Recombinant photoreactive tRNA molecules as probes for cross-linking studies

Photoreactive tRNA derivatives have been used extensively for investigating the interaction of tRNA molecules with their ligands and substrates. Recombinant RNA technology facilitates the construction of such tRNA probes through site-specific incorporation of photoreactive nucleosides. The general strategy involves preparation of suitable tRNA fragments and their ligation either to a photoreactive nucleotide or to each other. tRNA fragments can be prepared by site-specific cleavage of native tRNAs, or synthesized by enzymatic and chemical means. A number of photoreactive nucleosides suitable for incorporation into tRNA are presently available. Joining of tRNA fragments is accomplished either by RNA ligase or by DNA ligase in the presence of a DNA splint. The application of this methodology to the study of tRNA binding sites on the ribosome is discussed, and a model of the tRNA-ribosome complex is presented.     

The nature of bile salt micelles as studied by deuterium NMR

Deuterium NMR of 3α,12α-dihydroxy-7,7-dideutero-5β-cholanoic acid was studied. Molecular sizes obtained from deuterium spin-lattice relaxation time (T₁) data of 3α,12α-dihydroxy-7,7-dideutero-5β-cholanoic acid in methanol and in water are in accordance with monomeric and tetrameric structures in the two media, respectively. The deuterium T₁ and intensity of 3α,12α-dihydroxy-7,7-dideutero-5β-cholanoic acid in aqueous solution at pH 8.0–8.8 were studied as functions of NaCl and lecithin concentrations. The results indicated that tetramers are in equilibrium with larger aggregates when secondary micelles are formed in the precense of NaCl, and that 3α, 12α-dihydroxy-7,7-dideutero-5β-cholanoic acid forms mixed micelles with lecithin with a molecular ratio of 2 : 3.     

The membrane-embedded segment of cytochrome b5 as studied by cross-linking with photoactivatable phospholipids

Vesicles were prepared from a 9:1 (mole/mol) mixture of dipalmitoyl phosphatidylcholine and the radioactively labeled phospholipids, 1-palmitoyl-2-omega-(m-diazirinophenoxy)undecanoyl-sn-glycero-3-phosphocholine (PC-I) or 1-palmitoyl-2-omega-(2-diazo-3,3,3-trifluropropionyloxy)lauroyl-sn- glycero-3-phosphocholine (PC-II). Rabbit liver cytochrome b5 was inserted into these vesicles spontaneously and the resulting vesicles containing the cytochrome b5 in the transferable form were photolyzed. Cytochrome b5 containing covalently cross-linked phospholipids was isolated by Sephadex LH-60 column chromatography using ethanol/formic acid as the solvent. Of the total radioactivity, 4.6% (PC-I) or 11.3% (PC-II) was linked to the protein; of the former, up to 51% was base-labile, while in the latter, 22% was base-labile. The sites of cross-linking of PC-I to the protein were investigated by fragmentation with trypsin, Staphylococcus aureas V8 protease, CNBr, and o-iodosobenzoic acid followed by Sephadex LH-60 chromatography and Edman sequencing (solid phase) of the appropriate fragments. The distribution of cross-linking was broad (Ser-104 to Met-130), showing a bell-shaped pattern with a significant peak at Ser-118. The labeling pattern is consistent with the previously proposed loop-back model for the membranous segment in the transferable form of cytochrome b5.     

Slow down of actin depolymerization by cross-linking molecules

The ability to control the assembly and disassembly dynamics of actin filaments is an essential property of the cellular cytoskeleton. While many different proteins are known which accelerate the polymerization of monomers into filaments or promote their disintegration, much less is known on mechanisms which guarantee the kinetic stability of the cytoskeletal filaments. Previous studies indicate that cross-linking molecules might fulfill these stabilizing tasks, which in addition facilitates their ability to regulate the organization of cytoskeletal structures in vivo. The effect of depolymerization factors on such structures or the mechanism which leads finally to their disintegration remain unknown. Here, we use multiple depolymerization methods in order to directly demonstrate that cross-linking and bundling proteins effectively suppress the actin depolymerization in a concentration dependent manner. Even the actin depolymerizing factor cofilin is not sufficient to facilitate a fast disintegration of highly cross-linked actin networks unless molecular motors are used simultaneously. The drastic modification of actin kinetics by cross-linking molecules can be expected to have wide-ranging implications for our understanding of the cytoskeleton, where cross-linking molecules are omnipresent and essential.     

The nature of bile salt micelles as studied by deuterium NMR.

