Interactions of tryptophan, tryptophan peptides, and tryptophan alkyl esters at curved membrane interfaces

Motivated by ongoing efforts to understand the mechanism of membrane protein crystallogenesis and transport in the lipidic cubic phase, the nature of the interaction between tryptophan and the bilayer/aqueous interface of the cubic phase has been investigated. The association was quantified by partitioning measurements that enabled the free energy of interaction to be determined. Temperature-dependent partitioning was used to parse the association free energy change into its enthalpic and entropic components. As has been observed with tryptophan derivatives interacting with glycerophospholipid bilayers in vesicles, tryptophan partitioning in the cubic phase is enthalpy driven. This is in contrast to partitioning into apolar solvents, which exhibits the classic hydrophobic effect whose hallmark is a favorable entropy change. These results with tryptophan are somewhat surprising given the simplicity, homogeneity, and curvature of the interface that prevails in the case of the cubic phase. Nevertheless, the interaction between tryptophan and the mesophase is very slight as revealed by its low partition coefficient. Additional evidence in support of the interaction was obtained by electronic absorption and fluorescence spectroscopy and fluorescence quenching. Partitioning proved insensitive to the lipid composition of the membrane, examined by doping with glycerophospholipids. However, the interaction could be manipulated in meaningful ways by the inclusion in the aqueous medium of salt, glycerol, or urea. The effects seen with tryptophan were amplified rationally when measurements were repeated using tryptophan alkyl esters and with tryptophan peptides of increasing length. These findings are interpreted in the context of the insertion, folding, and function of proteins in membranes.     

Free tryptophan pool and tryptophan biosynthetic enzymes in Saccharomyces cerevisiae

The free tryptophan pool and the levels of two enzymes of tryptophan biosynthesis (anthranilate synthase and indoleglycerolphosphate synthase) have been determined in a wild type strain of Saccharomyces cerevisiae and in mutants with altered regulatory properties.The tryptophan pool of wild type cells growing in minimal medium is 0.07 mole per g dry weight. Addition of anthranilate, indole or tryptophan to the medium produces a fifteen- to forty-fold increase in tryptophan pool, but causes no repression of the biosynthetic enzymes. Inclusion of 5-methyltryptophan in the growth medium causes a reduction in growth rate and a derepression of the biosynthetic enzymes, and this is shown here not to be correlated with a decrease in the free tryptophan pool.Mutants with an altered anthranilate synthase showing decreased sensitivity to inhibition by l-tryptophan or by the analogue dl-5-methyltryptophan have a tryptophan pool far higher than the wild type strain, but no repression of indoleglycerolphosphate synthase was observed. Mutants with an anthranilate synthase more sensitive to tryptophan inhibition show a slightly reduced tryptophan pool, but no derepression of indoleglycerolphosphate synthase was found.A mutant with constitutively derepressed levels of the biosynthetic enzymes shows a considerably increased tryptophan pool. Addition of 5-methyltryptophan to the growth medium of non-derepressible mutants causes a decrease in growth rate accompanied by a decrease in the tryptophan pool.Abbreviations CDRP 1-(o-carboxyphenylamino)-1-deoxyribulosephosphate - paba paraaminobenzoic acid - PRA N-(5-phosphoribosyl)-anthranilate - tRNA transfer ribonucleic acid; trp1 to trp5 refer to the structural genes for corresponding tryptophan biosynthetic enzymes     

Post-translational isoprenylation of tryptophan

Bacillus subtilis and related bacilli produce a post-translationally modified oligopeptide, ComX pheromone, that stimulates natural genetic competence controlled by quorum sensing. The ComX pheromones are formed by geranylation or farnesylation on a tryptophan residue at the 3 position of its indole ring. This results in the formation of a tricyclic structure including, a newly formed five-membered ring, similar to proline. Isoprenylation of ComX to form ComX pheromones is essential for pheromonal activity, and is functionally more crucial than its amino acid sequence. The ComX pheromone is the first example of isoprenoidal modifiations of tryptophan residues in living organisms and post-translational isoprenylation of any amino acid in prokaryotes. Because the presence of geranylated compounds is unusual in primary and secondary metabolites outside the plant kingdom, post-translational geranylation in bacilli is unprecedented in nature.     

Tryptophan catabolism by tryptophan pyrrolase in rat liver. The effect of tryptophan loads and changes in tryptophan pyrrolase activity.

We investigated how changes in tryptophan pyrrolase activity and tryptophan loads affect the breakdown of tryptophan was estimated by injecting rats with [ring-2-14-C]tryptophan and measuring respiratory 14-CO2. We concluded, contrary to previous reports, that induction of tryptophan pyrrolase definitely will increase the rate of tryptophan breakdown. Tryptophan loads also increase tryptophan breakdown even in circumstances where there is no increase in tryptophan pyrrolase activity, presumably by increasing the saturation of the enzyme. After a tryptophan load (50 mg per kg) the increase in liver tryptophan concentration lasts only 30 min. The rapid return of liver tryptophan to normal may be due partly to the high turnover rate of liver tryptophan. We estimate that tryptophan pyrrolase degrades tryptophan in vivo at a rate that is equivalent to the whole liver tryptophan concentration in 7.5 min or less.     

Characterization of tryptophan and coenzyme luminescence in tryptophan synthase from Salmonella typhimurium.

