Tryptophan, protein emission

The width of the 0-0 line in single-tryptophan proteins at 77 K has been interpreted to reflect inhomogeneous broadening arising because the protein exists as a distribution of conformations. 30 34 The width of the 0-0 band of liver alcohol dehydrogenase is 500 cm-1 at 22°C.(10 31 35) The widths of the 0-0 transition for other proteins are somewhat greater. In many cases for the spectra taken at room temperature, low-resolution optics were used (as in Figure 3.2), and hence the published spectra may overestimate the width of the emission band. 

For single-tryptophan proteins there is some correlation between blue-shifted fluorescence emission maximum and phosphorescence lifetime (Table 3.2). Another correlation is that three of the proteins which exhibit phosphorescence, azurin, protease (subtilisin Carlsberg), and ribonuclease Tlt are reported to show resolved fluorescence emission at 77 K. Both blue-shifted emission spectra and resolved spectra are characteristic of indole in a hydrocarbon-like matrix. 

Table 3.2. Correlation between Fluorescence Emission Maximum and Phosphorescence Lifetime in Single-Tryptophan Proteins 1...

Mazhul et alP have reported that long-lived luminescence could be detected in intact human erythrocytes and white blood cells at ambient temperature. They have shown by emission spectra and pH dependency that this emission arises from tryptophan. The emission was not singleexponential, suggesting that more than one population of tryptophan emitted. Identification of the emitting species has not yet been conclusively made, but the white blood cell protein content is about 10% actin, a protein known to phosphorescence.(91)... 

Since protein emission spectra are generally rather broad, larger emission bandwidths can usually be tolerated. Only where it is important to resolve tyrosine fluorescence from tryptophan fluorescence and the Rayleigh scattering peak is it necessary to minimize the bandwidth. In cases where only low concentrations of material are available, it is necessary to strike a balance between resolution and light intensity in order to obtain the best possible signal-to-noise ratio. Settings of 2.5 to 10 nm are normal. 

In general, binding of the peptide to a protein will result in a shift in the tryptophan fluorescence emission maximum from approximately 355 nm (free peptide) to shorter wavelength as the tryptophan enters a more hydrophobic environment, and an overall intensification of the fluorescence emission intensity. The extent of the wavelength shift gives some information about the environment of the tryptophan in the complex. More importantly, the fluorescence enhancement on binding of the peptide may be used to determine the dissociation constant (K ) at the low protein/peptide concentrations required to study high affinity interactions. 

The comparison of the fluorescence spectra of p-lactoglobulin in an aqueous solution and in 50% ethanol (v/v) (not shown) demonstrates that the maximum of the tryptophan fluorescence emission is shifted from 332 nm to 338 nm, respectively. Additionally, a concomitant increase in the maximum fluorescence intensity may be observed. Red shift of the emission maximum implies that under the influence of alcohol the tryptophan residues, which in aqueous solutions are sheltered in the hydrophobic interior of a protein molecule [77], become more exposed to a polar environment. 

Intrinsic fluorescence is relatively rare in biological molecules. Most of the naturally occurring nucleic acids, carbohydrates and lipids show little or no useful fluorescence in the normal UV/visible region. In proteins, fluorescence can only be seen from tryptophan residues and, to a lesser extent, tyrosine side chains. This intrinsic protein fluorescence can be used in a number of practical applications. Typical Trp and protein emission spectra are shown in Figures 2.24 and 2.25. Some proteins contain intrinsically fluorescent prosthetic groups such as reduced pyridine nucleotides and flavoproteins, and the chlorophylls from green plants show a red fluorescence emission. 

Even in proteins that contain tyrosine but no tryptophan residues, emission of tyrosine residues occurs at very low quantum yield compared to free tyrosine. For example, in insulin that contains 4 tyrosines but no tryptophan, the fluorescence quantum yield is 0.037 at pH 7. The strong quenching of tyrosine at pH 7 was interpreted as coming from the fact that most of the tyrosine hydroxyl groups are involved in strong hydrogen bonds (Truong et al. 1967). 

