Tryptophan Fluorescence Emission Spectra Research Articles

PASylated Urate Oxidase Enzyme: Enhancing Biocatalytic Activity, Physicochemical Properties, and Plasma Half-Life.

Recombinant urate oxidase (UOX, E.C.1.7.3.3) is an important therapeutic enzyme used in preventing and treating chemotherapy-induced hyperuricemia and severe gout. However, UOX use is limited due to the poor stability and short plasma half-life. To solve this problem, we designed three PASylated variants of Aspergillus flavus UOX with different PAS sequences at the C- or N-terminus. The genes of native and PASylated variants (UOX-PAS20, PAS24-UOX, and UOX-PAS100) were designed and produced in Escherichia coli strain BL21 (DE3). The expressed recombinant native and PASylated enzymes were compared in terms of biophysical properties, kinetics parameters, and pharmacokinetics behavior using standard methods. PASylation of UOX with PAS100 polymer caused a 1.24-fold reduction in K m to 52.61 μM, and a 3.87-fold increase in K cat/K m for uric acid compared to the native variant. UOX-PAS100 retained its activity in different temperatures (20-55 °C); however, other variants lost nearly 50% of their original activity at 55 °C. UOX-PAS100 exhibited a 1.78-fold increase in hydrodynamic radius and a 1.64-fold larger apparent molecular size in comparison to the native UOX. Circular dichroism (CD) spectroscopy demonstrated that the addition of the PAS tag does not change the secondary structure of the fusion enzyme. The tryptophan fluorescence emission spectra for PASylated enzymes showed a significant modification in the conformational state of UOX by the PAS polymer presence. UOX-PAS100 retained 89.0% of the original activity following 72 h incubation in the presence of plasma at 37 °C. However, only about 61.0%, 57.0%, 50.0%, and 52.0% of activity from PAS24-UOX, UOX-PAS20, native UOX, and rasburicase (Fasturtec, Italy) remained, respectively, at the identical time. UOX-PAS100 had an increased biological half-life (8.21 h) when compared with the rasburicase (3.12 h) and native UOX (2.87 h) after being injected into a rat. Having considering everything, our results suggest that the UOX-PAS100, an A. flavus UOX fused with a C-terminally 100 amino acid PAS-residue, is a proper candidate with enhanced biological activity and extended plasma half-life for clinical therapy in patients suffering from hyperuricemia.

Bluetongue virus viral protein 7 stability in the presence of glycerol and sodium chloride

PurposeThe Orbivirus Bluetongue virus (BTV) is an economically significant disease that affects mainly wild and domestic ruminants. BTV is most often seen symptomatically in sheep, but is easily carried by goats, cattle, and wild ruminants. To date there are several problems with the vaccines currently available for BTV, and one of the most promising candidates to increase vaccine efficacy is a protein-based vaccine, for which viral protein 7 (VP7) is a great candidate to be included in it. In order to further these studies, the stability of BTV VP7 in common vaccine additives needs to be investigated.Materials and MethodsRecombinant BTV VP7 was expressed in a bacterial cell system and purified before being analysed using spectroscopic techniques including far-ultraviolet (UV) circular dichroism and intrinsic tryptophan fluorescence. BTV was analysed in a number of different buffer conditions.ResultsWe report here that BTV VP7 maintains its native secondary structure until at least 52℃ and native-like tertiary structure to at least 80℃. Far-UV circular dichroism and intrinsic tryptophan fluorescence emission spectra indicate significant secondary and tertiary structure remaining even at 90℃, respectively. Six M guanidinium chloride is able to unfold BTV VP7 while 8 M urea could not.ConclusionTwenty percent glycerol and 300 mM sodium chloride appear to have a protective effect on BTV VP7's structure, as significantly more structure is seen at 90℃ when compared to BTV VP7 without the addition of these chemicals. Both glycerol and sodium chloride are common vaccine additives.

