Amino acids Tryptophan Trp

The aim of this Chapter is to review a method by which fluorescence properties of organic dyes can, in general, be predicted and understood at a microscopic (nm scale) by interfacing quantum methods with classical molecular dynamics (MD) methods. Some review of our extensive applications [1] of this method to the widely exploited intrinsic fluorescence probe in proteins, the amino acid tryptophan (Trp) will be followed by a discussion of electrochromic membrane voltagesensing dyes. 

While the far UV region has received the most attention in the study of protein CD, there are intrinsic chromophores in proteins which give nse to signals in the near U V (A. = 250-350 nm). These include the side chains of aromatic amino acids (tryptophan, Trp, tyrosine, Tyr, and phenylalanine, Phe) and the disulfide moiety of cystine. The exact position of these bands depends on the extent of exposure to the solvent, the solvent polarity and pH, and their proximity to other groups. The analysis of the near UV CD spectra of proteins has been reviewed [7, 8],... 

The three aromatic amino acids, tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe), are the only native amino acids with useful fluorescence properties. Figure la shows their absorption and fluorescence spectra. Their fluorescence properties are summarized in Table 1. Note that the relative absorption coefficients increase in the order Phe < Tyr < Trp. The fluorescence quantum yields increase in the same order. The product of absorption coefficient and fluorescence quantum yield can be taken as a measure of the brightness of the fluorophore. By the standards of fluorescent dyes (see below), the brightness of aU three amino acids is poor. Trp is the brightest of the three, and for proteins with a small number of Trp residues it may be possible to assign fluorescence decays to specific Trp residues. As a result, of the three fluorescent amino acids, Trp is by far the most widely exploited for its fluorescent properties. Fluorescence from Tyr is also detectable but may be masked by Trp fluorescence. Proteins often contain many Tyr residues, so it is often not possible to isolate the fluorescence from individual Tyr residues. Fluorescence from Phe is weak and not often used in fluorescence studies. 

Intrinsic protein fluorescence originates with the aromatic amino acids. tryptophan (trp), tyrosine (tyr). and phenylalanine (phe) (Figure 3.1). The indole groups of tryptophan... 

The aromatic amino acids - tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) - have strong deep-UV absorption bands (A, < 230 nm) corresponding to Sq —> S2 transitions, but commonly are excited to the Sj state in fluorescence studies (Agx 260-280 nm) to minimize photoreaction and enhance fluorescence quantum yields (f). At these longer wavelengths, Trp has the largest molar extinction coefficient ( max 5600) and quantum yield (Of 0.2) of the three amino acids for Phe, the values of and Of are so poor that this species is rarely useful in fluorescence studies. When subjected to UV irradiation, proteins with both Trp and Tyr (Figure 1) typically exhibit emission spectra whose shape is characteristic of Trp residues (A ,3x 50 nm) because of nonradiative energy transfer from Tyr to Trp. 

Transformation of B. subtilis cells is easily studied in the laboratory if the genes under observation lead to a readily selectable phenotype. For example, consider competent recipient cells that are auxotrophic for a required metabolite, such as the amino acid tryptophan that is, they have a genetic defect in a gene (trp gene) that specifies one of the... 

The physiological signal controlling the lac and ara operons is the utilization of carbon sources for metabolic energy. In contrast, the tryptophan trp) operon is sensitive to the need for biosynthetic processes and is transcribed under conditions where intracellular concentrations of the amino acid tryptophan are below an optimal level for efficient protein synthesis. The trp operon consists of a promoter and operator region which controls the expression of a polycistronic mRNA encoding five proteins needed for tryptophan biosynthesis. 

The E. coli tryptophan (trp) operon (Fig. 28-19) includes five genes for the enzymes required to convert chorismate to tryptophan. Note that two of the enzymes catalyze more than one step in the pathway. The mRNA from the trp operon has a half-life of only about 3 min, allowing the cell to respond rapidly to changing needs for this amino acid. The Trp repressor is a homodimer, each subunit containing 107 amino acid residues (Fig. 

The trp operon governs the production of enzymes responsible for the synthesis of the amino acid, tryptophan, and operates when an adequate supply is not available from the culture medium. Therefore, tryptophan acts to inhibit the transcription of synthetic enzymes. In addition to structural genes, trpA to trpE, and promoter and operator sites (Figure 17.10a), there are two regions called... 

Chymotrypsin shows a strong preference for hydrolyzing peptide bonds formed by the carboxyl groups of the aromatic amino acids, phenylalanine, tyrosine, and tryptophan. Flowever, over time chymotrypsin also hydrolyzes amide bonds involving amino acids other than Phe, Tyr, or Trp. Peptide bonds having leucine-donated carboxyls become particularly susceptible. Thus, the specificity... 

