ESR and optical absorption studies on the copper (II) interaction with aromatic amino acids

Electron spin resonance and optical absorption studies have been done in order to investigate the interaction between Cu²⁺ and aromatic amino acids in aqueous solution at 77 K and at room temperature as well. Depending on the concentration each aromatic amino acid can form two different kinds of complexes with Cu²⁺ which can be characterized by its ESR pattern. Additional information was obtained from optical d-d and CT transitions.     

Molecular recognition of nucleic acids: coralyne binds strongly to poly(A)

The small molecule coralyne was found to bind preferentially and strongly to single-stranded poly(A) with an apparent association constant (Ka) of (1.8+/-0.3) x 10(6)M(-1). Binding of coralyne to poly(A) is predominantly enthalpically driven with a stoichiometry of one coralyne per four adenine bases. Poly(A) forms a coralyne dependent secondary structure with a melting temperature of 60 degrees C, for the conditions of our study.     

ESR and optical absorption studies on the copper(II) interaction with small peptides containing aromatic amino acids

The interaction of Cu(II) with di- and tripeptides each containing phenylalanine, tryptophan or histidine in the amino acid chain has been investigated by means of electron spin resonance (ESR) and optical absorption spectroscopy. Cu(II) complexes of dipeptides and tripeptides exhibit different magnetic and optical parameters. Dipeptide complexes have larger g -values and smaller {A –values than tripeptide complexes. When compared to dipeptide complexes, the d-d band of the central metal ion is blue shifted for tripeptide complexes. There are no significant differences in the behavior of Cu(II) peptide complexes containing phenylalanine or tryptophan. Complexes of histidine containing peptides, however, show modified spectra caused by the participation of the imidazole nitrogen in the coordination to Cu(II). The imidazole nitrogen seems to coordinate in-plane with other coordinating atoms or in an axial position depending on the kind of peptide.Part of the Ph.D. thesis of L.S., D-26Dedicated to Prof. Dr. H. Glubrecht on the occasion of his 60th birthday     

Mona/Gads SH3C binding to hematopoietic progenitor kinase 1 (HPK1) combines an atypical SH3 binding motif, R/KXXK, with a classical PXXP motif embedded in a polyproline type II (PPII) helix

Hematopoietic progenitor kinase 1 (HPK1) is implicated in signaling downstream of the T cell receptor. Its non-catalytic, C-terminal half contains several prolinerich motifs, which have been shown to interact with different SH3 domain-containing adaptor proteins in vitro. One of these, Mona/Gads, was also shown to bind HPK1 in mouse T cells in vivo. The region of HPK1 that binds to the Mona/Gads C-terminal SH3 domain has been mapped and shows only very limited similarity to a recently identified high affinity binding motif in SLP-76, another T-cell adaptor. Using isothermal titration calorimetry and x-ray crystallography, the binding of the HPK1 motif to Mona/Gads SH3C has now been characterized in molecular detail. The results indicate that although charge interactions through an RXXK motif are essential for complex formation, a PXXP motif in HPK1 strongly complements binding. This unexpected binding mode therefore differs considerably from the previously described interaction of Mona/Gads SH3C with SLP-76. The crystal structure of the complex highlights the great versatility of SH3 domains, which allows interactions with very different proteins. This currently limits our ability to categorize SH3 binding properties by simple rules.     

ESR an optical absorption studies on the copper(II) interaction with small peptides containing aromatic amino acids.

The interaction of Cu(II) with di- and tripeptides each containing phenylalanine, tryptophan or histidine in the amino acid chain has been investigated by means of electron spin resonance (ESR) and optical absorption spectroscopy. Cu(II) complexes of dipeptides and tripeptides exhibit different magnetic and optical parameters. Dipeptide complexes have larger gparallel-values and smaller A parallel values than tripeptide complexes. When compared to dipeptide complexes, the d-d band of the central metal ion is blue shifted for tripeptide complexes. There are no significant difference in the behavior of Cu(II) peptide complexes containing phenylalanine or tryptophan. Complexes of histidine containing peptides, however, show modified spectra caused by the participation of the imidazole nitrogen in the coordination to Cu(II). The imidazole nitrogen seems to coordinate in-plane with other coordinating atoms or in an axial position depending on the kind of peptide.     

