Spectra aromatic amino acids

Precursors of phenylpropanoids are synthesized from two basic pathways the shikimic acid pathway and the malonic pathway (see Fig. 3.1). The shikimic acid pathway produces most plant phenolics, whereas the malonic pathway, which is an important source of phenolics in fungi and bacteria, is less significant in higher plants. The shikimate pathway converts simple carbohydrate precursors into the amino acids phenylalanine and tyrosine. The synthesis of an intermediate in this pathway, shikimic acid, is blocked by the broad-spectrum herbicide glyphosate (i.e., Roundup). Because animals do not possess this synthetic pathway, they have no way to synthesize the three aromatic amino acids (i.e., phenylalanine, tyrosine, and tryptophan), which are therefore essential nutrients in animal diets. 

Native fluorescence of a protein is due largely to the presence of the aromatic amino acids tryptophan and tyrosine. Tryptophan has an excitation maximum at 280 nm and emits at 340 to 350 nm. The amino acid composition of the target protein is one factor that determines if the direct measurement of a protein s native fluorescence is feasible. Another consideration is the protein s conformation, which directly affects its fluorescence spectrum. As the protein changes conformation, the emission maximum shifts to another wavelength. Thus, native fluorescence may be used to monitor protein unfolding or interactions. The conformation-dependent nature of native fluorescence results in measurements specific for the protein in a buffer system or pH. Consequently, protein denatur-ation may be used to generate more reproducible fluorescence measurements. 

In addition to the effect of mutations at Phe-82 on the stability of the cytochrome c active site, the intense, negative Soret Cotton effect in the circular dichroism spectrum of ferricytochrome c is profoundly affected by the presence of non-aromatic amino acid residues at this position [115]. Recent examination of six position-82 iso-l-ferricytochrome c mutants establishes that while Tyr-82 exhibits a Soret CD spectrum closely similar to that of the wild-type protein, the intensity of the negative Soret Cotton affect varies with the identity of the residue at this position in the order Phe > Tyr > Gly > Ser = Ala > Leu > He, though the Ser, Ala, He, and Leu variants have effectively no negative Soret Cotton effect [108]. 

PMR studies have been performed on a number of other ribosomal proteins isolated by the acetic acid/urea method (Morrison etal., 1977a). The results of these studies have shown that acedc acid/urea-extracted proteins contain little tertiary structure. However, some structure was seen in protein S4 and especially in protein S16 as indicated by the appearance of ring-current shifted resonances in the apolar region of the spectrum (Morrison et al., 1977b). These are due to the interaction of apolar methyl groups with aromatic amino acids in the tertiary structure of the protein. The PMR spectra were recorded either in water or in dilute phosphate buffer at pH 7.0—conditions under which the proteins were soluble. 

The helix contents of five peptide fragments from the protein thermolysin have been determined by CD and NMR in both water and 30% TFE. 85 The helix content was obtained from CD by the method of Chen et a I.162 and the NMR method utilized chemical shifts. 84 Four of the five peptides correspond to helical regions in the intact protein, and one corresponds to an Q-loop. 86 The rms difference between CD and NMR helix contents for the five peptides under the two conditions is 7.5%. One peptide shows the largest deviations (0 vs 13% in water, 45 vs 62% in 30% TFE). If it is excluded, the rms deviation decreases to 4%. The peptide showing the largest deviation, residues 258-276 from the thermolysin sequence, has two Tyr and one Phe (with the Phe adjacent to one of the Tyr in the sequence), and therefore it has an above-average content of aromatic amino acids, which can perturb both the CD spectrum and NMR chemical shifts. Of the other four peptides, three have a single aromatic residue and the fourth has two aromatics. 

Side chains of the three aromatic amino acids phenylalanine, tyrosine, and tryptophan absorb ultraviolet light in the 240- to 300-nm region, while histidine and cystine absorb to a lesser extent. Figure 3-13 shows the absorption spectrum of a "reference compound" for tyrosine. There are three major absorption bands, the first one at 275 nm being a contributor to the well-... 

Determination of protein concentration (unitbu) requires an absorbance spectrum to be recorded on a good quality spectrophotometer from 240 to 350 nm. Aromatic amino acid residues do not absorb above 320 nm, so the spectrum between 320 and 350 nm should be only marginally above baseline. The presence of turbidity will result in finite attenuance in this region that increases toward lower wavelengths. 

Second-order derivatives of the spectrum of Phe, Tyr, and Trp present characteristic absorption minima at 257, 280, and 290 nm. In addition, each aromatic amino acid has other minima of lower intensity 250 and 264 nm for Phe, 272 nm for Tyr, and 268 and 278 nm for Trp (81). In peptides composed only of Tyr and Phe, the spectral contribution of each aromatic amino acid to the derivative of the peptide spectrum can be distinguished quickly by the Phe (257 nm) and Tyr (278 nm) absorption minima. However, identification of Tyr in the presence of Trp is unclear,... 

If the side chains have strongly chromophoric groups near the backbone, such as in polymers of aromatic amino acids like poly(L-phenylalanine), poly(L-tyrosine), and poly(L-tryptophan), the CD spectrum is strongly dependent on the side chains and is totally different from the standard spectra of polypeptides lacking chromophoric groups in the side chains. This is due to the interactions between amide and aro-... 

Absorption spectrum of ai-acid glycoprotein displays two peaks at 225 and 278 nm (Figure 2.3). This feature is characteristic for all proteins. The peak at 278 nm originates from the three aromatic amino acids of the proteins, tyrosine, tryptophan, and phenylalanine. The e for a protein is generally calculated at 278 nm. 

Structural correlations on the basis of CD spectra provide good information about the stereochemistry of chiral molecules. The structure of (—)-tetrahydrobiopterin, the cofactor for hydroxyl-ations of aromatic amino acids, was determined by x-ray crystallographic analysis as (6R,l, 2 5)-6-(L -dihydroxypropyO-S J -tetrahydropterin (135). Its CD spectrum exhibits a negative Cotton... 

The CD spectra of peroxidases in the heme absorption region are highly sensitive to the conformation of the heme group inside the protein cavity and to the dipole-dipole coupling interactions between the porphyrin and the surrounding aromatic amino acid side chains [122, 123]. In this region, GS horseradish A2 peroxidase presents a band at 311 and the intense CD Soret band at 412 nm with a shoulder at 354 nm (Fig. 11.3). The main differences between the CD spectrum of CIII compared to that of GS are a reduction of the CD band at 311 nm and a red-shift of the Soret CD band to 430 nm with the disappearance of the 354 nm shoulder. Additionally a broad band, possibly composed by a series of shoulders, appears from 350 to 400 nm. During spontaneous decay, the CD spectrum of CIII slowly returns to that of GS, with the characteristic reduction of the Soret band due to porphyrin destruction (see above). 

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