Polynuclear zinc(II) complexes of thiosemicarbazone: Synthesis, X-ray structure and biological evaluation

Two new dimeric Zn(II) ([{ZnL1(DMSO2)}2]·DMSO (1), [{ZnL2Cl}2] (2)) and a novel tetrameric Zn(II) complex ([(Zn2L3)2(μ–OAc)2(μ3–O)2] (3)), where H2L1 = 4–(p–methoxyphenyl) thiosemicarbazone of o–hydroxynapthaldehyde, HL2 = 4–(p–methoxyphenyl)thiosemicarbazone of benzoyl pyridine and H2L3 = 4–(p–chlorophenyl)thiosemicarbazone of o–vanillin are reported. Ligands and their complexes were characterized by spectroscopic and single crystal X–ray diffraction techniques. In addition, the complexes exhibited good binding affinity towards HSA (1012 M−1), which is supported by their ability to quench the tryptophan fluorescence emission spectra of HSA. The complexes were also screened for their DNA binding propensity through UV–vis absorption titration, circular dichroism and fluorescence spectral studies. Results show that they effectively interact with CT–DNA through an intercalative mode of binding, with binding constants ranging from 103 to 104 M−1. Among the three complexes 1 has the highest binding affinity towards CT–DNA. Further, the phosphatase activity was evaluated using bis(2,4–dinitrophenyl)phosphate (BDNPP) as substrate, however, the complexes did not yield any measurable catalytic activity. Nevertheless the complexes showed significant cytotoxic potential against HeLa and HT–29 cancer cell lines that was assessed through MTT assay and DAPI staining. Remarkably, complex 1 showed better activity than cisplatin against HT–29 cell line.

Elucidating the role of surfactant dispersed CNTs towards HSA fibrillation in vitro — A multiple spectroscopic approach

Nanoparticles could act as the efficient catalyst or the inhibitor towards the fibrillation pathway of biomolecules. In the present work, spectroscopic approaches including Thioflavin T fluorescence, Congo red dye absorbance, tryptophan fluorescence emission, three-dimensional fluorescence and circular dichroism spectra has been used to detect and evaluate the fibrillation induced conformational changes in HSA in the existence of CNTs. CR dye absorbance showed the red shift from 495 to 500nm upon binding with fibrils. Similarly, ThT fluorescence emission upon the excitation at 450nm showed the gradual increase in the intensity that conveyed the enhanced formation of fibrils in the presence of CNTs. Trp emission maxima obtained at 344nm gradually decreased with no prominent shift in the fibrillar samples. Changes in the 3D spectral peaks 1 and 2 of native and fibrillar solutions suggested the alteration in the aromatic amino acid residues micro-environment. CD results showed increased percentage contents of beta-sheet structures in the fibrillar solutions interacted with CNTs. Hence, the research outcome of the study conveys a promoting role of surfactant detached SW and MWCNTs against HSA fibrillation in vitro.

Characterization of Membrane Interactions of Antimicrobial Lactoferricin Peptides with Central Residue Substitutions

The rise in antibiotic-resistant bacteria has led to an active search for new and more effective antimicrobial drugs. A hexapeptide (LfB6: RRWQWR-NH2) derived from the iron-binding protein lactoferrin exhibits antimicrobial activity (Tomita, Acta Paediatr Jpn, 1994, 36:585). A related heptapeptide produced in our lab, with 4 positively charged arginines and 2 methylated tryptophans (RRMeWQMeWRR-NH2; MeTrp-LfB7), exhibits enhanced activity against gram negative and gram positive bacteria. Substitutions of the central glutamine (Gln4;Q) residue that may alter peptide conformational flexibility are now being investigated. When Gln4 was changed to glycine (Gly;G) or proline (Pro;P), significant changes in peptide-membrane interactions were observed, although the antimicrobial activity was not increased. We now examine the effects of replacing Gln4 with gamma amino butyric acid (GABA), to introduce more flexibility; or D-Pro-Gly, to constrain the backbone into a β-hairpin turn (Stanger and Gellman, J. Am. Chem. Soc. 1998, 120:4236). The increased positive ellipticity at ∼230 nm observed in the CD spectrum of the D-Pro-Gly peptide in anionic membranes suggests significant Trp-Trp interactions. Tryptophan fluorescence emission spectra indicate that peptides with either GABA or D-Pro-Gly substitutions are more deeply buried in anionic membranes. Although the GABA and D-Pro-Gly peptides were both more active against gram negative (E. coli), compared to gram positive (S. aureus) bacteria, they were not as active as the Gln4 peptide. Investigations of the peptide-lipid interactions are being continued by means of solid-state 2H and 31P NMR spectra.

Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein’s regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The kcat/Kphe value is down 104 for the mutant enzyme, and the Km value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

Characterization and Solution Structure of Mouse Myristoylated Methionine Sulfoxide Reductase A

Methionine sulfoxide reductase A is an essential enzyme in the antioxidant system which scavenges reactive oxygen species through cyclic oxidation and reduction of methionine and methionine sulfoxide. The cytosolic form of the enzyme is myristoylated, but it is not known to translocate to membranes, and the function of myristoylation is not established. We compared the biochemical and biophysical properties of myristoylated and nonmyristoylated mouse methionine sulfoxide reductase A. These were almost identical for both forms of the enzyme, except that the myristoylated form reduced methionine sulfoxide in protein much faster than the nonmyristoylated form. We determined the solution structure of the myristoylated protein and found that the myristoyl group lies in a relatively surface exposed "myristoyl nest." We propose that this structure functions to enhance protein-protein interaction.

Structural and Functional Relationships between the Lectin and Arm Domains of Calreticulin

Calreticulin and calnexin are key components in maintaining the quality control of glycoprotein folding within the endoplasmic reticulum. Although their lectin function of binding monoglucosylated sugar moieties of glycoproteins is well documented, their chaperone activity in suppressing protein aggregation is less well understood. Here, we use a series of deletion mutants of calreticulin to demonstrate that its aggregation suppression function resides primarily within its lectin domain. Using hydrophobic peptides as substrate mimetics, we show that aggregation suppression is mediated through a single polypeptide binding site that exhibits a K(d) for peptides of 0.5-1 μM. This site is distinct from the oligosaccharide binding site and differs from previously identified sites of binding to thrombospondin and GABARAP (4-aminobutyrate type A receptor-associated protein). Although the arm domain of calreticulin was incapable of suppressing aggregation or binding hydrophobic peptides on its own, it did contribute to aggregation suppression in the context of the whole molecule. The high resolution x-ray crystal structure of calreticulin with a partially truncated arm domain reveals a marked difference in the relative orientations of the arm and lectin domains when compared with calnexin. Furthermore, a hydrophobic patch was detected on the arm domain that mediates crystal packing and may contribute to calreticulin chaperone function.

Evidence for the Involvement of Zinc in the Association of CRISP1 with Rat Sperm During Epididymal Maturation

Rat epididymal protein CRISP1 (cysteine-rich secretory protein 1) associates with sperm during maturation and participates in fertilization. Evidence indicates the existence of two populations of CRISP1 in sperm: one loosely bound and released during capacitation, and one strongly bound that remains after this process. However, the mechanisms underlying CRISP1 binding to sperm remain mostly unknown. Considering the high concentrations of Zn(2+) present in the epididymis, we investigated the potential involvement of this cation in the association of CRISP1 with sperm. Caput sperm were coincubated with epididymal fluid in the presence or absence of Zn(2+), and binding of CRISP1 to sperm was examined by Western blot analysis. An increase in CRISP1 was detected in sperm exposed to Zn(2+), but not if the cation was added with ethylenediaminetetra-acetic acid (EDTA). The same results were obtained using purified CRISP1. Association of CRISP1 with sperm was dependent on epididymal fluid and Zn(2+) concentrations and incubation time. Treatment with NaCl (0.6 M) removed the in vitro-bound CRISP1, indicating that it corresponds to the loosely bound population. Flow cytometry of caput sperm exposed to biotinylated CRISP1/avidin-fluorescein isothiocyanate revealed that only the cells incubated with Zn(2+) exhibited an increase in fluorescence. When these sperm were examined by epifluorescence microscopy, a clear staining in the tail, accompanied by a weaker labeling in the head, was observed. Detection of changes in the tryptophan fluorescence emission spectra of CRISP1 when exposed to Zn(2+) supported a direct interaction between CRISP1 and Zn(2+). Incubation of either cauda epididymal fluid or purified CRISP1 with Zn(2+), followed by native-PAGE and Western blot analysis, revealed the presence of high-molecular-weight CRISP1 complexes not detected in fluids treated with EDTA. Altogether, these results support the involvement of CRISP1-Zn(2+) complexes in the association of the loosely bound population of CRISP1 with sperm during epididymal maturation.