Deuterium NMR of 3alpha,12alpha-dihydroxy-7,7dideutero-5beta-cholanic acid was studied. Molcular sizes obtained from deuterium spin-lattice relaxation time (T1) data of 3alpha,12alpha-dihydroxy-7,7-dideutero-5beta-cholanoic acid in methanol and in water are in accordance with monometic and tetrameric structures in the two media, respectively. The deuterium T1 and intensity of 3alpha,12alpha-dihydroxy-7,7-dideutero-5beta-cholanoic acid in aqueous solution at pH 8.0--8.8 were studied as functions of NcC1 and lecithin concentrations. The results indicated that tetramers are in equilibrium with larger aggregates when secondary micelles are formed in the precense of NaC1, and that 3alpha,12alpha-dihydroxy-7,7-dideutero-5beta-cholanoic acid forms mixed micelles with lecithin with a molecular ratio of 2 : 3.     

Polypeptide modification and cross-linking by oxidized 3-hydroxykynurenine

3-Hydroxykynurenine (3OHKyn) is present in the mammalian lens as a UV filter and is formed from kynurenine in the tryptophan metabolic pathway. 3OHKyn is a readily autoxidized o-aminophenol which binds to proteins in vitro. The lens, particularly its central region, the nucleus, becomes increasingly oxidized with age. Under such conditions, the oxidation products of 3OHKyn may bind to lens proteins and contribute to nuclear cataract formation. The purpose of this study was to determine the structures of in vitro reaction products of 3OHKyn with model peptides as a general model for 3OHKyn modification of proteins. 3OHKyn was incubated with the dipeptide glycylglycine (GG) and the tetrapeptide tuftsin (sequence TKPR) under oxidizing conditions, and the reaction products were characterized by a variety of spectroscopic techniques. The major 3OHKyn-GG reaction product involves formation of a benzimidazole moiety between the GG N-terminus and the oxidized amino and/or phenol groups of 3OHKyn. In contrast, tuftsin, which has an N-terminal threonine, forms predominantly a cross-linked dimer with oxidized 3OHKyn. This product is analogous in structure to the dimeric reaction product, quinilinobenzoxamine, formed between oxidized 3OHKyn and glycyllysine [Aquilina, J. A., et al. (1999) Biochemistry 38, 11455-11464], which contains a benzoxazole moiety. The identification of a tuftsin dimer suggests that 3OHKyn can react with any peptide having a free alpha-amino group, via a general side chain elimination mechanism. The identification of both benzimidazole and benzoxazole adducts in peptides with a free N-terminus suggests that peptide amino groups can react initially at either the aromatic amino or hydroxyl group of oxidized 3OHKyn. The proportion of each adduct may change, however, depending on the amino acid sequence at the N-terminus.     

Evidence for an arginine residue at the substrate binding site of Escherichia coli adenylosuccinate synthetase as studied by chemical modification and site-directed mutagenesis

Chemical modification of adenylosuccinate synthetase from Escherichia coli with phenylglyoxal resulted in an inhibition of enzyme activity with a second-order rate constant of 13.6 M-1 min-1. The substrates, GTP or IMP, partially protected the enzyme against inactivation by the chemical modification. The other substrate, aspartate, had no such effect even at a high concentration. In the presence of both IMP and GTP during the modification, nearly complete protection of the enzyme against inactivation was observed. Stoichiometry studies with [7-14C]phenylglyoxal showed that only 1 reactive arginine residue was modified by the chemical reagent and that this arginine residue could be shielded by GTP and IMP. Sequence analysis of tryptic peptides indicated that Arg147 is the site of phenylglyoxal chemical modification. This arginine has been changed to leucine by site-directed mutagenesis. The mutant enzyme (R147L) showed increased Michaelis constants for IMP and GTP relative to the wild-type system, whereas the Km for aspartate exhibited a modest decrease as compared with the native enzyme. In addition, kcat of the R147L mutant decreased by a factor of 1.3 x 10(4). On the bases of these observations, it is suggested that Arg147 is critical for enzyme catalysis.     

