Calculated electrostatic gradients in recombinant human H-chain ferritin

Calculations to determine the electrostatic potential of the iron storage protein ferritin, using the human H-chain homopolymer (HuHF), reveal novel aspects of the protein. Some of the charge density correlates well with regions previously identified as active sites in the protein. The three-fold channels, the putative ferroxidase sites, and the nucleation sites all show expectedly negative values of the electrostatic potential. However, the outer entrance to the three-fold channels are surrounded by regions of positive potential, creating an electrostatic field directed toward the interior cavity. This electrostatic gradient provides a guidance mechanism for cations entering the protein cavity, indicating the three-fold channel as the major entrance to the protein. Pathways from the three-fold channels, indicated by electrostatic gradients on the inner surface, lead to the ferroxidase center, the nucleation center and to the interior entrance to the four-fold channel. Six glutamic acid residues at the nucleation site give rise to a region of very negative potential, surrounding a small positively charged center due to the presence of two conserved arginine residues, R63, in close proximity (4.9 A), suggesting that electrostatic fields could also play a role in the nucleation process. A large gradient in the electrostatic potential at the 4-fold channel gives rise to a field directed outward from the internal cavity, indicating the possibility that this channel functions to expel cations from inside the protein. The 4-fold channel could therefore provide an exit pathway for protons during mineralization, or iron leaving the protein cavity during de-mineralization.     

Expression and characterization of recombinant Manduca sexta serpin-1B and site-directed mutants that change its inhibitory selectivity

Hemolymph of Manduca sexta contains a number of serine proteinase inhibitors from the serpin superfamily. During formation of a stable complex between a serpin and a serine proteinase, the enzyme cleaves a specific peptide bond in an exposed loop (the reactive-site region) at the surface of the serpin. The amino acid residue on the amino-terminal side of this scissile bond, the P1 residue, is important in defining the selectivity of a serpin for inhibiting different types of serine proteinases. M. sexta serpin-1B, with alanine at the position predicted from sequence alignments to be the P1 residue, was previously named alaserpin. This alanyl residue was changed by site-directed mutagenesis to lysine (A343K) and phenylalanine (A343F). The serpin-1B cDNA and its mutants were inserted into an expression vector, H6pQE-60, and the serpin proteins were expressed in Escherichia coli. Affinity-purified recombinant serpins selectively inhibited mammalian serine proteinases: serpin-1B inhibited elastase; serpin-1B(A343K) inhibited trypsin, plasmin, and thrombin; serpin-1B(A343F) inhibited chymotrypsin as well as trypsin. All three serpins inhibited human cathepsin G. This insect serpin and its site-directed mutants associated with mammalian serine proteinases at rates similar to those reported for mammalian serpins. Serpin-1B and its mutants formed SDS-stable complexes with the enzymes they inhibited. The scissile bond was determined to be between residues 343 and 344 in wild-type serpin-1B and in serpin-1B with mutations at residue 343. These results demonstrate that the P1 alanine residue defines the primary selectivity of serpin-1B for elastase-like enzymes, and that this selectivity can be altered by mutations at this position.     

Aggregation of recombinant human interferon gamma: kinetics and structural transitions

Protein aggregation is a complex phenomenon that can occur in vitro and in vivo, usually resulting in the loss of the protein's biological activity. While many aggregation studies focus on a mechanism due to a specific stress, this study focuses on the general nature of aggregation. Recombinant human interferon-gamma (rhIFN-gamma) provides an ideal model for studying protein aggregation, as it has a tendency to aggregate under mild denaturing stresses (low denaturant concentration, temperature below the Tm, and below pH 5). All of the aggregates induced by these stresses have a similar structure (high in intermolecular beta-sheet content and a large loss of alpha-helix) as determined by infrared and circular dichroism spectroscopy. Thermally induced and denaturant-induced aggregation processes follow first-order kinetics under the conditions of this study. Spectroscopic and kinetic data suggest that rhIFN-gamma aggregates through an intermediate form possessing a large amount of residual secondary structure. In contrast to the aggregates formed under denaturing stresses, the salted-out protein has a remarkably nativelike secondary structure.     

