The fluorescence decay of tryptophan residues in native and denatured proteins.

The fluorescence decay kinetics at different ranges of the emission spectrum is reported for 17 proteins. Out of eight proteins containing a single tryptophan residue per molecule, seven proteins display multiexponential decay kinetics, suggesting that variability in protein structure may exist for most proteins. Tryptophan residues whose fluorescence spectrum is red shifted may have lifetimes longer than 7 ns. Such long lifetimes have not been detected in any of the denatured proteins studied, indicating that in native proteins the tryptophans having a red-shifted spectrum are affected by the tertiary structure of the protein. The fluorescence decay kinetics of ten denatured proteins studied obey multiexponential decay functions. It is therefore concluded that the tryptophan residues in denatured proteins can be grouped in two classes. The first characterized by a relatively long lifetime of about 4 ns and the second has a short lifetime of about 1.5 ns. The emission spectrum of the group which is characterized by the longer lifetime is red shifted relative to the emission spectrum of the group characterized by the shorter lifetime. A comparison of the decay data with the quantum yield of the proteins raises the possibility that a subgroup of the tryptophan residues is fully quenched. It is noteworthy that despite this heterogeneity in the environment of tryptophan residues in each denatured protein, almost the same decay kinetics has been obtained for all the denatured proteins studied in spite of the vastly different primary structures. It is therefore concluded that each tryptophan residue interacts in a more-or-less random manner with other groups on the polypeptide chain, and that on the average the different tryptophan residues in denatured proteins have a similar type of environment.     

Nitration and nitrosation of N-acetyl-L-tryptophan and tryptophan residues in proteins by various reactive nitrogen species

Proteins are targets of reactive nitrogen species such as peroxynitrite and nitrogen dioxide. Among the various amino acids in proteins, tryptophan residues are especially susceptible to attack by reactive nitrogen species. We carried out experiments on the reactions of peroxynitrite and other reactive nitrogen species with N-acetyl-L-tryptophan under various conditions. Four major products were identified as 1-nitroso-N-acetyl-L-tryptophan, 1-nitro-N-acetyl-L-tryptophan, 6-nitro-N-acetyl-L-tryptophan, and N-acetyl-N'-formyl-L-kynurenine on the basis of their mass and UV spectra. The reactions with SIN-1 (a peroxynitrite generator), Angeli's salt (a nitroxyl donor), and spermine NONOate (a nitric oxide donor) generated the nitroso derivative but not the nitro derivatives. A myeloperoxidase-H(2)O(2)-NO(2)(-) system generated the nitro derivatives but not the nitroso derivative. Under physiological conditions 6-nitro-N-acetyl-L-tryptophan was stable, whereas the 1-nitroso and 1-nitro derivatives decomposed with half-lives of 1.5 and 18 h, respectively. After treatment with various reactive nitrogen species, bovine serum albumin was enzymatically hydrolyzed and analyzed for 6-nitro-L-tryptophan and 3-nitro-L-tyrosine by HPLC with electrochemical detection. Levels of 6-nitro-L-tryptophan and 3-nitro-L-tyrosine were similar in the nitrated protein. 6-Nitro-L-tryptophan in proteins can be measured as an additional biomarker of protein nitration.     

Maltose chemotaxis involves residues in the N-terminal and C-terminal domains on the same face of maltose-binding protein.

The periplasmic maltose-binding protein (MBP) of Escherichia coli is the recognition component of the maltose chemoreceptor and of the active transport system for maltose. It interacts with the Tar chemotactic signal transducer and the integral cytoplasmic-membrane components (the MalF and MalG proteins) of the maltose transport system. Maltose binds in a cleft between the globular N-terminal and C-terminal domains of MBP, which are connected by a moveable hinge. The two domains undergo a large motion relative to one another as the protein moves from the open, unbound state to the closed, ligand-bound state. We generated, by doped-primer mutagenesis, amino acid substitutions that specifically disrupt the chemotactic function of MBP. These substitutions cluster in two well-defined regions that are nearly contiguous on the surface of MBP in its closed conformation. One region is in the N-terminal domain and one is in the C-terminal domain. The distance between the two regions is expected to change substantially as the protein goes from the open to the closed form. These results support a model in which ligand binding brings two recognition sites on MBP into the proper spatial relationship to interact with complementary sites on Tar. Mutations in MBP that appear to cause defects in interaction with MalF and MalG are distributed differently from mutations that primarily affect maltose taxis. We conclude that the regions of MBP that contact Tar and those that contact MalF and MalG are adjacent on the face of the protein opposite the hinge connecting the two domains and that those regions are largely, although perhaps not entirely, distinct.     

