Tryptophan Residues In Proteins Research Articles

Simultaneous determination of tyrosine and tryptophan residues in proteins by second-derivative spectroscopy

The mutual interference between the second-derivative bands of tyrosine and tryptophan in proteins has been evaluated in terms of the ratio r between two peak-to-peak distances. The r values have been found to be well related, although not linearly, to the tyrosine/tryptophan ratio in both model compound mixtures and proteins. A method for the simultaneous determination of the two major protein chromophores at neutral pH has been developed.

Direct recording of the initially excited and the solvent relaxed fluorescence emission spectra of tryptophan by phase sensitive detection of fluorescence.

Abstract Phase sensitive detection of fluorescence was used to directly record the initially excited and the solvent‐relaxed emission spectra of N‐acetyl‐L‐tryptophanamide in propylene glycol. Emission from the initially excited state was suppressed by adjusting the phase sensitive detector to be out of phase with the emission on the short wavelength side of the fluorescence spectrum. Then, the phase sensitive intensities revealed the emission spectrum of the solvent relaxed state. Similarly, the emission from the solvent relaxed state was suppressed by adjusting the detector to be out of phase with the emission on the long wavelength side of the spectrum, allowing the spectrum of the initially excited state to be directly recorded. Distinct emission spectra could be recorded when the solvent relaxation time was comparable to the fluorescence lifetime. At higher or lower temperatures, emission occurs predominantly from a single state, and suppression of the fluorescence signal at any arbitrary wavelength resulted in suppression of the entire emission. A simple theory is described which allows the spectral relaxation times to be estimated from the phase sensitive intensities. From this analysis we obtained an activation energy for spectral relaxation of 3 kcal/mol. This activation energy is smaller than that found for the temperature dependence of fluorescence depolarization, 7.8 kcal/mol. We attribute this difference to the smaller molecular motions required for spectral relaxation.The method of phase sensitive detection of fluorescence shows excellent resolving power and sensitivity, and this method should facilitate measurement of spectral relaxation around tryptophan residues in proteins.

Investigation of membrane structure using fluorescence quenching by spin-labels. A review of recent studies.

Fluorescence quenching is the loss of fluorescence intensity which is observed when a fluorescent molecule or group interacts with another molecule or group, called the quencher. By use of tryptophan residues of proteins, together with specific probe molecules, quenching can be applied to problems of biological and model membrane structure. Quenching interactions are short range (less than 50 A) so that structure on the scale of molecular dimensions can be examined. This review summarizes the recent applications of fluorescence quenching by spin (nitroxide)-labeled molecules to problems of membrane structure, including determination of the distance of membrane-bound molecules from the membrane surface, the strength of lipid-protein interactions and the strength of protein-protein interactions within membranes. The unique advantages and the limitations of this powerful method are examined.

Linear mapping of tryptophan residues in Vesiculovirus M and N proteins by partial chemical cleavage.

Nonlimit chemical cleavage at tryptophan residues of protein labeled at the amino terminus afforded a simple procedure for generating specific fragments and for mapping tryptophan positions. A comparison of the matrix (M) and nucleocapsid (N) proteins of four members of the Vesiculovirus group by this procedure suggests considerable conservation of tryptophan number and location in the four serotypes examined.

Use of fluorescent probes that form intramolecular excimers to monitor structural changes in model and biological membranes