Amylobarbitone hydroxylation kinetics in small samples of rat and human liver

1. Two micro methods for determining amylobarbitone hydroxylase activity from less than 3 mg liver are described. One is based on single-ion monitoring g.l.c.-mass spectrometry and the other on t.l.c. separation of 14C-labelled product. 2. Km and Vmax have been determined with rat liver and both needle and open biopsy samples of human liver. 3. Both methods are sufficiently sensitive and reproducible for use with 20 mg needle biopsies.     

Probing the folate-binding site of human thymidylate synthase by site-directed mutagenesis. Generation of mutants that confer resistance to raltitrexed, Thymitaq, and BW1843U89

Human thymidylate synthase (TS) contains three highly conserved residues Ile-108, Leu-221, and Phe-225 that have been suggested to be important for cofactor and antifolate binding. To elucidate the role of these residues and generate drug-resistant human TS mutants, 14 variants with multiple substitutions of these three hydrophobic residues were created by site-directed mutagenesis and transfected into mouse TS-negative cells for complementation assays and cytotoxicity studies, and the mutant proteins expressed and characterized. The I108A mutant confers resistance to raltitrexed and Thymitaq with respective IC50 values 54- and 80-fold greater than wild-type but less resistance to BW1843U89 (6-fold). The F225W mutant displays resistance to BW1843U89 (17-fold increase in IC50 values), but no resistance to raltitrexed and Thymitaq. It also confers 8-fold resistance to fluorodeoxyuridine. Both the kinetic characterization of the altered enzymes and formation of antifolate-resistant colonies in mouse bone marrow cells that express mutant TS are in accord with the IC50 values for cytotoxicity noted above. The human TS mutants (I108A and F225W), by virtue of their desirable properties, including good catalytic function and resistance to antifolate TS inhibitors, confirm the importance of amino acid residues Ile-108 and Phe-225 in the binding of folate and its analogues. These novel mutants may be useful for gene transfer experiments to protect hematopoietic progenitor cells from the toxic effects of these drugs.     

Viability and preliminary in vivo characterization of site-directed mutants of Escherichia coli single-stranded DNA-binding protein

Site-directed mutations involving selected amino acids of Escherichia coli single-stranded DNA-binding protein (SSB) were tested for their in vivo functionality when introduced into a chromosomal ssb deletion strain on a plasmid. All mutants complemented the ssb deletion for viability when present on a pSC101 derivative. The generation time with ssbW54S doubled in comparison to the ssb+ control, and both the ssbW54S- and ssbH55K-containing strains exhibited temperature sensitivity. ssbH55K, ssbW54S, ssbW88T, and ssbH55Y (ssb-1) strains displayed reduced survival to ultraviolet irradiation, while ssbW40T and ssbF60L strains were comparable to the ssb+ control strain. This study represents the first investigation of the in vivo properties of ssb mutations constructed for in vitro analysis of DNA binding by SSB.     

Distinct deactivation and desensitization kinetics of recombinant GABAA receptors

The functional role of the large heterogeneity in GABAA receptor subunit genes and its role in setting the properties of inhibitory synapses in the CNS is poorly understood. A kinetic comparison between currents elicited by ultra-rapid application with a piezoelectric translator of 1 mM GABA to mammalian cells transfected with cDNAs encoding distinct GABAA receptor subunits revealed that the intrinsic deactivation and desensitization properties depend on subunit combination. In particular, receptors containing alpha 6 with beta 2 gamma 2 subunits were endowed with a significantly slower deactivation as compared to those receptors containing alpha 1 with beta 2 gamma 2 subunits. While desensitization produced by prolonged GABA applications on alpha 1 beta 2 gamma 2 receptors was characterized by a rapid exponential decay followed by a slower decay and a steady state response, alpha 6 beta 2 gamma 2 receptors lacked desensitization. Furthermore, GABAA receptors lacking the gamma 2 subunit were characterized by a much larger non-desensitization component and a very rapid deactivation. Lastly, analysis of GABA-activated currents in cells cotransfected with alpha 1 and alpha 6 together with beta 2 gamma 2 subunit revealed unique kinetic properties. Our results suggest that distinct subunit composition confers specific deactivation and desensitization properties that may profoundly affect synaptic decay kinetics and the capability to sustain high frequency synaptic inputs.     