Interaction of Extracellular Domain 2 of the Human Retina-specific ATP-binding Cassette Transporter (ABCA4) with All-trans-retinal

The retina-specific ATP-binding cassette (ABC) transporter, ABCA4, is essential for transport of all-trans-retinal from the rod outer segment discs in the retina and is associated with a broad range of inherited retinal diseases, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. A unique feature of the ABCA subfamily of ABC transporters is the presence of highly conserved, long extracellular loops or domains (ECDs) with unknown function. The high degree of sequence conservation and mapped disease-associated mutations in these domains suggests an important physiological significance. Conformational analysis using CD spectroscopy of purified, recombinant ECD2 protein demonstrated that it has an ordered and stable structure composed of 27 +/- 3% alpha-helix, 20 +/- 3% beta-pleated sheet, and 53 +/- 3% coil. Significant conformational changes were observed in disease-associated mutant proteins. Using intrinsic tryptophan fluorescence emission spectrum of ECD2 polypeptide and fluorescence anisotropy, we have demonstrated that this domain specifically interacts with all-trans-retinal. Furthermore, the retinal interaction appeared preferential for the all-trans-isomer and was directly measurable through fluorescence anisotropy analysis. Our results demonstrate that the three macular degeneration-associated mutations lead to significant changes in the secondary structure of the ECD2 domain of ABCA4, as well as in its interaction with all-trans-retinal.

Replacement of Phe274 with conserved residue Tyr274 for reactive center loop expulsion in antithrombin.

The reactive center loop (RCL) of native antithrombin is partially inserted in the main serpin body. It must be fully exposed for optimal inhibitory function. To test the hypothesis that P(14)-s2B interaction affects loop insertion in antithrombin. By mutating Phe(274) to Tyr(274), the objective was to introduce P(14)-s2B interaction in antithrombin. Site-directed mutagenesis and affinity chromatography were used to obtain purified recombinant protein. Antithrombin's ability to form sodium dodecyl sulfate (SDS)-stable complex with thrombin, stoichiometry of thrombin inhibition, second-order rate constant for thrombin and factor Xa (fXa) inhibition (M(-1) s(-1)), and heparin dissociation constant (K(D); tryptophan fluorescence emission spectra) were determined. A marginal, but inconclusive, difference between the wild type and the mutant was observed. The result highlights the variable effect of P(14)-s2B interaction in different serpins. Alternate hypothesis for achieving loop expulsion is proposed.

Biorecognition of Chemically Modified Bovine Serum Albumin with Lactose Prepared under Different Conditions

Glycoconjugates consist of glycans attached to proteins or lipids. Glycans are involved in important biological functions such as trafficking of glycoconjugates, mediation, and modulation of cell adhesion and signaling. This study was conducted to obtain neoglycoconjugates containing a large number of carbohydrates, added through the condensation of reducing sugars with protein amino groups, whose structures were recognized by lectins. Neoglycoconjugates (BSA-Lac) of bovine serum albumin (BSA) with d-lactose were obtained using two sets of glycation conditions, each previously selected by its ability to glycate proteins extensively. The conditions included dry heat at 60 degrees C (for 7, 14, 21, and 28 days) and wet heat in 43% relative humidity (RH) at 50 degrees C (for 5, 10, 15, and 20 h). Products were characterized by gel electrophoresis, tryptophan fluorescence emission spectra, mass spectrometry, free amino group analysis, and their biological recognition established by a galactose-specific lectin and Escherichia coli K88 adhesins. BSA-Lac when compared to BSA revealed an increase in monomer mass due to addition of either 13 (dry heat) or 14 (wet heat) lactoses and formation of polymers (in wet conditions). All BSA-Lac products showed reduced intensity of intrinsic fluorescence, decreased amino groups' availability, and were recognized by Ricinus communis I lectin (RCAI) and by E. coli K88 adhesins. Overall, glycation using both conditions was time-dependent, but greater biorecognition was observed with wet-heat products, due to a higher global glycation and/or to the carbohydrate accessibility. The strategy used in this work represents a simple procedure to obtain glycoconjugates that could be used for recognition studies in biological systems.

