Tryptophan in proteins

Protein Hydrolysis. Acid hydrolysis of protein by 6 MHQ in a sealed tube is generally used (110°C, 24-h). During hydrolysis, slight decomposition takes place in serine (ca 10%) and threonine (ca 5%). Cystine and tryptophan in protein cannot be deterruined by this method because of complete decomposition. 

The above discussion sets the stage for the type of QM-MM studies we have performed for tryptophan in proteins. The use of MD simulations to study membranes is now a mature field, and has recently been reviewed in the context of the present book by Demchenko and Yesylevskyy [82]. A number of questions might be answered regarding details of the mechanism of voltage-sensitive dyes by... 

Broos J, Tveen-Jensen K, de Waal E, Hesp BH, Jackson JB, Canters GW, Callis PR (2007) The emitting state of tryptophan in proteins with highly blue-shifted fluorescence. Angew Chem Int Ed Engl 46(27) 5137-5139... 

Callis PR, Liu T (2004) Quantitative prediction of fluorescence quantum yields for tryptophan in proteins. J Phys Chem B 108 4248 1259... 

Callis PR, Vivian JT (2003) Understanding the variable fluorescence quantum yield of tryptophan in proteins using QM-MM simulations. Quenching by charge transfer to the peptide backbone. Chem Phys Lett 369 409-414... 

Fluorescent probes can be divided into three classes (i) intrinsic probes-, (ii) extrinsic covalently bound probes and (iii) extrinsic associating probes. Intrinsic probes are ideal but there are only a few examples (e.g. tryptophan in proteins). The advantage of covalently bound probes over the extrinsic associating probes is that the location of the former is known. There are various examples of probes covalently... 

Formation of specific complexes in the excited states ( exciplexes )f 35 52 85) Exciplexes are complexes not present in the ground state that form due to the extensive redistribution of electron density that occurs upon excitation. Among exciplexes, there may be some whose formation does not require substantial nuclear rearrangements and thus occurs rather rapidly even at 77 K. The formation of exciplexes is accompanied by a spectral shift to longer wavelengths. It is postulated that the fluorescence from tryptophan in proteins in a variety of cases is fluorescence from tryptophan exciplexes)35 85) In studies of the effects of environmental dynamics on the spectra, the exciplexes may be considered as individual fluorophores. 

F. Tanaka and N. Mataga, Fluorescence quenching dynamics of tryptophan in proteins. Effect of internal rotation under potential barrier, Biophys. J. 51, 487-495 (1987). 

Differences between the spectra of fluorescence and phosphorescence are immediately obvious. For all tryptophans in proteins the phosphorescence spectrum, even at room temperature, is structured, while the fluorescence emission is not. (Even at low temperatures the fluorescence emission spectrum is usually not structured. The notable exceptions include a-amylase and aldolase, 26 protease, azurin 27,28 and ribonuclease 7, staphylococcal endonuclease, elastase, tobacco mosaic virus coat protein, and Drosophila alcohol dehydrogenase 12. )... 

Phosphorescence is readily detectable from most types of proteins at room temperature. Tryptophan phosphorescence lifetimes and yields are very sensitive to environment, and therefore phosphorescence is sensitive to conformational changes in proteins. Fundamental questions concerning exactly what parameters affect lifetime and spectra of tryptophan in proteins remain still to be answered. 

If a collisional quencher of the fluorophore is also incorporated into the membrane, the lifetime will be shortened. The time resolution of the fluorescence anisotropy decay is then increased,(63) providing the collisional quenching itself does not alter the anisotropy decay. If the latter condition does not hold, this will be indicated by an inability to simultaneously fit the data measured at several different quencher concentrations to a single anisotropy decay process. This method has so far been applied to the case of tryptophans in proteins(63) but could potentially be extended to lipid-bound fluorophores in membranes. If the quencher distribution in the membrane differed from that of the fluorophore, it would also be possible to extract information on selected populations of fluorophores possibly locating in different membrane environments. 