Characterization of recombinant soybean leghemoglobin a and apolar distal histidine mutants

The cDNA for soybean leghemoglobin a (Lba) was cloned from a root nodule cDNA library and expressed in Escherichia coli. The crystal structure of the ferric acetate complex of recombinant wild-type Lba was determined at a resolution of 2.2 A. Rate constants for O2, CO and NO binding to recombinant Lba are identical with those of native soybean Lba. Rate constants for hemin dissociation and auto-oxidation of wild-type Lba were compared with those of sperm whale myoglobin. At 37 degrees C and pH 7, soybean Lba is much less stable than sperm whale myoglobin due both to a fourfold higher rate of auto-oxidation and to a approximately 600-fold lower affinity for hemin. The role of His61(E7) in regulating oxygen binding was examined by site-directed mutagenesis. Replacement of His(E7) with Ala, Val or Leu causes little change in the equilibrium constant for O2 binding to soybean Lba, whereas the same mutations in sperm whale myoglobin cause 50 to 100-fold decreases in K(O2). These results show that, at neutral pH, hydrogen bonding with His(E7) is much less important in regulating O2 binding to the soybean protein. The His(E7) to Phe mutation does cause a significant decrease in K(O2) for Lba, apparently due to steric hindrance of the bound ligand. The rate constants for O2 dissociation from wild-type and native Lba decrease significantly with decreasing pH. In contrast, the O2 dissociation rate constants for mutants with apolar E7 residues are independent of pH, suggesting that hydrogen bonding to the distal histidine residue in the native protein is enhanced under acid conditions. All of these results support the hypothesis that the high affinity of Lba for oxygen and other ligands is determined primarily by enhanced accessibility and reactivity of the heme group.     

Time-resolved resonance Raman study on the binding of carbon monoxide to recombinant human myoglobin and its distal histidine mutants

Time-resolved resonance Raman (RR) spectra of the recombined species of photodissociated CO with recombinant human myoglobin (Mb) and several E7 mutants, in which distal His was replaced by Gly (H64G), Gln (H64Q), Ala (H64A), Ile (H64I), Val (H64V), and Leu (H64L) through site-directed mutagenesis, were observed in the time range -20 ns to 1 ms following photolysis. The Fe-CO stretching (VFe-CO) RR band was observed successfully with pulse excitation when the laser power was greatly reduced. H64H, H64G, and H64Q gave the VFe-CO band at 505-510 cm-1 in their stationary states. In their recovery processes 1-100 microseconds after photodissociation, a broad transient band was observed at slightly lower frequencies than those of their equilibrium structures for H64G and H64Q, but a transient VFe-CO band corresponding to the so-called "open" form was not identified around 490 cm-1 for any of the three species. A second group, H64A, H64I, H64V, and H64L, gave the main VFe-CO band at 490-495 cm-1 with a shoulder around 510 cm-1 (except for H64L) in the stationary state and exhibited a much faster recovery than the first group. These latter four species gave a broad transient band around 492-500 cm-1 in the time range of 100-1000 ns, while the approximately 510 cm-1 shoulder appeared much later. The equilibrium relative intensity of the two bands was attained at 500 microseconds, suggesting that the interconversion between the two forms is slower than 100 microseconds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steady state kinetics of 4-hydroxyphenylpyruvate dioxygenase from human liver (III)

Kinetic experiments have been made with an apparently homogenous preparation of human liver 4-hydroxyphenylpyruvate dioxygenase Form 3 (4-hydroxyphenylpyruvate: oxygen oxidoreductase (hydroxylating, decarboxylating), EC 1.13.11.27) at 37 degrees in 0.2 M Tris/HCL, pH 7.5, by measuring the evolved carbon dioxide from the 1-14C-labeled substrate or the formation of homogentisate from the U-14C-labeled substrate. The effect of variations in the concentrations of substrates, products, and metal chelators on the velocity of the forward reaction was studied. The results agree with an Ordered Bi Bi kinetic mechanism (Cleland, W. W. (1963) Biochim. Biophys. Acta 67, 104--137), where 4-hydroxyphenylpyruvate is added prior to oxygen and CO2 released before homogentisate. A Theorell-Chance mechanism has not been excluded.     