Characterization of the mechanism of action of the genetically modified Cry1AbMod toxin that is active against Cry1Ab-resistant insects

Bacillus thuringiensis Cry toxins are used in the control of insect pests. They are pore-forming toxins with a complex mechanism that involves the sequential interaction with receptors. They are produced as protoxins, which are activated by midgut proteases. Activated toxin binds to cadherin receptor, inducing an extra cleavage including helix α-1, facilitating the formation of a pre-pore oligomer. The toxin oligomer binds to secondary receptors such as aminopeptidase and inserts into lipid rafts forming pores and causing larval death. The primary threat to efficacy of Bt-toxins is the evolution of insect resistance. Engineered Cry1AMod toxins, devoid of helix α-1, could be used for the control of resistance in lepidopterans by bypassing the altered cadherin receptor, killing resistant insects affected in this receptor. Here we analyzed the mechanism of action of Cry1AbMod. We found that alkaline pH and the presence of membrane lipids facilitates the oligomerization of Cry1AbMod. In addition, tryptophan fluorescence emission spectra, ELISA binding to pure aminopeptidase receptor, calcein release assay and analysis of ionic-conductance in planar lipid bilayers, indicated that the secondary steps in mode of action that take place after interaction with cadherin receptor such as oligomerization, receptor binding and pore formation are similar in the Cry1AbMod and in the wild type Cry1Ab. Finally, the membrane-associated structure of Cry1AbMod oligomer was analyzed by electron crystallography showing that it forms a complex with a trimeric organization.

An Unconventional Form of Actin in Protozoan Hemoflagellate, Leishmania

Leishmania actin was cloned, overexpressed in baculovirus-insect cell system, and purified to homogeneity. The purified protein polymerized optimally in the presence of Mg2+ and ATP, but differed from conventional actins in its following properties: (i) it did not polymerize in the presence of Mg2+ alone, (ii) it polymerized in a restricted range of pH 7.0-8.5, (iii) its critical concentration for polymerization was found to be 3-4-fold lower than of muscle actin, (iv) it predominantly formed bundles rather than single filaments at pH 8.0, (v) it displayed considerably higher ATPase activity during polymerization, (vi) it did not inhibit DNase-I activity, and (vii) it did not bind the F-actin-binding toxin phalloidin or the actin polymerization disrupting agent Latrunculin B. Computational and molecular modeling studies revealed that the observed unconventional behavior of Leishmania actin is related to the diverged amino acid stretches in its sequence, which may lead to changes in the overall charge distribution on its solvent-exposed surface, ATP binding cleft, Mg2+ binding sites, and the hydrophobic loop that is involved in monomer-monomer interactions. Phylogenetically, it is related to ciliate actins, but to the best of our knowledge, no other actin with such unconventional properties has been reported to date. It is therefore suggested that actin in Leishmania may serve as a novel target for design of new antileishmanial drugs.

Rheology of whey protein–dextran conjugate films at the air/water interface

The film properties of whey protein isolate (WPI) and WPI–dextran Maillard conjugates with different levels of dextran attachment were examined using surface shear and dilatational rheology. The changes in film properties were assessed in relation to the changes in protein unfolding induced by various levels of attachment of dextrans of different molecular weight. Attachment of ∼1 dextran per mole of WPI (low conjugation) did not change the surface modulus or the surface dilatational modulus. At the low level of conjugation there was little effect on protein structure, as measured by the negligible effect of dextran attachment on tryptophan fluorescence emission and circular dichroism spectra. Increasing the number of dextrans attached to ∼5 dextrans per mole WPI (moderate conjugation), which caused substantial protein unfolding, markedly decreased both the surface and dilatational moduli. The results suggested that the loss of film strength was a result of protein unfolding. The consequences of this are discussed in terms of the origin of film strength and in relation to the functional behaviour of these interfacial films.

