Result Of Limited Proteolysis Research Articles

Rigidifying flexible sites: An approach to improve stability of chondroitinase ABC I

The stability of chondroitin ABC lyase I (cABC I) at physiological temperature is one of the current obstacles to its clinical application. In this study, we used a protein engineering approach; rigidify flexible sites, to improve stability of cABC I. B-factor analysis showed a flexible loop at the N-terminal domain of cABC I which may be involved in its thermal instability and five residues in this region were replaced with proline. Thermal inactivation and thermal denaturation analysis revealed that Glu138Pro mutation increased half-life and Tm of enzyme, respectively. The Km values of mutated enzymes were slightly increased compared to the wild type enzyme. The results of limited proteolysis indicated that Glu138Pro mutant was more resistant against trypsinolysis and this variant was less quenched in both acrylamide and KI quenching experiments. Moreover, intrinsic fluorescence intensity of Glu138Pro variant was increased and its ANS fluorescence intensity was decreased, whereas no considerable changes were observed in the far-UV CD spectra. The structural analyses indicated compactness of structure of Glu138Pro enzyme which can be related to moderately enhanced stability of this mutant. This study demonstrated that rigidifying flexible residues can be considered as a possible approach to increase the stability of the protein.

Structural Analysis of Baseplate Wedge Proteins of BacteriophageT4

Gp10, gp11 and gp7 constitute part of the wedges, six of which surround the hub to form a baseplate. These proteins interact to form a precursor oligomer in the initial assembly process of the wedge. Recent 3D-image reconstruction of the baseplate before and after contraction (hexagon and star, respectively) together with crystal structures of gp11 and part of gp10 has revealed that these proteins play a central role during the conformational change. In order to fully understand the mechanism of the structural change, we need to determine the whole structure of gp10 and gp7, but it has been difficult. As we have recently succeeded in isolating gp7, we applied limited proteolysis of gp10 and its complex with gp11 or gp7 by lysyl endopeptidase. Gp10 alone, which is a trimer, is cleaved at Lys289, but when complexed with gp11, gp10 is then cleaved at Lys194 where gp11 remained intact. Gp10 consists of four domains, I through IV, where gp11 binds to domain III. Based on the results of limited proteolysis, the region between His195 and Lys289 (95 residues) is likely to belong to domain III. On the other hand, when gp10 is complexed with gp7 and subjected to limited proteolysis,gp10 becomes resistant to the protease, although gp7 was cleaved as in the same way as is proteolysed alone. The results indicated that that the resulting C-terminal 124 residues of gp7 form a stable complex with gp10. Crystallization of the protease-resistant region of gp10-gp11 complex is under way.

Telomere resolution by Borrelia burgdorferi ResT through the collaborative efforts of tethered DNA binding domains

Borrelia burgdorferi, a causative agent of Lyme disease, has a highly unusual segmented genome composed of both circular molecules and linear DNA replicons terminated by covalently closed hairpin ends or telomeres. Replication intermediates of the linear molecules are processed into hairpin telomeres via the activity of ResT, a telomere resolvase. We report here the results of limited proteolysis and mass spectroscopy to identify two main structural domains in ResT, separated by a chymotrypsin cleavage site between residues 163 and 164 of the 449 amino acid protein. The two domains have been overexpressed and purified. DNA electrophoretic mobility shift assays revealed that the C-terminal domain (ResT(164-449)) displays sequence-specific DNA binding to the box 3,4,5 region of the telomere, while the N-terminal domain (ResT(1-163)) exhibits sequence-independent DNA binding activity. Further analysis by DNase I footprinting supports a model for telomere resolution in which the hairpin binding module of the N-terminal domain is delivered to the box 1,2 region of the telomere through its tethering to ResT(164-449). Conversely, ResT(1-164) may play an important regulatory role by modulating both sequence-specific DNA binding activity and catalysis by the C-terminal domain.

Assembly of an active group II intron-maturase complex by protein dimerization.