DNA-fiber EPR spectroscopy as a tool to study DNA-metal complex interactions: DNA binding of hydrated Cu(II) ions and Cu(II) complexes of amino acids and peptides

DNA-fiber EPR spectroscopy and its application to studies of the DNA binding orientation and dynamic properties of Cu(II) ions and their complexes with amino acids and peptides are reviewed. Cu(II) ions bind in at least two different binding modes; one mode was mobile while the other mode fixed the orientation of the coordination plane. The hydroxyl groups of L-Ser and L-Thr fixed the coordination plane of their respective Cu(II) complexes parallel to the DNA base pair plane, whereas Cu(II) complexes of Lys and Arg induced several binding modes, depending on the tertiary structure of the DNA and the chirality of the amino acids. Unusually broadened signals observed for the His complex were assigned to a mono-L-His complex stacked stereospecifically along the DNA double helix. In comparison, Cu(II). Xaa-Xaa' -His type complexes oriented in the minor groove with different affinities and extents of randomness depending on the Xaa-Xaa' sequence and the chirality of Xaa or Xaa' while the C-terminal Xaa residues in Cu(II).Arg-Gly-His-Xaa (Xaa=L-Leu or L-Glu) decreased the stereospecificity and the stability of the complexes bound to DNA. In contrast to Xaa-Xaa'- His complexes, the coordination planes of Cu(II).Gly-L-His-Gly and Cu(II).Gly-L-His-L-Lys complexes were found to lie parallel to the DNA-fiber axis. Dinuclear Cu(II).carnosine complexes were also shown to bind to DNA stereospecifically.     

Chitosan gel beads immobilized Cu (II) for selective adsorption of amino acids

Chitosan (CS) gel beads were prepared by using phase inversion and precipitation technique. The gel beads could bind copper (II), by which Cu (II) ion-immobilized chitosan gel beads (CS-Cu²⁺ gel beads) were prepared, and the amount of the immobilized Cu (II) was about 35 mg/g when the CS gel beads were incubated in 150 ppm cupric sulfate solution. The CS-Cu²⁺ gel beads could selectively adsorb histidine (His) from the mixed solution containing His and tryptophan (Trp); and the selective coefficient which was defined as the adsorbed amount ratio of His to Trp was about 8.0 at the pH value of 7.4. The effect of the pH value on the amino acid adsorption was also studied. In order to investigate the relationship of the amino acid adsorption and protein adsorption, the adsorbed amounts for IgG and albumin were determined; and the results indicated that the CS-Cu²⁺ gel beads could adsorb a larger amount of IgG than albumin due to the larger amount of the exposed residual His. The study provided a sorbent and a method to selectively remove His and IgG.     

A fluorophotometric method for the determination of amino acids by using pyridoxal and zinc(II) ion

A procedure for the determination of amino acids has been developed based on their interaction with pyridoxal and Zn²⁺ ion in pyridine methanol to yield highly fluorescent chelates. This fluorescence procedure is 20–100 times more sensitive than the colorimetric procedure. N-Pyridoxylidene amino acid-Zn(II) chelate contains a 1:1 molar ratio of ligand to metal ion.     

A computer method for finding common base paired helices in aligned sequences: application to the analysis of random sequences.