X-r dTTraction studies (Mcsserschmidt et al. 1989 1992) have shown that six tryptophan residues / tnonomcr are completely shielded from the solvent, six others are buried but not completely and two are exposed to the solvent. Figure 3.42 displays the position of the emission peak of protein emission with excitation wavelength, in absence and presence of 1.3 M CsCi. 

Human immunoefficiency virus (HIV) is the agent responsible for the acquired immunodeficiency syndrom (AIDS). HIV-1 protease is among the targets identified for chemotherapy. It is a homodimeric aspartyl protease of 99 amino acid residues per monomer. The protein contains two Trp residues (W6 and W42) per monomer located in different environment. The fluorescence emission spectrum of the protein shows a peak at 341 nm 215 nm) indicating that the Trp residues are responsible for the protein emission. Also, tlie position of this peak is blue-shifted compared to the fluorescence maximum of Tryptophan in water and thus the average exposure of the Trp residues to the solvent is not total. Trp residues are partly buried within the hydrophobic core of the protein. 

The intrinsic tryptophan fluorescence emission spectra of Sac7 and Sso7 (5 p, Af protein in 10 mM KH2PO4, pH 6.8) are obtained with excitation at 295 nm using 4 nm excitation and emission slit widths. Excitation at 295 nm prevents contributions from the two tyrosine and two phenylalanine residues. An emission maximum at 350 nm is similar to that of free tryptophan and indicates significant solvent exposure, consistent with the NMR solution structure. Addition of double-stranded DNA [e.g., duplex poly[d(GC)]] leads to quenching of tryptophan fluorescence by nearly 90%. A blue shift of the emission maximum to 340 nm is also observed. 

The conformation of bovine myelin basic protein (MBP) in AOT/isooctane/water reversed micellar systems was studied by Waks et al. 67). This MBP is an extrinsic water soluble protein which attains an extended conformation in aqueous solution 68 but is more density packed at the membrane surface. The solubilization of MBP in the AOT reversed micelles depends on the water/AOT-ratio w0 68). The maximum of solubilization was observed at a w0-value as low as 5.56. The same value was obtained for another major protein component of myelin, the Folch-Pi proteolipid 69). According to fluorescence emission spectra of MBP, accessibility of the single tryptophane residue seems to be decreased in AOT reversed micelles. From CD-spectra one can conclude that there is a higher conformational rigidity in reversed micelles and a more ordered aqueous environment. 

Additional evidence for conformational changes in the transporter has come from measurement of the intrinsic fluorescence of the protein tryptophan residues, of which there are six, in the presence of substrates and inhibitors of transport. The fluorescence emission spectrum of the transporter has a maximum at about 336 nm, indicating the presence of tryptophan residues in both non-polar environments (which would emit maximally at about 330 nm) and in polar environments (which would emit at 340-350 nm) [154], The extent of quenching by the hydrophilic quencher KI indicates that more than 75% of the fluorescence is not available for quenching, and so probably stems from tryptophan residues buried within the hydrophobic interior of the protein or lipid bilayer [155]. Fluorescence is quenched... 

Fluorescent probes are divided in two categories, i.e., intrinsic and extrinsic probes. Tryptophan is the most widely used intrinsic probe. The absorption spectrum, centered at 280 nm, displays two overlapping absorbance transitions. In contrast, the fluorescence emission spectrum is broad and is characterized by a large Stokes shift, which varies with the polarity of the environment. The fluorescence emission peak is at about 350 nm in water but the peak shifts to about 315 nm in nonpolar media, such as within the hydrophobic core of folded proteins. Vitamin A, located in milk fat globules, may be used as an intrinsic probe to follow, for example, the changes of triglyceride physical state as a function of temperature [20]. Extrinsic probes are used to characterize molecular events when intrinsic fluorophores are absent or are so numerous that the interpretation of the data becomes ambiguous. Extrinsic probes may also be used to obtain additional or complementary information from a specific macromolecular domain or from an oil water interface. 

The six major proteins of milk, asl-, o s2-, and /c-casein, jS-lactoglobulin, and a-lactalbumin, contain at least one tryptophan residue [57], the fluorescence of which allows the monitoring of the structural modifications of proteins and their physicochemical environment during the coagulation processes. Emission fluorescence spectra of the protein tryptophanyl residues were recorded for the milk coagulation kinetics induced by... 

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