Group II intron-encoded proteins promote both splicing and mobility of the intron RNA through formation of a specific RNA-protein complex. The Lactococcus lactis L1.LtrB intron encodes a maturase, LtrA, with reverse transcriptase homology and specific binding affinity for two domains of the intron RNA. The catalytically active ribonucleoprotein (RNP) has splicing, endonuclease, and reverse transcriptase activity, enabling efficient insertion of the intron sequence by a retro-homing mechanism. To determine the composition and assembly mechanism of the RNP complex, purified LtrA protein was analyzed for its ability to recognize a series of intron-derived RNAs. Equilibrium dissociation measurements show that LtrA recognizes two intronic domains, DI and DIV. However, distinct electrostatic requirements for binding imply different modes of molecular recognition in each case. Stoichiometric binding experiments show that the functional RNP complex consists of a dimeric protein species bound to a single intron RNA. Significant differences between the measured equilibrium dissociation constants and kinetically derived values suggest that conformational changes accompany assembly of the intron-maturase complex, and results of limited proteolysis and fluorescence spectroscopy experiments suggest that significant RNA-dependent structural changes within the maturase occur upon association with the intron. These results support a mutually induced fit model in which RNA-dependent conformational changes within LtrA enable stable association of the protein dimer with two independent intron domains to form a functional RNP.

Design and properties of human D-amino acid oxidase with covalently attached flavin

An "artificial flavinylation" approach was developed to replace a native noncovalent flavin prosthetic group with a covalently attached flavin analogue in recombinant human d-amino acid oxidase. The protein residue Gly-281 was replaced with Cys by site-directed mutagenesis, followed by reaction between mutated apoenzyme and the thiol-reactive flavin analogue, 8-methylsulfonyl FAD. The stoichiometric process of flavin attachment was accompanied by gain in enzymatic activity, reaching up to 26% activity of the recombinant native enzyme. The steady-state kinetic data together with the results of limited proteolysis and benzoate-binding studies suggest that, although mutation perturbs protein structural and catalytic properties, the flavinylation alone does not have any negative impact. We conclude that, despite the implemented restraints on its mobility, the covalently attached flavin is properly positioned within the protein active site and acts efficiently during d-amino acid oxidase catalytic turnover.

Design and properties of human D-amino acid oxidase with covalently attached flavin.

An "artificial flavinylation" approach was developed to replace a native noncovalent flavin prosthetic group with a covalently attached flavin analogue in recombinant human d-amino acid oxidase. The protein residue Gly-281 was replaced with Cys by site-directed mutagenesis, followed by reaction between mutated apoenzyme and the thiol-reactive flavin analogue, 8-methylsulfonyl FAD. The stoichiometric process of flavin attachment was accompanied by gain in enzymatic activity, reaching up to 26% activity of the recombinant native enzyme. The steady-state kinetic data together with the results of limited proteolysis and benzoate-binding studies suggest that, although mutation perturbs protein structural and catalytic properties, the flavinylation alone does not have any negative impact. We conclude that, despite the implemented restraints on its mobility, the covalently attached flavin is properly positioned within the protein active site and acts efficiently during d-amino acid oxidase catalytic turnover.

Reconstitution into liposomes of highly purified PO glycoprotein from avian peripheral nerve myelin

To explore the adhesive, possibly fusogenic properties of the major protein of peripheral nerve myelin (PO protein), three methods were employed for the reconstitution of this glycoprotein into liposomes of various lipid compositions. The most successful technique for the preparation of proteoliposomes was a detergent solubilization that employed Bio-Beads for the removal of Triton X-100 from the PO protein – lipid – detergent mixture. This method yielded a similar level of PO protein relative to lipids in the liposomes to that found in myelin. The protein to lipid ratio of the reconstituted proteoliposomes was only 1.4-fold lower than the initial protein to lipid ratio employed to prepare the liposomes. In contrast, the solvent evaporation and reverse-phase evaporation methods provided liposomes with 30 times lower protein to lipid ratio when compared with PO protein to lipid ratio initially used for the preparation of proteoliposomes. The incorporation of PO protein was dependent on the lipid composition of the vesicles and on the degree of purification of the protein. Proteolytic enzyme treatment showed that proteoliposomes containing the highly purified form of PO protein were partially or completely sensitive to trypsin treatment, whereas in liposomes containing a partially purified form of PO protein, a 22.4-kilodalton fragment was completely protected from trypsin digestion, indicating a transmembrane orientation of the partially purified polypeptide. Endoglycosidase F treatment of the liposomes established that all of the oligosaccharide moieties were removed from the liposome-associated protein, indicating the oligosaccharide moiety was exposed to the outside surface of the liposomes. Taken together, the results of limited proteolysis and endoglycosidase treatments did not provide strong evidence for the transmembrane orientation of highly purified PO in the liposomes, but were consistent with a surface of partial hairpin configuration, where the extracellular domains are facing the outside milieu. Electron microscopic examinations revealed highly convoluted, elongated shapes arid extensive fusion of PO liposomes containing lipids capable of adopting the hexagonal (H11) phase. Proteoliposomes prepared with the simple lipid composition of dipalmitoyl phosphatidylcholine -cholesterol (10:1 molar ratio) yielded stable, unilamellar liposomes of 80 nm average diameter, that encapsulated water-soluble drugs efficiently into the intravesicular compartment.Key words: liposomes, PO glycoprotein, myelin, peripheral nerve.