1,3-dipyrenylpropane (PC3P) and bis(4-biphenylmethyl)ether, two molecules that form intramolecular excimers, were embedded in phospholipid vesicles and biological membranes to monitor dynamic properties of membrane lipids. Excimer formation was evaluated from determinations of excimer to monomer emission intensity ratios (ID/IM). ID/IM values of PC3P and bis(4-biphenylmethyl)ether were reduced when cholesterol was added to egg lecithin vesicles. PC3P was sensitive to the temperature-induced crystalline to liquid-crystalline phase transition in dimyristoyl phosphatidylcholine vesicles. For studies of cellular membranes, membranes, PC3P was used exclusively, because of the fluorescence of tryptophan residues of membrane proteins interferes with the responses bis(4-biphenylmethyl)ether. Microviscosities of membrane interiors were calculated from standard curves of IM/ID plotted against solvent viscosity. Microviscosity values of egg lecithin vesicles and biological membranes, especially those obtained with PC3P, were more than an order of magnitude lower than values obtained by other techniques. We concluded that the intramolecular process leading to the formation of the excimer is influenced differently in isotropic solvents than in anisotropic environments, such as lipid bilayers. Although distinguishable ID/IM ratios can be obtained for different biological membranes (mitochondrial, microsomal, and plasma membranes were studied), this parameter may be phenomenological and not simply related to membrane microviscosity. As such, fluorescent probes that form intramolecular excimers are of value in making qualitative comparisons of different membranes and in studying the relative effects of physical changes and chemical agents on membrane structure. These probes may also be valuable for studying structural anisotropy of biological membranes.

The rates of photodestruction of tryptophan residues in human and bovine ocular lens proteins

The relative rates of photodestruction of tryptophan residues in α-, β-, and γ-crystallin fractions from both human and bovine lenses have been measured. The destruction of tryptophan was monitored by observing loss of tryptophan fluorescence, changes in the UV absorption spectra, and changes in the near-UV CD spectra. Photolysis experiments using monochromatic 290 nm radiation were performed on air-equilibrated and vacuum-degassed 1·0 mg/ml protein solutions at 25°C. Both neutral aqueous buffer solutions and the denaturing solvents 8 m-urea and 5 m-guanidine hydrochloride were used in the photolysis studies. The observed rates of destruction of tryptophan residues in all three human crystallins were the same. Denaturing the human crystallin fractions with urea or guanidine hydrochloride did not significantly alter the photolysis rates. The same photolysis rates, and the same lack of effect of denaturation, were also observed for the bovine proteins. Vacuum degassing the lens protein solutions, prior to UV irradiation, resulted in reduction in the photolysis rates by a factor of about one-half.

State of aromatic amino acid residues in soybean 11S globulin heated in the presence of N-ethylmaleimide by derivative spectrophotometry.

The heating of 11S globulin in the presence of N-ethylmaleimide (NEM) caused changes in the intensities of the second derivative spectra, but scarcely affected the positions of the peaks and troughs.The difference-derivative absorption spectra indicated that half of tyrosine residues burried in native 11S globulin was exposed by heating, but tryptophan residues were hardly done. Furthermore, the tryptophanyl fluorescence spectra gave the same information as that obtained by the difference-derivative absorption spectra. The second derivative spectro-photofluorimetry may be used to discriminate between burried and exposed forms of tryptophan residues in protein depending on solvent polarity. These results suggested that the blocking of sulfhydryl residues on heating caused them to retain a hydrophobic region burried in the protein.

Nanosecond segmental mobilities of tryptophan residues in proteins observed by lifetime-resolved fluorescence anisotropies

Steady-state and lifetime-resolved fluorescence anisotropy measurements of protein fluorescence were used to investigate the depolarizing motions of tryptophan residues in proteins. Lifetime resolution was achieved by oxygen quenching. The proteins investigated were carbonic anhydrase, carboxypeptidase A, alpha-chymotrypsin, trypsin, pepsin, and bovine and human serum albumin. When corrected for overall protein rotation, the steady state anisotropies indicate that, on the average, the tryptophan residues in these proteins rotate 29 degrees +/- 6 degrees during the unquenched excited state lifetimes of these proteins, which range from 1.7 to 6.1 ns. The lifetime-resolved anisotropies reveal correlation times for these displacements ranging from 1 to 12 ns. On the average these correlation times are tenfold shorter than that expected for overall protein rotation. We conclude that the tryptophan residues in these proteins display remarkable freedom of motion within the protein matrix, which implies that these matrices are highly flexible on the nanosecond time scale.