Triazoles have been used extensively in analytical chemistry. Nitron (182) has been used for the determination of boron, rhenium, and tungsten. Nitron has been bonded to a polymer and used for the removal of nitrate from water <79Mi 402-oi >. Other triazoles have been used for the spectrophoto-metric assay of cobalt, rhodium, and platinum <68Mi 402-01,72ZAK2209). Hydroxyphenylazotriazoles form brilliant lakes with a number of metal ions <73AJC1585>. 5-" C-3-Diazotriazole couples only to tryptophane below pH 6.8 and can used for the specific determination of tryptophane in proteins <84M1 402-02). 

The constant K is known as the Stem-Volmer quenching constant /cQ is the rate constant for the quenching reaction, and t0 the lifetime in the absence of quencher. Fluorescence quenching of tryptophan in proteins by acrylamide or 02 has been used to determine whether tryptophan side chains are accessible to solvent or are "buried" in the protein.141 142 The long-lived phosphorescence of tryptophan can be studied in a similar... 

Burstein et al. (1973) classified tryptophan in proteins into three categories, according to the position of their fluorescence maximum (Xrnax) and the bandwidth (AX) of their spectrum ... 

Experiment 1. Quantitative Determination of Tryptophan in Proteins in 6 M Guanidine... 

Kierdaszuk, B., Gryc27nski, I , Modrak Wojcik, A., Bzowska, A., Shugar, D , and Lakowicz, J. R. (1995) fluorescence of tyrosine and tryptophan in proteins using one- and two-photon excitation. Photochemistry and Photobioiogy. 61 319-324. 

In the titration (Table XlXa) and cleavage (Table XlXb) experiments both NBS and iV-bromoacetamide (NBA) have been used. The oxidation by NBS of tryptophan in proteins and peptides, as judged from spectral changes, is almost always instantaneous during gradual addition, while with NBA, within the ratio of 2 moles to one of tryptophan, as long as 20 min are needed before the absorbance at 280 m/i reaches a steady minimum. 

Holiday, E. R. Spectrophotometry of Proteins. I. Absorption Spectra of Tyrosine, Tryptophan and their Mixtures. II. Estimation of Tyrosine and Tryptophan in Proteins. Biochem. J. 30, 1795 (1936). 

Schafer K, Determination of the amino acid tryptophan in protein fibres . Journal of the Society of Dyers and Colourists, 1997, 113, 275-280. 

Total internal reflection fluorescence spectroscopy has been used to assay the fluorescence of tryptophan in proteins or of fluorescence markers. Morphine has been determined in this manner with a detection limit of 0.2 pmol/l on a quartz support bearing immobilized fluorescein-labeled antihapten (Kronick and Little, 1973). 

Fluorescence of tryptophans in proteins also decayed with non-exponential functions, even though the proteins contained single tryptophan. The concept of rotamer was applied to explain the non-exponential decay profiles observed in proteins. 

This explanation for the origin of the non-exponential decays of tryptophan fluorescence, however, cannot rationalize the results of time-resolved fluorescence anisotropy in these proteins. Beec-hem and Brand review the time-resolved fluorescence of tryptophans in proteins (32). The following conclusions concerning time-resolved fluorescence can be derived i) fluorescence decays with at least two-lifetime components, when internal rotation of tryptophan is observed from the time-resolved fluorescence anisotropy, ii) fluorescence decays... 

To explain the above behaviors of the fluorescence decay function and time-resolved fluorescence anisotropy of single tryptophan in proteins, a model was proposed (33), in which an angular-dependent quenching constant was introduced into a rotational analogue of Smoluchowski equation given by Favro (34) for the internal rotation of tryptophan, as expressed by [1]. Rotational motion of tryptophan in a spherical macromolecule is illustrated in Fig.9. 

The study of fluorescence depolarization of tryptophans in proteins is likely to develop into a powerful tool for analyzing the internal motions.4593 Clarification of the photophysics of tryptophans by jet measurements,460 extension of the time scale of observation into the picosecond and femtosecond range,461 and utilization of normal or modified proteins with only a single tryptophan462 should lead to a significant increase in the utility of this experimental approach. 

S Tryptophan, which offen occurs af only one or a few places in a protein, is a useful fluorescent probe for study of protein dynamics. The optical properties of 7-azatrypfophan, 2-azotrypfophan, and 5-hydrox-ytryptophan are even better because their absorption maxima occur at longer wavelengths. These amino acids can be biosynthetically introduced in place of tryptophan in proteins. maximum fluores-... 

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