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.     

Studies on the photooxidation of tryptophan

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1. From the study of the photooxidation of tryptophan in three different aqueous systems, it has been concluded that the primary reaction is one which leads to the formation of formylkynurenine. Secondary reactions result in the formation of a variety of simpler amino acids together with a complex mixture of aromatic and polymeric materials.     

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     

Pressure effects on tryptophan and its derivatives

The high pressure effects on fluorescence of free tryptophan (Trp) and its derivatives, N-acetyl-tryptophan (AT), N-acetyl-tryptophanamide (NATA), tryptophanamide (TA), and tryptophan, containing 6-polypeptides in aqueous solution, were investigated in a pressure range from 0.1 to 650 MPa. It was found by analyzing the center of spectral mass in the wavelength range from 300 to 450 nm that high pressure shifted the fluorescence spectra of all these species to red direction: 421 cm(-1) for Trp, 305 cm(-1) for AT, 310 cm(-1) for NATA, 265 cm(-1) for TA, and 220 cm(-1) for single tryptophan containing 6-polypeptides. All the fluorescence efficiencies (i.e., quantum yield) of the compounds were reduced with pressure except free tryptophan where its fluorescence efficiency was enhanced with pressure. Glycerol, ethanol, and pH obviously influenced the pressure effects on their fluorescence characteristics. Since the tryptophan fluorescence is usually used as a probe for protein structural investigation, these findings suggested that the intrinsic pressure effect on tryptophan (or its derivatives) must be taken in consideration to explain the phenomenon observed in high pressure study on biomolecules when using the usual fluorospectroscopic approaches. In the present investigation, the mechanisms involved for pressure effects on tryptophan and its derivatives were explored and discussed.     

The effects of maintained tryptophan levels on ergot alkaloid production and overall tryptophan economy inClaviceps purpurea

Summary Measurements of alkaloid production, dryweight, and total tryptophan requirement in batch cultures ofClaviceps purpurea maintained at predetermined levels of extracellular tryptophan by repeated measured additions of the aminoacid confirm previous accounts of the overall regulatory effects even in the absence of major fluctuations in the tryptophan level.     

Effects of pyrazole on rat liver tryptophan oxygenase

The effects of pyrazole administration on rat liver tryptophan oxygenase have been studied both under basal conditions and after induction by cortisol or activation by tryptophan.Pyrazole administration is followed by a decrease of the basal holoenzyme and total enzyme activities. It induces furthermore a considerable inhibition of the cortisol mediated tryptophan oxygenase induction. These effects are not mediated by a modification of a tryptophan oxygenase effector, as shown by mixed homogenate experiments. The tryptophan enhancement of total tryptophan oxygenase activity is not affected by pyrazole administration contrary to the holoenzyme activity. Pyrazole added $\text{in vitro}$ inhibits liver tryptophan oxygenase activity only when used at concentrations which are considerably higher that those occuring $\text{in vivo}$ after pyrazole administration.