We describe a new computer program that identifies conserved secondary structures in aligned nucleotide sequences of related single-stranded RNAs. The program employs a series of hash tables to identify and sort common base paired helices that are located in identical positions in more than one sequence. The program gives information on the total number of base paired helices that are conserved between related sequences and provides detailed information about common helices that have a minimum of one or more compensating base changes. The program is useful in the analysis of large biological sequences. We have used it to examine the number and type of complementary segments (potential base paired helices) that can be found in common among related random sequences similar in base composition to 16S rRNA from Escherichia coli. Two types of random sequences were analyzed. One set consisted of sequences that were independent but they had the same mononucleotide composition as the 16S rRNA. The second set contained sequences that were 80% similar to one another. Different results were obtained in the analysis of these two types of random sequences. When 5 sequences that were 80% similar to one another were analyzed, significant numbers of potential helices with two or more independent base changes were observed. When 5 independent sequences were analyzed, no potential helices were found in common. The results of the analyses with random sequences were compared with the number and type of helices found in the phylogenetic model of the secondary structure of 16S ribosomal RNA. Many more helices are conserved among the ribosomal sequences than are found in common among similar random sequences. In addition, conserved helices in the 16S rRNAs are, on the average, longer than the complementary segments that are found in comparable random sequences. The significance of these results and their application in the analysis of long non-ribosomal nucleotide sequences is discussed.     

Identification of two amino acids in activin A that are important for biological activity and binding to the activin type II receptors

Activins are members of the transforming growth factor-beta family of growth and differentiation factors. In this paper, we report the results of a structure-function analysis of activin A. The primary targets for directed mutagenesis were charged, individual amino acids located in accessible domains of the protein, concentrating on those that differ from transforming growth factor-beta2, the x-ray crystal structure of which is known. Based on the activities of the recombinant activin mutants in two bioassays, 4 out of 39 mutant proteins (D27K, K102A, K102E, and K102R) produced in a vaccinia virus system were selected for further investigation. After production in insect cells and purification of these four mutants to homogeneity, they were studied in bioassays and in cross-linking experiments involving transfected receptor combinations. Mutant D27K has a 2-fold higher specific bio-activity and binding affinity to an ActRIIA/ALK-4 activin receptor complex than wild type activin, whereas mutant K102E had no detectable biological activity and did not bind to any of the activin receptors. Mutant K102R and wild type activin bound to all the activin receptor combinations tested and were equipotent in bioassays. Our results with the Lys-102 mutants indicate that the positive charge of amino acid 102 is important for biological activity and type II receptor binding of activins.     

A role for aromatic amino acids in the binding of Xenopus ribosomal protein L5 to 5S rRNA

The formation of the Xenopus L5-5S rRNA complex depends on nonelectrostatic interactions. Fluorescence assays with 1-anilino-8-naphthalenesulfonate demonstrate that a hydrophobic region on L5 becomes exposed upon removal of bound 5S rRNA by treatment with ribonucleases. Several conserved aromatic amino acids, mostly tyrosines, were identified by comparative sequence analysis and changed individually to alanine. Substitution with alanine at any of three positions, Y86, Y99, or Y226, essentially abolishes RNA-binding activity, whereas those made at Y95 and Y207 have more modest effects. Replacement with phenylalanine at Y86 and Y226 does not change binding affinity, indicating that the aromatic ring of the side chain, not the hydroxyl group, is the critical functionality for binding. Alternatively, the phenolic hydroxyls at Y99 and Y207 do contribute to binding. The structural integrity of the mutant proteins was assessed using thermal denaturation and limited digestion with proteases. The T(m) of Y99A is 10 degrees C lower than that of the wild-type protein, and there are some differences in the protease digestion patterns that together indicate the structure of this mutant has been significantly perturbed. The structures of the other variants are not detectably different from the wild-type protein. These results provide evidence that intermolecular stacking interactions involving at least two tyrosine residues, Y86 and Y226, are necessary for formation of the L5-5S rRNA complex and can account, at least in part, for the contribution nonelectrostatic interactions make to the free energy of binding.     

Thermodynamics of binding to SH3 domains: the energetic impact of polyproline II (PII) helix formation

Although numerous biophysical studies have focused on elucidating the structural and thermodynamic determinants that govern the free energy of binding between various SH3 domains and their putative recognition sequences, a quantitative accounting of the energetics of this interaction has proven enigmatic. Specifically, the binding results in a large and negative change on the standard enthalpy and entropy functions, a result which is inconsistent with the positive values for these quantities that is expected from the hydrophobic nature of the binding pocket. Here, the binding of the C-terminal SH3 domain of Sem-5 to its putative recognition peptide on the Sos (Son of Sevenless) protein is investigated using isothermal titration calorimetry under a variety of temperature and pH conditions. In addition, the energy associated with folding the Sos peptide into the binding competent polyproline II conformation is quantitatively evaluated. These results provide a rationale for the observed discrepancy between the experimental and predicted behavior and indicate that the determinants of binding in this system cannot be ascertained from a static structural representation of the binding process.     

