) s(-1) for Cys (as determined by Lapidus et al.), 0.8-8 x 10(6) s(-1) for His+, 0.37-3.7 x 10(6) s(-1) for Tyr and 0.2-2 x 10(5) s(-1) for Phe and His. In the cases of His and Tyr, k(obs) drops sharply with increasing pH, with midpoint transitions about 1 pH unit above the pKa, indicating that quenching is almost exclusive to the protonated form. From the temperature dependence of the rate, obtained in 50/50 propylene glycol/water between -20 degrees C and 20 degrees C, the reaction is characterized by activation energies of about 5 kcal.M(-1) for His+ and Tyr and 8 kcal.M(-1) for Phe. An analysis of the groups in contact with Trp residues in proteins that exhibit long phosphorescence lifetimes at ambient temperature leads to the conclusion that the contact rate of the peptide group and of the remaining side chains is lower than 0.1 s(-1), showing that these moieties are practically inert with respect to the triplet-state lifetime. It shows further that the immobilization of the aromatic side chains within the globular fold cuts their quenching effectiveness drastically to contact rates < 2 s(-1), a phenomenon attributed to the low probability of forming a stacked exciplex with the indole ring. All evidence suggests that, except in the case of nearby Cys or Trp residues, whose interaction with the triplet state reaches beyond van der Waals contact, the emission of buried Trp residues is unlikely to be quenched by surrounding protein groups.

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“…In comparison, when these macromolecules (or the free chromophores) are embedded in rigid matrices, such as glycol/water glasses, τ assumes a uniform value between 5 and 7 s. In moving from rigid to fluid solutions the main contributions to the shortening of τ are from the enhancement of radiationless transitions from the excited triplet state, determined by the increase in protein/solvent mobility (1, 2), plus the onset of intramolecular quenching reactions by local protein moieties (3). Further, in low‐viscosity solutions bimolecular reactions may occur with solutes in the solvent (long range) or with solutes diffusing inside the macromolecule to the site of chromophores (4), both of which are often a cause of spurious reduction of τ by trace impurities. Thus, τ is a sensitive probe of the protein structure surrounding the chromophore reporting on the flexibility of the site (3, 5), the proximity of indole to quenching side chains or prosthetic groups (6, 7) and its accessibility to small solutes (4, 8, 9).…”

Section: Introductionmentioning

confidence: 99%

Intramolecular Quenching of Tryptophan Phosphorescence in Short Peptides and Proteins^¶

Gonnelli

Strambini

2005

Photochem & Photobiology

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The phosphorescence lifetime (τ) of tryptophan (Trp) residues in proteins in aqueous solutions at ambient temperature can vary several orders of magnitude depending on the flexibility of the local structure and the rate of intramolecular quenching reactions. For a more quantitative interpretation of τ in terms of the local protein structure, knowledge of all potential quenching moieties in proteins and of their reaction rates is required. The quenching effectiveness of each amino acid (X) side chain and of the peptide backbone was investigated by monitoring their intramolecular quenching rate (kobs) in tripeptides of the form acetyl‐Trp‐Gly‐X‐CONH2 (WGX), where Trp is joined to × by a flexible Gly link. The results indicate that among the various groups present in proteins only the side chains of Cys, His, Tyr and Phe are able to quench Trp phosphorescence at a detectable rate (kobs > 40 s−1), with the quenching effectiveness for rotationally unrestricted side chains ranking in the order Cys >> His+ > Tyr >> Phe ∼ His. For the aromatic side chains the corresponding contact rate at 20°C is estimated to be between 3‐4 × 109 s−1 for Cys (as determined by Lapidus et al.), 0.8‐8 × 106 s−1 for His+, 0.37‐3.7 × 106 s−1 for Tyr and 0.2‐2 × 105 s−1 for Phe and His. In the cases of His and Tyr, kobs drops sharply with increasing pH, with midpoint transitions about 1 pH unit above the pKa, indicating that quenching is almost exclusive to the protonated form. From the temperature dependence of the rate, obtained in 50/50 propylene glycol/water between −20°C and 20°C, the reaction is characterized by activation energies of about 5 kcal·M−1 for His+ and Tyr and 8 kcal ·M−1 for Phe. An analysis of the groups in contact with Trp residues in proteins that exhibit long phosphorescence lifetimes at ambient temperature leads to the conclusion that the contact rate of the peptide group and of the remaining side chains is lower than 0.1 s−1, showing that these moieties are practically inert with respect to the triplet‐state lifetime. It shows further that the immobilization of the aromatic side chains within the globular fold cuts their quenching effectiveness drastically to contact rates < 2 s−1, a phenomenon attributed to the low probability of forming a stacked exciplex with the indole ring. All evidence suggests that, except in the case of nearby Cys or Trp residues, whose interaction with the triplet state reaches beyond van der Waals contact, the emission of buried Trp residues is unlikely to be quenched by surrounding protein groups.

show abstract

Tryptophan Phosphorescence from Proteins at Room Temperature

Cited by 32 publications

References 77 publications

Light-induced aggregation of type I soluble tumor necrosis factor receptor

Light-induced aggregation of type I soluble tumor necrosis factor receptor

Intramolecular Quenching of Tryptophan Phosphorescence in Short Peptides and Proteins¶

Intramolecular Quenching of Tryptophan Phosphorescence in Short Peptides and Proteins^¶

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