Folding transitions in calpain activator peptides studied by solution NMR spectroscopy

Calpastatin, the endogenous inhibitor of calpain, a cysteine protease in eukaryotic cells, is an intrinsically unstructured protein, which upon binding to the enzyme goes through a conformational change. Peptides calpA (SGKSGMDAALDDLIDTLGG) and calpC (SKPIGPDDAIDALSSDFTS), corresponding to the two conserved subdomains of calpastatin, are known to activate calpain and increase the Ca²⁺ sensitivity of the enzyme. Using solution NMR spectroscopy, here we show that calpA and calpC are disordered in water but assume an α‐helical conformation in 50% CD₃OH. The position and length of the helices are in agreement with those described in the literature for the bound state of the corresponding segments of calpastatin suggesting that the latter might be structurally primed for the interaction with its target. According to our data, the presence of Ca²⁺ induces a backbone rearrangement in the peptides, an effect that may contribute to setting the fine conformational balance required for the interaction of the peptides with calpain. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

The three-dimensional structure and function of Escherichia coli ribosomal RNA, as studied by cross-linking techniques

A large number of intra-RNA and RNA-protein cross-link sites have been localized within the 23S RNA from E. coli 50 S ribosomal subunits. These sites, together with other data, are sufficient to constrain the secondary structure of the 23 S molecule into a compact three-dimensional shape. Some of the features of this structure are discussed, in particular, those relating to the orientation of tRNA on the 50 S subunit as studied by site-directed cross-linking techniques. A corresponding model for the 16S RNA within the 30 S subunit has already been described, and here a site-directed cross-linking approach is being used to determine the path followed through the subunit by messenger RNA.     

A novel cross-linking reagent for bovine hemoglobin modification

A novel cross-linking reagent, methoxypolyethelene glycol-glutamic acid, was synthesized and used to modify bovine hemoglobin. Bis-tetrameric hemoglobin with moderate affinity for O₂ was obtained under the controlled reaction conditions.     

Potential participation of calpain in platelet activation studied by use of a cell penetrating calpain inhibitor (calpeptin)

Employing a cell penetrating calpain inhibitor (calpeptin), the role of calpain in platelet activation was examined. In washed platelets (WPs) both thrombin and collagen-induced platelet aggregation were dose-dependently inhibited by calpeptin. The addition of plasma to WPs interfered with the action of calpeptin, however more than 3 min preincubation of calpeptin with WPs completely abolished the influence of plasma. In thrombin-activated WPs with calcium, the increase of intracellular calcium concentration, [Ca2+]i, and the production of inositol triphosphate (IP3) were dose-dependently inhibited by calpeptin. The generation of thromboxane B2 (TxB2) was inhibited by calpeptin in collagen and thrombin-activated WPs. In [3H]-arachidonic acid (AA)-labelled platelets, calpeptin increased the amount of [3H]-AA liberated by inhibiting [3H]-AA degradation after collagen or thrombin stimulation. When [14C]-AA degradation by the platelet suspension was observed, calpeptin inhibited TxB2 and hydroxyheptadecatrienoic acid (HHT) generation but increased prostaglandin (PG) E1, E2, 12-hydroxyeicosatetraenoic acid (12HETE) and AA. Based on these findings, calpain may be involved in the activation phospholipase C and thromboxane synthetase.     

Evidence for modification of protein phosphorylation by cytokinins

Kinetin stimulated phosphorylation of protein in floated Chinese-cabbage leaf discs, but inhibited protein phosphorylation in nuclei+chloroplast extracts from Chinese-cabbage or tobacco leaves. Kinetin also inhibited protein phosphorylation in isolated tobacco nuclei or nuclei from carrot secondary-phloem tissue. Purified Chinese-cabbage leaf ribosomes exhibited protein kinase activity which was inhibited by kinetin and zeatin. The ribosome-associated kinase responded to kinetin and zeatin differently from that associated with nuclei+chloroplast preparations. Protein phosphorylation in vitro was not affected by adenosine 3':5'-cyclic monophosphate, indol-3-ylacetic acid or gibberellic acid. It was only inhibited by N(9)-unsubstituted purines, among which the known cytokinins were the most effective inhibitors. The results are discussed in relation to possible similarities between the effects of cytokinins in plant tissues and the effects of adenosine 3':5'-cyclic monophosphate in animal tissues. Both compounds appear to modify the activity of protein kinases and both affect many different cellular processes.     

Increased thermal stability of glucose dehydrogenase by cross-linking chemical modification

A hetero-oligomeric glucose dehydrogenase (GDH) from a moderate thermophilic bacterium, SM4 was cross-linked with glutaraldehyde (GA) and it now showed only one optimum temperature for reaction at around 65°C, which approximately follows the Arrhenius equation. The native enzyme had shown optima at both 45°C and 75°C. In addition to the alteration of the optimum temperature for reaction, GA cross-linked GDH retained more than 90% of its initial activity even after 30 min of incubation at 65°C.     