Importance in Catalysis of the 6-Phosphate-binding Site of 6-Phosphogluconate in Sheep Liver 6-Phosphogluconate Dehydrogenase

The 6-phosphate of 6-phosphogluconate (6PG) is proposed to anchor the sugar phosphate in the active site and aid in orientating the substrate for catalysis. In order to test this hypothesis, alanine mutagenesis was used to probe the contribution of residues in the vicinity of the 6-phosphate to binding of 6PG and catalysis. The crystal structure of sheep liver 6-phosphogluconate dehydrogenase shows that Tyr-191, Lys-260, Thr-262, Arg-287, and Arg-446 contribute a mixture of ionic and hydrogen bonding interactions to the 6-phosphate, and these interactions are likely to provide the majority of the binding energy for 6PG. All mutant enzymes, with the exception of T262A, exhibit an increase in K(6PG) that ranges from 5- to 800-fold. There is also a less pronounced increase in K(NADP), ranging from 3- to 15-fold, with the exception of T262A. The R287A and R446A mutant enzymes exhibit a dramatic decrease in V/E(t) (600- and 300-fold, respectively) as well as in V/K(6PG)E(t) (10(5) - and 10(4)-fold), and therefore no further characterization was carried out with these two mutant enzymes. No change in V/E(t) was observed for the Y191A mutant enzyme, whereas 20- and 3-fold decreases were obtained for the K260A and T262A mutant enzymes, respectively, resulting in a decrease in V/K(6PG)E(t) range from 3- to 120-fold. All mutant enzymes also exhibit at least an order of magnitude increase in 13C-isotope effect -1, indicating that the decarboxylation step has become more rate-limiting. Data are consistent with significant roles for Tyr-191, Lys-260, Thr-262, Arg-287, and Arg-446 in providing the binding energy for 6PG. In addition, these residues also likely ensure proper orientation of 6PG for catalysis and aid in inducing the conformation change that precedes, and sets up the active site for, catalysis.

Betaine aldehyde dehydrogenase from Pseudomonas aeruginosa: cloning, over-expression in Escherichia coli, and regulation by choline and salt

In the human pathogen Pseudomonas aeruginosa, betaine aldehyde dehydrogenase (BADH) may play a dual role assimilating carbon and nitrogen from choline or choline precursors--abundant at infection sites--and producing glycine betaine, which protects the bacteria against the high-osmolarity stress prevalent in the infected tissues. We cloned the P. aeruginosa BADH gene and expressed the BADH protein in Escherichia coli. The recombinant protein appears identical to its native counterpart, as judged by Western blot, N-terminal amino acid sequence, tryptophan-fluorescence emission spectra, circular-dichroism spectroscopy, size-exclusion chromatography, and kinetic properties. Computational analysis indicated that the promoter sequence of the putative operon that includes the BADH gene has a consensus-binding site for the choline-sensing transcription repressor BetI, and putative boxes for ArcA and Lrp transcription factors but no known elements of response to osmotic stress. This is consistent with the strong induction of BADH expression by choline and with the lack of effect of NaCl. As there were significant amounts of BADH protein and activity in P. aeruginosa cells grown on glucose plus choline, as well as the BADH activity exhibiting tolerance to salt, it is likely that glycine betaine is synthesized in vivo and could play an important osmoprotectant role under conditions of infection.