Some regularities of dynamic accessibility of buried fluorescent residues to external quenchers in proteins

The temperature and viscosity dependences of quenching of buried tryptophan residues of several proteins by external ionic (iodide) and neutral (acrylamide) quenchers have been studied. The effective quenching rate constant is shown to be proportional to the diffusion coefficient of the free solvent (water or 20 vol% glycerol). This fact supports the idea that the accessibility of buried chromophores of proteins to quenchers is based on the dynamic perturbations in protein structure (the dynamic accessibility). These structural perturbations are assumed to be governed by some diffusion-limited processes in the solvent surrounding the protein molecule.

Phosphorescence and optically detected magnetic resonance studies of a class of anomalous tryptophan residues in globular proteins.

Phosphorescence and optically detected magnetic resonance studies of a class of anomalous tryptophan residues in globular proteins.

Study of reactivity of tryptophan residues in serum albumins and lysozyme by N-bromosuccinamide fluorescence quenching

The kinetics of quenching by N-bromosuccinamide of the fluorescence of tryptophan residues in various proteins and peptides were studied using the stopped-flow technique. Human serum albumin, which has a single tryptophan residue located in a hydrophobic fold, showed biphasic kinetics; one component was second order and the other first order, the rate for the latter component being independent of the concentration of quencher. Bovine serum albumin, which has in addition a tryptophan residue at the surface of the molecule, showed triphasic kinetics; two components corresponded to those for human serum albumin, and there was a faster second-order component resulting from the surface tryptophan. It is concluded that reaction with the buried tryptophan involves the initial second-order formation of a complex in which the quencher is located at the mouth of a hydrophobic fold, and that this is followed by a first-order conformational change which allows interaction to occur between the quencher and the tryptophan. The kinetics of lysozyme fluorescence quenching at pH 5.4 showed two relaxations whose rates were proportional to the N-bromosuccinamide concentration. The results of kinetic and titration experiments suggest that a molecule of lysozyme contains at least two groups of tryptophan residues of significantly different reactivities. The faster component probably reflects the bromination of the tryptophan residues at the active site. An octapeptide consisting of residues 22 to 29 of glucagon showed essentially the same quenching kinetics as glucagon itself, and Leu-Trp-Met showed the same behavior as Gly-Trp-Gly. The results indicate that quenching by N-bromosuccinamide provides a useful indication of the accessibility of tryptophan residues, and that side reactions do not significantly affect the kinetics.

A reinvestigation of chlortetracycline fluorescence: effect of pH, metal ions, and environment

The pH dependence of chlortetracycline fluorescence has been reinvestigated. Below pH 7.5, excitation at 400 nm results in an emission band at 530 nm. Above pH 7.5, in addition to the above band, a strong fluorescence is observed at 430 nm on excitation at 345 nm. p K a values of 3.5 and 7.7 have been determined for the 530- and 430 nm bands, permitting their assignment to the ring A and ring BCD chromophores, respectively. Both bands are shown to be sensitive to divalent metal binding. Excitation energy transfer from the 430-bband to the 530-nm band is observed in metal complexes, with transfer efficiency being greater for the Mg 2+ chelate than the Ca 2+ chelate. The effect of solvent and lipid on band intensities and transfer efficiencies is reported. The interaction of chlortetracycline with BSA in the presence of Mg 2+ leads to the observation of energy transfer from protein tryptophan residues to the 520-nm band of the complex via the intermediacy of the 430-nm band.