Tryptophan synthase from Salmonella typhimurium is a bifunctional alpha 2 beta 2 complex that catalyzes the formation of L-tryptophan. We have characterized over the temperature range from 160 to 293 K the fluorescence and phosphorescence properties of the single tryptophan present at position 177 of the beta-subunit and of the pyridoxal 5'-phosphate bound through a Schiff's base in the beta-active site. The comparison between the fluorescence of the pyridoxal phosphate bound either to the protein or to valine free in solution indicates substantial protection for the coenzyme against thermal quenching and a greater intensity of the ketoenamine tautomer band. Trp-177 is highly luminescent, and its proximity to the pyridoxal moiety leads to an over 50% quenching of its fluorescence with both reduced and native coenzyme. The Trp phosphorescence spectrum possesses a narrow, well-defined, 0-0 vibrational band centered at 418.5 nm, a wavelength that indicates strong polar interactions with neighboring charges. The observation of delayed fluorescence in the native complex implies that the excited triplet state is involved in a process of triplet-singlet energy transfer to the ketoenamine tautomer. The rate of energy transfer, heterogeneous in low-temperature glasses with rate constants of 2.26 and 0.07 s-1, becomes homogeneous in fluid solutions as the coenzyme tautomer interconversion is likely faster than the phosphorescence decay. In both apo- and holo-alpha 2 beta 2, the phosphorescence from Trp-177 is long-lived even at ambient temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of tryptophan and tryptophan residues in proteins by reactive nitrogen species.

Formation of 3-nitrotyrosine by the reaction between reactive nitrogen species (RNS) and tyrosine residues in proteins has been analyzed extensively and it is used widely as a biomarker of pathophysiological and physiological conditions mediated by RNS. In contrast, few studies on the nitration of tryptophan have been reported. This review provides an overview of the studies on tryptophan modifications by RNS and points out the possible importance of its modification in pathophysiological and physiological conditions. Free tryptophan can be modified to several nitrated products (1-, 4-, 5-, 6-, and 7-), 1-N-nitroso product, and several oxidized products by reaction with various RNS, depending on the conditions used. Among them, 1-N-nitrosotryptophan and 6-nitrotryptophan (6-NO(2)Trp) have been found as the abundant products in the reaction with peroxynitrite, and 6-NO(2)Trp has been the most abundant product in the reaction with the peroxidase/hydrogen peroxide/nitrite systems. 6-NO(2)Trp has also been observed as the most abundant nitrated product of the reactions between peroxynitrite or myeloperoxidase/hydrogen peroxide/nitrite and tryptophan residues both in human Cu,Zn-superoxide dismutase and in bovine serum albumin, as well as the reaction of peroxynitrite with myoglobin and hemoglobin. Several oxidized products have also been identified in the modified Cu,Zn-SOD. However, no 1-N-nitrosotryptophan and 1-N-nitrotryptophan has been observed in the proteins reacted with peroxynitrite or the myeloperoxidase/H(2)O(2)/nitrite system. The modification of tryptophan residues in proteins may occur at a more limited number of sites in vivo than that of tyrosine residues, since tryptophan residues are more buried inside proteins and exist less frequently in proteins, generally. However, surface-exposed tryptophan residues tend to participate in the interaction with the other molecules, therefore the modification of those tryptophans may result in modulation of the specific interaction of proteins and enzymes with other molecules.     

The photodecomposition of tryptophan peptides

Aqueous solutions of the four trytophan peptides, Ala-Trp-Val, Val-Trp-Ala, Ala-Ala-Trp-Val and Ala-Gly-Trp-Leu, have been irradiated with light of wavelengths >295 n. The major changes were destruction of the tryptophan residue, liberation of ammonia and the formation of photoproducts of increased molecular weight. Up to 40% of Ala-Ala-Trp-Val and Ala-Gly-Trp-Leu were converted to products with molecular weights ranging from two to four times those of the original tetrapeptides. Most of the yellow material formed during irradiation in air was present in the high molecular weight fractions. Irradiation of Ala-Gly-[¹⁴C]Trp-Leu gave the following identifiable photoproducts: Ala-Gly-Asp-Leu, Ala-Gly-(N′-formyl)Kyn-Leu, Ala-Gly-Oia-Leu, and ammonia, where Kyn means kynurenine and Oia, β-(3-oxidolyl)alanine.     

The hydroxylation of tryptophan.

Products of the chemical hydroxylation of tryptophan by Fenton and Udenfriend reactions are similar to those obtained by ionizing radiation. When tryptophan is exposed to either of these systems, a mixture of four hydroxytryptophans, oxindole-3-alanine, and N-formylkynurenine is formed. This observation indicates that the hydroxyl radical attacks the aromatic nucleus as well as the 2 and 3 positions of the pyrrole ring. During gamma-radiolysis of nitrous oxide-saturated tryptophan solution and in the absence of oxygen or ferric edta, the hydroxyl radical adduct (or hydroxycyclohexadienyl radical) of tryptophan undergoes dimerization and polymerization, which results in a yellow product with maximal absorbance at 425 nm. In the presence of ferric edta, or in a Fenton system, the hydroxyl radical adduct disproportionates, and hydroxylated derivatives are formed. The yields of the hydroxytryptophans are proportional to the concentration of ferric edta to a limiting yield of 54% of the theoretical yield, which is taken to be one hydroxylated product per two hydroxyl radicals. Under these conditions, 4-, 5-, 6-, and 7-hydroxy-derivatives of tryptophan are found in the proportion 4:2:2:3, respectively. The presence of dioxygen during gamma-radiolysis increases the yield of N-formylkynurenine, but does not affect the total yield of hydroxytryptophans. Similarly, tryptophan subjected to the Udenfriend reaction yields 4-, 5-, 6-, and 7-hydroxytryptophan and N-formylkynurenine in approximately equal amounts.