Tyrosine Modification Is Not Required for Myeloperoxidase-induced Loss of Apolipoprotein A-I Functional Activities

Apolipoprotein A-I (apoAI), the major protein of high density lipoprotein, plays an important role in reverse cholesterol transport via its activity as an ABCA1-dependent acceptor of cellular cholesterol. We reported recently that myeloperoxidase (MPO) modification of apoAI inhibits its ABCA1-dependent cholesterol acceptor activity (Zheng, L., Nukuna, B., Brennan, M. L., Sun, M., Goormastic, M., Settle, M., Schmitt, D., Fu, X., Thomson, L., Fox, P. L., Ischiropoulos, H., Smith, J. D., Kinter, M., and Hazen, S. L. (2004) J. Clin. Invest. 114, 529-541). We also reported that MPO-mediated chlorination preferentially modifies two of the seven tyrosines in apoAI, and loss of parent peptides containing these residues dose-dependently correlates with loss in ABCA1-mediated cholesterol acceptor activity (Zheng, L., Settle, M., Brubaker, G., Schmitt, D., Hazen, S. L., Smith, J. D., and Kinter, M. (2005) J. Biol. Chem. 280, 38-47). To determine whether oxidative modification of apoA-I tyrosine residues was responsible for the MPO-mediated inactivation of cholesterol acceptor activity, we made recombinant apoAI with site-specific substitutions of all seven tyrosine residues to phenylalanine. ApoAI and the tyrosine-free apoAI were equally susceptible to dose-dependent MPO-mediated loss of ABCA1-dependent cholesterol acceptor activity, as well as lipid binding activity. MPO modification altered the migration of apoAI on SDS gels and decreased its alpha-helix content. MPO-induced modification also targeted apoAI tryptophan and lysine residues. Specifically, we detected apoAI tryptophan oxidation to mono- and dihydroxytryptophan and apoAI lysine modification to chlorolysine and 2-aminoadipic acid. Thus, tyrosine modification of apoAI is not required for its MPO-mediated inhibition of cholesterol acceptor activity.

Biochemical properties of Hsp70 chaperone system from Meiothermus ruber

The guanidine-hydrochloride (Gdn-HCl) and thermally induced unfolding of Hsp70 from Meiothermus ruber (Mru.Hsp70) were analysed using tryptophan fluorescence and 8-anilino-1-naphthalenesulfonic acid (ANS) binding. The ANS binding to Mru.Hsp70 showed both the increase in fluorescence intensity and a shift in emission maximum. Analysis of the unfolding profile of Mru.Hsp70 indicated that Gdn-HCl induced unfolding of Mru.Hsp70 occurred through intermediate species. The tryptophan and ANS fluorescence emission spectra revealed that ATP induced conformational change increased the thermal stability of Mru.Hsp70. The data obtained are similar to those of Escherichia coli DnaK. The ATP-ase activity of chaperones is fundamental for their biological activity. It this paper we demonstrate that, in contrast to Thermus thermophilus, both Mru.Hsp40 and Mru.Hsp22 co-chaperones affect the ATP-ase activity of Mru.Hsp70. The use of truncated Mru.Hsp40 proteins showed that full-length Mru.Hsp40 is required for stimulation of ATP-ase activity of Mru.Hsp70. E. coli GrpE could act as nucleotide exchange factor the in thermophilic Hsp70 ATP hydrolysis reaction. However, the role of E. coli DnaJ in the M. ruber ATP cycle needs further analysis. We selected the new substrate laccA suitable for determination of refolding activity of thermophilic chaperones.

Hydrophobic Core Variant Ubiquitin Forms a Molten Globule Conformation at Acidic pH

The conformational properties of hydrophobic core variant ubiquitin (Val26 to Ala mutation) in an acidic solution were studied. The intrinsic tryptophan fluorescence emission spectrum, far-UV and near-UV circular dichroic spectra, the fluorescence emission spectrum of 8-anilinonaphthalene-1-sulfonic acid in the presence of V26A ubiquitin, and urea-induced unfolding measurements indicate this variant ubiquitin to be in the partially folded molten globule conformation in solution at pH 2. The folding kinetics from molten globule to the native state was nearly identical to those from the unfolded state to the native state. This observation suggests that the equilibrium molten globule state of hydrophobic core variant ubiquitin is an on-pathway folding intermediate.