The identification of tryptophan residues in proteins as oxidised derivatives during amino acid sequence determinations

The identification of tryptophan residues in proteins as oxidised derivatives during amino acid sequence determinations

Effect of temperature on the fluorescence quenching by N-bromosuccinamide of tryptophan residues in proteins

The quenching of tryptophan fluorescence by N-bromosuccinamide was studied by the fluorescence stopped-flow technique over a temperature range of 5 to 35 °C in order to compare the reactivities of tryptophan residues in various molecules. The highest rates and lowest activation energies were found with the simple molecule N-acetyltryptophanamide. The hormone glucagon, which contains a single tryptophan residue, showed a somewhat lower rate and higher activation energy. α-Chymotrypsin, which has three groups of tryptophan residues in different environments, gave three rate constants which were lower than those with the other compounds, and the activation energies were correspondingly higher. There was a linear relationship between enthalpy of activation and the Gibbs energy of activation, the proportionality factor being 6.7. This indicates a ΔH≠ − ΔS≠ compensation, with an isokinetic temperature of 350 K. The compensation is discussed in the light of the steric effects.

The reactivity of tryptophan residues in proteins. Stopped-flow kinetics of fluorescence quenching

The quenching of tryptophan fluorescence by N-bromosuccinamide, studied by the fluorescence stopped-flow technique, was used to compare the reactivities of tryptophan residues in protein molecules. The reaction of N-bromosuccinamide with the indole group of N-acetyltryptophanamide, a model compound for bound tryptophan, followed second-order kinetics with a rate constant of (7.8 ± 0.8) · 10 5 dm 3 · mol −1 · s −1 at 23°C. The rate does not depend on the ionic strength or on the pH near neutrality. The non-fluorescent intermediate formed from N-acetyltryptophanamide on the reaction with N-bromosuccinamide appears to be a bromohydrin compound. The second-order rate constant for fluorescence quenching of tryptophan in Gly-Trp-Gly by N-bromosuccinamide was very similar, (8.8 ± 0.8) · 10 5 dm 3 · mol −1 · s −1. Apocytochrome c has the conformation of a random coil with the single tryptophan largely exposed to the solvent. The rate constant for the fluorescence quenching of the tryptophan in apocytochrome c by N-bromosuccinamide was (3.7 ± 0.3) · 10 5 dm 3 · mol −1 s −1. The fluorescence quenching by N-bromosuccinamide of the tryptophan residues incorporated in α-chymotrypsin at pH 7.0 showed three exponential terms from which the following rate constants were derived: 1.74 · 10 5, 0.56 · 10 5 and 0.11 · 10 5 dm 3 · mol −1 · s −1. This protein is known to have eight tryptophan residues in the native state, six residues at the surface, and two buried. Three of the surface tryptophans have the indole rings protruding out of the molecule and may account for the fastest kinetic phase of the quenching process. The intermediate phase may be due to three surface tryptophans whose indole rings point inwards, and the slowest to the two interior tryptophan residues.

Subnanosecond motions of tryptophan residues in proteins

The dynamics of protein molecules in the subnanosecond and nanosecond time range were investigated by time-resolved fluorescence polarization spectroscopy. Synchrotron radiation from a storage ring was used as a pulsed light source to excite the single tryptophan residue in a series of proteins. The full width at half maximum of the detected light pulse was 0.65 nsec, making it feasible to measure emission anisotropy kinetics in the subnanosecond time range and thereby to resolve internal rotational motions. The proteins investigated exhibit different degrees of rotational freedom of their tryptophan residue, ranging from almost no mobility to nearly complete freedom in the subnanosecond time range. The tryptophan residue of Staphylococcus aureus nuclease B (20,000 daltons) has a single rotational correlation time (varphi) of 9.9 nsec at 20 degrees C, corresponding to a rotation of the whole protein molecule. By contrast, bovine basic A1 myelin protein (18,000 daltons) exhibits varphi of 0.09 and 1.26 nsec, showing that the tryptophan residue in this protein is highly flexible. The single tryptophan of human serum albumin (69,000 daltons) has almost no rotational freedom at 8 degrees C (varphi = 31.4 nsec), whereas at 43 degrees C it rotates rapidly (varphi(1) = 0.14 nsec) within a cone of semiangle 26 degrees in addition to rotating together with the whole protein (varphi(2) = 14 nsec). Of particular interest in the large angular range (semiangle, 34 degrees ) and fast rate (varphi(1) = 0.51 nsec) of the rotational motion of the tryptophan residue in Pseudomonas aeruginosa azurin (14,000 daltons). This residue is known to be located in the hydrophobic interior of the protein. The observed amplitudes and rates of these internal motions of tryptophan residues suggest that elementary steps in functionally significant conformational changes may take place in the subnanosecond time range.