Inhibition, reactivation and aging kinetics of cyclohexylmethylphosphonofluoridate-inhibited human cholinesterases

Cyclohexylmethylphosphonofluoridate (cyclosarin) is a highly toxic organophosphate, which was shown to be rather resistant to conventional oxime therapy. To give more insight into the inhibition, reactivation and aging kinetics, human acetyl-(AChE) and butyrylcholinesterase (BChE) were inhibited by cyclosarin (k2 of 7.4 and 3.8 x 10(8) M(-1) min(-1), respectively; pH 7.4, 37 degrees C) and reactivated with obidoxime, pralidoxime and three experimental oximes. The new oxime HL? 7 (1-[[[4-aminocarbonyl)-pyridinio]-methoxy]-methyl]-2,4-bis-[ (hydroxyimino)methyl] pyridinium dimethanesulphonate) was shown to be superior to the other oximes. At oxime concentrations anticipated to be relevant in humans, obidoxime and pralidoxime were extremely weak reactivators of AChE. Aging velocity of BChE was almost fourfold higher compared to AChE (ka of 0.32 h(-1) and 0.08 h(-1), respectively). A substantial spontaneous reactivation was observed with AChE. These results support previous in vivo findings that obidoxime and pralidoxime are insufficient antidotes in cyclosarin poisoning. By contrast, HL? 7 was shown to be an extremely potent reactivator of human AChE and BChE, which supports its position as a broad-spectrum oxime.     

Molecular structures of the S124A, S124T, and S124V site-directed mutants of UDP-galactose 4-epimerase from Escherichia coli

UDP-galactose 4-epimerase plays a critical role in sugar metabolism by catalyzing the interconversion of UDP-galactose and UDP-glucose. Originally, it was assumed that the enzyme contained a "traditional" catalytic base that served to abstract a proton from the 4'-hydroxyl group of the UDP-glucose or UDP-galactose substrates during the course of the reaction. However, recent high-resolution X-ray crystallographic analyses of the protein from Escherichia coli have demonstrated the lack of an aspartate, a glutamate, or a histidine residue properly oriented within the active site cleft for serving such a functional role. Rather, the X-ray crystallographic investigation of the epimerase.NADH.UDP-glucose abortive complex from this laboratory has shown that both Ser 124 and Tyr 149 are located within hydrogen bonding distance to the 4'- and 3'-hydroxyl groups of the sugar, respectively. To test the structural role of Ser 124 in the reaction mechanism of epimerase, three site-directed mutant proteins, namely S124A, S124T, and S124V, were constructed and crystals of the S124A.NADH.UDP, S124A.NADH.UDP-glucose, S124T. NADH.UDP-glucose, and S124V.NADH.UDP-glucose complexes were grown. All of the crystals employed in this investigation belonged to the space group P3221 with the following unit cell dimensions: a = b = 83.8 A, c = 108.4 A, and one subunit per asymmetric unit. X-ray data sets were collected to at least 2.15 A resolution, and each protein model was subsequently refined to an R value of lower than 19.0% for all measured X-ray data. The investigations described here demonstrate that the decreases in enzymatic activities observed for these mutant proteins are due to the loss of a properly positioned hydroxyl group at position 124 and not to major tertiary and quaternary structural perturbations. In addition, these structures demonstrate the importance of a hydroxyl group at position 124 in stabilizing the anti conformation of the nicotinamide ring as observed in the previous structural analysis of the epimerase.NADH. UDP complex.     