Amino acid abbreviations aa, generic amino acid Ala, L-alanine Aib, 2-aminoisobutyric acid Cys, L-cysteine Asp, L-aspartic acid Glu, L-glutamic acid Gly, glycine Lys, L-ly-sine Ser, L-serine Trp, L-tryptophan Ser, 2-amino-3-(5-methyl-2,4-dioxo-3,4-dihydro-2H-... 

The 3-amino-1 -mcthyl-5//-pyrido[4,3-b]indolc derivatives (31 Trp-P-1) and (32 Trp-P-2) were found as tryptophane pyrolysates in broiled fish and meat and in pyrolysates of protein and amino acids by Sugimura and coworkers198. These mutagens are heterocyclic amines and exhibit mutagenicity in the Ames test supplemented with S-9 mix198. The pyridoindole derivatives Trp-P-1 and Trp-P-2 are /V-hydroxylated at the exocyclic amino group to form proximate reactive compounds. 

The Lac operon is but one example of the genetic adaptations which allow bacteria to respond to their environment. Other examples are to be found in amino acid metabolism, for example the TRP operon which regulates tryptophan metabolism. 

Aryl side chain containing L-a-amino acids, such as phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp), are derived through the shikimate pathway. The enzymatic transformation of phosphoenolpyr-uvate (PEP) and erythro-4-phosphate, through a series of reactions, yields shikimate (Scheme 2). Although shikimate is an important biosynthetic intermediate for a number of secondary metabolites, this chapter only describes the conversion of shikimate to amino acids containing aryl side chains. In the second part of the biosynthesis, shikimate is converted into chorismate by the addition of PEP to the hydroxyl group at the C5 position. Chorismate is then transformed into prephenate by the enzyme chorismate mutase (Scheme 3). 

The code is degenerate. More than one codon can specify a single amino acid. All amino adds, except Met and tryptophan (Trp), have more than one codon. 

The reference 28 authors continue to detail experimental observations that place voltage sensor helices in positions within the membrane. Miller and coworkers conducted site-directed mutagenesis for all residues of helices Sl-S3. ° In these experiments, tryptophan (trp) residues were substituted for each amino acid in turn to determine which residues would be trp-tolerant. These experiments confirmed a-helical conformations for SI and S2 and showed that K+ channel function was altered when trp residues were placed in some (labeled non-trp-tolerant), but not all, positions. The same treatment for helix S3 yielded complex results. At S3 s N-terminal end the distribution of trp-tolerant positions were consistent with an a-helical structure, however, this was not the case at S3 s C-terminal end. Other tests indicated that S3 might be helical for its entire length and that the N-terminal end interfaces with both lipid and protein while the C-terminal end interfaces with water. Comparisons of trp-tolerant or trp-intolerant residues over several different Kv channel... 

Formation of the bis-histidyl-heme complex also produces characteristic alterations in the protein s conformation, particularly in the environment of aromatic amino acids, notably tryptophan. Exposure of Trp residues to solvent decreases (113), Trp fluorescence is quenched (111), and an unusual band of positive ellipticity at 230 nm attributable to Trp is nearly doubled in intensity (Fig. 4) (104, 111, 124). 

Fig. S6. Separation of D,L-dansyl amino acids. Conditions 0.65 oiAf L 2>isopropyl-dien-Zn(II) 0.17 A NH,Ac to pH 9.0 with aqueous NI 35/65 CH,CN/H,0 T - 30 flowrate 2 tnl/min column 15 cm by 4.6 mm i.d. S iun Hypersil C solutes CySO H -cysteic acid Ser - serine Trp - tryptophan thr - threonine Norval - norvaline Leu w leucine Norleu - norleucine Phe phenylalanine. Detection at 254 nm. Reprinted with permission from LePage ef at. C246), Am/. Chem. Copyright 1979 by the American Chemical Society.

Fio. 38. Plot of the algorithm of the retention factor, k, and log P, the water-/i>octanol partition coefficient of eight amino acids. The chromatographic data were obtained on 3 ftm LiChrosorb kP-8, 230 x 4.6 mm i.d. eluierit 0.1 M aqueous phosphate buffer, pH 6.7, T 70 C. Eluites Trp, tryptophan Phe, phenylalanine Leu, leudne Val. valine Tyr, tyrosine Lys, lysine Ala, alanine Gly, glycine. Reprinted with permission from Molnar and Horvith QOS). 

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