A spectroscopic probe of stacking interactions between nucleic acid bases and tryptophan residues of proteins.

The external heavy atom effect of mercury on the spectroscopic properties of the indole ring has been used to investigate stacking interactions of tryptophan with mercurinucleotides in mixed aggregates formed in frozen aqueous solutions as well as in oligopeptide-polynucleotide complexes. This effect is characterized at 77 K by a quenching of the tryptophan fluorescence, an enhancement of the phosphorescence emission and a drastic shortening of the phosphorescence lifetime. These phenomena result from an enhanced spin-orbit coupling due to a close contact between the mercury atom and the indole ring. Dissociation of the complexes leads to a recovery of the spectroscopic properties of the free tryptophan ring. The possible use of this spin-orbit probe to provide evidence for stacking interactions in protein-nucleic acid complexes is discussed.

Fluorescence spectra of tryptophan residues in human and bovine lens proteins

The water soluble proteins from human and bovine lenses have been studied using fluorescence spectroscopy. Excitation of aqueous solutions of α, β and γ-crystallins at 290 nm produced fluorescence emission due solely to tryptophan residues. No emission attributable to tyrosine or phenylalanine residues could be detected. The fluorescence emission maxima for all of the human and bovine crystallins in aqueous solution were in the range 332±2 nm, and the bandwidths were in the range 52±2 nm. These results indicate that the tryptophan residues in these proteins exist mainly in hydrophobic environments in aqueous solution. All of the human and bovine crystallin fractions could be denatured by addition of 8 m-urea, 5 m-guanidine hydrochloride, or 1% sodium dodecyl sulfate (SDS). The former two reagents shifted the fluorescence maxima into the range 349±2 nm, indicating that the majority of tryptophan residues are exposed to water in these solutions. Addition of SDS produced only a small spectral shift to 335±1 nm, but produced a marked spectral broadening to 59±1 nm, again indicating significant protein denaturation. The fluorescence data for lens crystallins have been compared with corresponding data for α-chymotrypsin in an effort to classify the lens crystallin data into discrete spectral classes based on specific fluorescence properties of three types of tryptophan residues.

Photochemical modifications of the tryptophan residues of wheat-germ agglutinin in the presence of trichloroethanol.

Trichloroethanol is an efficient quencher of indole fluorescence of model compounds and proteins [Eftink, M. R. and Ghiron, C. A. (1976) J. Phys. Chem. 80, 486--493]. At low quencher concentrations, the quenching follows the classical Stern-Volmer law. Bimolecular rate constants calculated from measured quenching constants and lifetimes are equal to 6 X 10(9) M-1s-1 and 1.2 X 10(9) M-1s-1 for N-acetyltrypotophanamide and wheat germ agglutinin, respectively. Upon ultraviolet irradiation in the presence of trichloroethanol, transformation of fluorescent tryptophan occurs, leading to a fluorescent photoproduct. This can be easily used as a method for the quantitative determination of fluorescent tryptophan residues in proteins. In good agreement with previous results, two fluorescent tryptophan residues per polypeptide chain are found in wheat germ agglutinin. Concomitantly with the photochemical reactions, the hemagglutinating protein activity and its affinity constant towards chitin oligomers are reduced. A probable location of tryptophan residues in the binding sites of wheat germ agglutinin is proposed.

An enzymatic approach to the labeling of tryptophan residues in peptides and proteins

An enzymatic approach to the labeling of tryptophan residues in peptides and proteins