Kinetic studies on the removal of iron and aluminum from recombinant and site-directed mutant N-lobe half transferrins

Kinetic studies have been conducted in pH 7.4 Hepes buffer at 25 degreesC on the removal of Fe(III) and Al(III) from the recombinant N-lobe half molecule of human serum transferrin (Tf/2N) and from the R124A, K206A, and K296A mutants of this protein. The rates of iron removal from Tf/2N by 3-hydroxypyridin-4-one (deferiprone) and nitrilotriacetic acid (NTA) are essentially identical with previous results on N-terminal monoferric transferrin (Tf-FeN). For both Tf/2N and Tf-FeN, iron removal by deferiprone follows simple saturation kinetics, while iron removal by NTA follows simple first-order kinetics. There is some discrepancy between the two proteins with respect to iron removal by PPi, but this may be due to differences in the chloride concentrations among different studies. The addition of Fe(NTA)2 to R124A at ambient bicarbonate concentrations forms the Fe-NTA-Tf ternary complex, but the usual Fe-CO3-Tf complex can be formed by adding ferrous ion in the presence of a larger excess of bicarbonate. This complex releases its iron very rapidly by a mechanism that is first-order with respect to the ligand. This suggests that the first-order component of metal release from transferrin involves the displacement of the synergistic carbonate anion. Since iron removal from K206A and K296A at pH 7.4 is extremely slow, studies have been conducted on the more labile Al3+ complexes of Tf/2N, K206A, and K296A. The removal of Al3+ from Tf/2N by PPi follows the same complex kinetic order with respect to the ligand concentration that is observed for iron removal, while the removal of Al3+ from both K206A and K296A reverts to a simple saturation process. The addition of perchlorate retards the removal of Al3+ from both K206A and K296A, suggesting that these lysine residues are not associated with the allosteric effects of inorganic anions on the rates of metal removal.     

Imidazole binding to Rhodobacter capsulatus cytochrome c2. Effect of site-directed mutants on ligand binding

Although ligand binding in c-type cytochromes is not directly related to their physiological function, it has the potential to provide valuable information on protein stability and dynamics, particularly in the region of the methionine sixth heme ligand and the nearby peptide chain that has been implicated in electron transfer. Thus, we have measured the equilibrium and kinetics of binding of imidazole to eight mutants of Rhodobacter capsulatus cytochrome c2 that differ in overall protein stability. We found that imidazole binding affinity varies 70-fold, but does not correlate with overall protein stability. Instead, each mutant exerts an effect at the local level, with the largest change due to mutant G95E (glycine substituted by glutamate), which shows 30-fold stronger binding as compared with the wild-type protein. The kinetics of imidazole binding are monophasic and reach saturation at high ligand concentrations for all the mutants and wild-type protein, which is attributed to a rate-limiting conformational change leading to breakage of the iron-methionine bond and providing a binding site for imidazole. The mutants show as much as an 18-fold variation in the first-order rate constant for the conformational change, with the largest effect found with mutant G95E. The kinetics also show a lack of correlation with overall protein stability, but are consistent with localized effects on the dynamics of hinge region 88-102 of the protein, which changes conformation to permit ligand binding. These results are consistent with R. capsulatus cytochrome c2 stabilizing the complex through hydrogen bonding to the imidazole. The larger effects of mutant G95E on equilibrium and kinetics are likely to be due to its location within the hinge region adjacent to heme ligand methionine 96, which is displaced by imidazole.     

Rat liver catechol-O-methyltransferase kinetics and assay methodology

In mammals, catechol-O-methyltransferase (COMT) is distributed throughout various organs, the highest activities being found in the liver and kidney. However, comparisons of the kinetic parameters are difficult to perform, since the experimental procedures in the enzyme assay vary quite considerably. The present work was aimed at studying the optimal liver COMT assay conditions for determining the kinetics of the enzyme. The COMT assay was performed with liver homogenates from 60 days old male Wistar rats with adrenaline (AD) as the substrate. Time course experiments using 100 microM S-adenosyl-L-methionine (SAMe) and 300 microM AD showed linearity of O-methylation reaction upto 10 min. Using 100 microM SAMe, Vmax (nmol mg protein-1 h-1) and Km (microM) values progressively decreased respectively from 22.1 and 104.8 at 5 min down to 5.8 and 24.62 at 60 min incubation periods. This decrease was not due to end-product inhibition. Using 2500 microM AD, Km values (microM) for the methyl donor SAMe increased progressively from 174 at 5 min upto 1192.5 at 60 min; upto 30 min of incubation Vmax values did not change. When a 5 min incubation period and 500 microM SAMe were used, Vmax and Km values for liver COMT were 63.4 nmol mg protein-1 h-1 and 261.1 microM, respectively. It is concluded that an incubation period of 5 min and a SAMe concentration of 500 microM provide optimal conditions for the liver homogenate COMT assay.     