ATP: citrate lyase of Rhodotorula gracilis: purification and properties

ATP: citrate lyase was purified from the oleaginous yeast Rhodotorula gracilis to homogeneity as judged by polyacrylamide gel electrophoresis, using a novel citrate-Sepharose procedure. The enzyme was found to have a molecular weight of 520 000 and consisted of four identical subunits ( M r = 120 000). Two minor low molecular weight bands were observed on SDS-PAGE ( M r 51 000 and M r 49 000). Trypsin digestion experiments indicated that these could have been the result of limited proteolysis by an endogenous trypsin-like proteinase . In this respect, it resembles the mammalian ATP:citrate lyase. The enzyme was stimulated by NH 4 + ions and inhibited by palmitoyl, lauroyl, oleoyl, myristoyl and stearoyl-CoA esters, glutamate and glucose 6-phosphate but not by acetyl-CoA or shorter chain fatty acyl-CoA esters. The enzyme exhibited normal Michaelis-Menten kinetics for citrate; however there was a 3-fold increase in K m with a high concentration of Cl − ions (0.25 M). The possible regulatory roles of ATP:citrate lyase in R. gracilis are discussed in the light of these findings.

Activation of a D-form of rabbit muscle glycogen synthase by Ca 2+-activated protease

Glycogen synthase isolated from rabbit skeletal muscle as a D-form (synthase D 2) is activated by a rat liver cytosolic protein differing from any of the known protein phosphatases (D 2 activase). Although reversible by phosphorylation by cyclic AMP-dependent protein kinase, the activation is a result of limited proteolysis by D 2 activase, which has been identified as the Ca 2+-activated protease.

Single-chain structure of human ceruloplasmin.

In many reports human ceruloplasmin has been shown to contain several polypeptide chains. Their molecular weights have been estimated to 17000 and 59000. These findings formed the basis for proposed models of ceruloplasmin quarternary structure. Recently it was however observed, that the low molecular weight peptide chains found in commercial samples of human ceruloplasmin are absent if the protein is prepared directly from fresh serum, and evidence was presented that the earlier observed subunits were the result of limited proteolysis.The subunit structure of human ceruloplasmin has now been reconsidered using the new preparation. The reduced and cyanoethylated protein has been studied in solutions of 6 M guanidine hydrochloride. Its intrinsic viscosity and sedimentation coefficient, as well as data from gel filtration on calibrated columns of agarose gels are consistent with the presence of a single continuous polypeptide chain of about 1050 amino acid residues with a molecular weight around 120000. Together with the carbohydrate this accounts for the molecular weight of the native protein as determined by sedimentation equilibrium.

Studies on the activation of purified human factor XIII

1. 1. Factor XIII was purified approximately 1000-fold from human plasma. 2. 2. A quantitative assay for transamidase activity is described and was used to follow the activation of Factor XIII. 3. 3. Activation by thrombin (EC 3.4.4.13) was time-dependent, was inhibited by N a-p- toluenesulphonyl- l-arginine methyl ester, had a pH optimum at 8.0, and gave a value of 1.2·10 −6 M for K m . 4. 4. Unlike bovine Factor XIII, thrombin activation of the human material proceed in the absence of calcium. 5. 5. Activation by thrombin may be the result of limited proteolysis somewhat analogous to that responsible for the fibrinogen to fibrin conversion; the ability of reptilase to activate Factor XIII supports this idea though another coagulant enzyme, Arvin, was ineffective.