Stabilization of pyrroloquinoline quinone glucose dehydrogenase by cross-linking chemical modification

Summary Cross-linking chemical modification of pyrroloquinoline quinone (PQQ) glucose dehydrogenase (GDH) by glutaraldehyde was carried out and its stability was analyzed. Although native PQQGDH was inactivated within 30 min at a higher temperature than 50 °C, cross-linked PQQGDH retained more than 40% of initial activity even after 30 min of incubation at 54 °C. In addition to the increase in thermal stability, cross-linked PQQGDH gained high EDTA tolerance. The stabilization may be achieved by increased the rigidity of PQQGDH holo enzyme conformation.     

Dependence of DNA-protein cross-linking via guanine oxidation upon local DNA sequence as studied by restriction endonuclease inhibition

Oxidative damage plays a causative role in many diseases, and DNA-protein cross-linking is one important consequence of such damage. It is known that GG and GGG sites are particularly prone to one-electron oxidation, and here we examined how the local DNA sequence influences the formation of DNA-protein cross-links induced by guanine oxidation. Oxidative DNA-protein cross-linking was induced between DNA and histone protein via the flash quench technique, a photochemical method that selectively oxidizes the guanine base in double-stranded DNA. An assay based on restriction enzyme cleavage was developed to detect the cross-linking in plasmid DNA. Following oxidation of pBR322 DNA by flash quench, several restriction enzymes (PpuMI, BamHI, EcoRI) were then used to probe the plasmid surface for the expected damage at guanine sites. These three endonucleases were strongly inhibited by DNA-protein cross-linking, whereas the AT-recognizing enzyme AseI was unaffected in its cleavage. These experiments also reveal the susceptibility of different guanine sites toward oxidative cross-linking. The percent inhibition observed for the endonucleases, and their pBR322 cleavage sites, decreased in the order: PpuMI (5'-GGGTCCT-3' and 5'-AGGACCC-3') > BamHI (5'-GGATCC-3') > EcoRI (5'-GAATTC-3'), a trend consistent with the observed and predicted tendencies for guanine to undergo one-electron oxidation: 5'-GGG-3' > 5'-GG-3' > 5'-GA-3'. Thus, it appears that in mixed DNA sequences the guanine sites most vulnerable to oxidative cross-linking are those that are easiest to oxidize. These results further indicate that equilibration of the electron hole in the plasmid DNA occurs on a time scale faster than that of cross-linking.     

Synergistic transmembrane insertion of the heterodimeric PGLa/magainin 2 complex studied by solid-state NMR

The skin secretions of amphibians are a rich source of antimicrobial peptides. The two antimicrobial peptides PGLa and magainin 2, isolated from the African frog Xenopus laevis, have been shown to act synergistically by permeabilizing the membranes of microorganisms. In this report, the literature on PGLa is extensively reviewed, with special focus on its synergistically enhanced activity in the presence of magainin 2. Our recent solid state ²H NMR studies of the orientation of PGLa in lipid membranes alone and in the presence of magainin 2 are described in detail, and some new data from 3,3,3-²H₃-L-alanine labeled PGLa are included in the analysis.     

Evidence for involvement of calpain in c-Myc proteolysis in vivo

Precise control of the level of c-Myc protein is important to normal cellular homeostasis, and this is accomplished in part by degradation through the ubiquitin-proteasome pathway. The calpains are a family of calcium-dependent proteases that play important roles in proteolysis of some proteins, and their possible participation in degradation of intracellular c-Myc was therefore investigated. Activation of calpain with the cell-permeable calcium ionophore A23187 in Rat1a-myc or ts85 cells in culture induced rapid cleavage of c-Myc. This degradation was both calpain- and calcium-dependent since it was inhibited by preincubation with either the calpain-inhibitory peptide calpeptin or the calcium-chelating agent EGTA. A23187-induced c-Myc cleavage occurred in a time-dependent manner comparable to that of FAK, a known calpain substrate, and while calpeptin was able to significantly protect c-Myc from degradation, inhibitors of the proteasome or caspase proteases could not. Exposure of Rat1a-myc or ts85 cells in culture to calpeptin, or to the thiol-protease inhibitor E64d, resulted in the accumulation of c-Myc protein without an impact on ubiquitin-protein conjugates. Using an in vitro assay, calpain-mediated degradation occurred rapidly with wild-type c-Myc as the substrate, but was significantly prolonged in some c-Myc mutants with increased transforming activity derived from lymphoma patients. Those mutants with a prolonged half-life in vitro were also more resistant to A23187-induced cleavage in intact cells. These studies support a role for calpain in the control of c-Myc levels in vivo, and suggest that mutations impacting on sensitivity to calpain may contribute to c-Myc-mediated tumorigenesis.