Effects of ammonia on hydrolysis of proteins and lipids from fish residues

This study investigated the influence of ammonia on the hydrolysis rates of proteins and lipids in fish residues under mesophilic anaerobic incubation at a neutral pH. The hydrolysis kinetics of the fish residues, which contained primarily proteins and lipids, were examined at initial ammonia concentrations of 0–16 g N l⁻¹. Carbon hydrolysis was suppressed more by ammonium in the acidogenesis phase than in the acidogenesis/methanogenesis period of a single-stage anaerobic digestion. Conversely, hydrolysis of compounds containing nitrogen was similarly suppressed by ammonia during acidogenesis and acidogenesis/methanogenesis phases of a single-stage anaerobic digestion. Parameter uncertainty analysis demonstrated that the proteins fraction in the fish residues was entirely biodegradable. Model fitting demonstrated that two fractions of lipid substrates exist, namely, easy and hard to biodegrade with hydrolysis rates that were affected differently by ammonia content.     

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.     

Biosynthesis of the C-terminal amide in peptide hormones

Recent developments in the study of peptide amidation are reviewed. The main areas covered are assay procedures, purification of amidating enzymes, co-fact0rs and regulation; mechanism and specificity of the amidating reaction, and multiple forms of the amidating enzyme and glycosylation. Discussion is presented on aspects that are poorly understood and new areas open to investigation are indicated.     

Structure-function studies on red pigment-concentrating hormone, II. The significance of the C-terminal tryptophan amide

The significance of the C-terminal tryptophan residue of the red pigment-concentrating hormone (RPCH: Glu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH2) regulating the blanching of the crustacean chromatophores has been investigated. RPCH and a number of analogues that differ only in the C-terminal part of the hormone, have been synthesized and assayed for biological activity on the shrimp Leander adspersus. It has been shown that the indole skeleton of tryptophan is an absolute requirement for the biological activity of the hormone. To provide maximum response the tryptophan must be blocked as the amide. The activity of synthetic [Tyr4]RPCH and adipokinetic hormone (AKH) purified from Schistocerca gregaria has been compared with the activity of synthetic RPCH.     

Hybrid native partitioning of interactions among nonconserved residues in chimeric proteins

Given any operational criterion for pairwise interatomic interactions, for a pair of structurally homologous proteins there exists for both proteins a unique equivalent partitioning of the nonconserved residue positions into mutually non-interacting clusters. In the formation of a chimeric protein derived from these two parental sequences, if nonnative-like interactions are to be avoided in its tertiary structure, then all of the nonconserved residues of each cluster must necessarily be either maintained or interchanged simultaneously. This hybrid native partitioning criterion is applied to known gene shuffling results. When the degree of estimated disruption is modest, the HybNat algorithm provides an efficient predictor of structural integrity. This supports the expectation that a substantial fraction of sequences that conform to the hybrid native partitioning criterion will yield tertiary structures that largely preserve the native-like interactions of the parental proteins.     

Second-derivative spectroscopy of proteins: Studies on tyrosyl residues

Ionization of the phenolic group of N-acetyltyrosynamide has been studied using second-derivative spectroscopy. At pH 12.5 the second-derivative spectrum of the model compound revealed the presence of derivative bands in a spectral region (between 250 and 270 nm) where interference coming from other ultraviolet-absorbing chromophores is negligible. One of these peaks (260-nm peak) has been employed for the determination of tyrosyl groups in mixtures containing the aromatic amino acids.