Coprophagy in marmosets due to insufficient protein (amino acid) intake

In the course of experiments to study the minimal protein requirement of common marmosets by nitrogen balance methods, coprophagy was observed. It occurred in most animals fed diets either with a protein content below 6% or lacking histidine/arginine. The protein level of 6% had previously been evaluated as being the minimal protein requirement for maintenance of marmosets.     

Conserved charged amino acids are key determinants for fatty acid binding proteins (FABPs)-membrane interactions. A multi-methodological computational approach

Based on the analysis of the mechanism of ligand transfer to membranes employing in vitro methods, Fatty Acid Binding Protein (FABP) family has been divided in two subgroups: collisional and diffusional FABPs. Although the collisional mechanism has been well characterized employing in vitro methods, the structural features responsible for the difference between collisional and diffusional mechanisms remain uncertain. In this work, we have identified the amino acids putatively responsible for the interaction with membranes of both, collisional and diffusional, subgroups of FABPs. Moreover, we show how specific changes in FABPs’ structure could change the mechanism of interaction with membranes. We have computed protein–membrane interaction energies for members of each subgroup of the family, and performed Molecular Dynamics simulations that have shown different configurations for the initial interaction between FABPs and membranes. In order to generalize our hypothesis, we extended the electrostatic and bioinformatics analysis over FABPs of different mammalian genus. Also, our methodological approach could be used for other systems involving protein–membrane interactions.     

Thallium (I) interactions in biological fluids: a potentiometric investigation of thallium(I) complex equilibria with some sulphur-containing amino acids

Formation constants for thallium(I) complexes of L-cysteine (CysH2), DL-penicillamine (PenH2), N-acetyl-L-cysteine (AcyH2), and N-acetyl-DL-penicillamine (ApeH2) in aqueous solution have been determined in 150 mmol dm-3 NaCl medium at 37 degrees C by potentiometric titrations using a glass electrode. Glycine has been used as a model for simple amino acids. The experimental data may be explained by the formation of the complexes T1(Cys)-, T1(Cys)H, T1(Pen)-, T1(Pen)H, T1(Acy)-, and T1(Ape)- with log formation constants 3.26, 11.28, 3.60, 12.05, 2.27, and 2.45, respectively. Analysis of the results obtained and comparison of thallium(I) complexing ability with that of dimethyl-thallium(III) seem to indicate that thallium(I) toxicity does not directly stem from its interference with the metabolism of sulphur-containing compounds.     

The lower cytotoxicity of cinnamomin (a type II RIP) is due to its B-chain

The cytotoxicity of intact cinnamomin (a type II ribosome-inactivating protein, RIP) and the RNA N-glycosidase activity of cinnamomin A-chain have been studied and compared with those of ricin. Cinnamomin A-chain exhibits a similar RNA N-glycosidase activity in inhibiting in vitro protein synthesis compared with that of ricin, whereas the cytotoxicity to BA/F3beta cells of intact cinnamomin is markedly lower than intact ricin. In order to demonstrate that it is the B-chains of the two RIPs that bear the difference in cytotoxicity, two hybrid RIPs are prepared from the purified A-/B-chains of cinnamomin and ricin by the disulfide exchange reaction. It has been found that hybrid RIP constructed from cinnamomin A-chain and ricin B-chain is more toxic to BA/F3beta cells than the native cinnamomin, and equivalent to the native ricin. However, the cytotoxicity to BA/F3beta cells of the hybrid RIP constructed from the ricin A-chain and cinnamomin B-chain is lower than ricin, equivalent to the native cinnamomin. Furthermore, the bound amounts of two B-chains on the cell surface are determined by the method of direct cellular ELISA and Scatchard analysis of the binding of the two B-chains indicates that cinnamomin and ricin share similar binding sites with different affinity.