Stimulation of asialoglycoprotein uptake by recombinant human hepatocyte growth factor in normal and damaged rat liver

BACKGROUND: Hepatocyte growth factor (HGF) is a strong mitogen of hepatocytes. However, little is known about the effect of HGF on the asialoglycoprotein receptors (ASGPR) of hepatocytes. The aim of this study was to identify alterations in binding of ligand to ASGPR by recombinant human HGF (rhHGF) infusion. METHODS: RhHGF was administered to rats with either normal or dimethylnitrosamine (DMN)-damaged livers. Technetium-99m-diethylenetriaminepentaacetic acid-galactosyl-human serum albumin (GSA) blood clearance was used to measure ASGPR activity. RESULTS: In normal and damaged rats, liver weight, hepatocyte nuclear size, and number of hepatocytes (cells/mm2) were not altered by rhHGF, but GSA blood clearance after rhHGF infusion was significantly increased over the preinfusion rate. CONCLUSIONS: Independent of proliferation of hepatocytes, rhHGF stimulates a hepatocytic function of the receptor-mediated uptake of ASGP.     

Purification and characterization of human topoisomerase I mutants

A system for rapid purification and characterization of eukaryotic topoisomerase-I mutants has been developed. The system utilizes six-histidine tagging of human topoisomerase I expressed in Saccharomyces cerevisiae to enable purification by nickel-affinity chromatography. Virtually homogenous mutant proteins are then tested for their ability to relax supercoiled DNA plasmids and their capacity for binding, cleaving and religating short defined DNA substrates. Relaxation-deficient mutants were obtained by site-directed mutagenesis of selected highly conserved amino acids. The mutants Tyr723Phe (active site mutation), Arg488Gln and Lys532Glu were inert in relaxation of DNA, whereas Lys720Glu showed a 50-fold reduction in specific relaxation activity. Accordingly, only Lys720Glu showed low, but detectable cleavage activity on suicide DNA substrates, uncoupling the cleavage and religation events of topoisomerase I. The relative religation efficiency of Lys720Glu comparable to that of wild-type topoisomerase I, indicating that Lys720 is involved in interactions important for normal DNA cleavage, but not for the religation reaction. All mutants could be cross linked by ultraviolet light to bromo-dUTP-substituted DNA oligonucleotides carrying a topoisomerase-I-binding site, indicating that the deficiency of Tyr723Phe, Arg488Gln and Lys532Glu in DNA relaxation and cleavage is not due to an inability of these mutants to bind DNA non-covalently.     

Characterization of folded, intermediate, and unfolded states of recombinant human interstitial collagenase

Recombinant interstitial collagenase (rMMP-1) forms insoluble inclusion bodies when over-expressed in Escherichia coli. We surveyed conditions for renaturation of purified rMMP-1 in 6 M guandine hydrochloride (GdnHCl) and found that optimal folding occurred when the denatured protein was diluted at 4 degrees C in approximately 2 M guanidine HCl, 20% glycerol, 2.5 mM reduced and oxidized glutathione, and 5 mM CaCl2, followed by buffer exchange to remove denaturant and thiols. The circular dichroism spectrum and catalytic constants of the refolded enzyme were similar to those of native MMP-1. The propeptide, which comprises approximately 20% of the mass of proMMP-1, was not required for folding to a functional enzyme. Size exclusion chromatography and spectroscopic measurements at intermediate [GdnHCl] revealed two intermediate folding states. The first, observed at 1 M GdnHCl, had a slightly larger Stokes' radius than the folded protein. CD and fluorescence analysis showed that it contained ordered tryptophan residues with a higher quantum yield than the fully folded state. The second intermediate, which appeared between 2 and 4 M GdnHCl, exhibited properties consistent with the molten globule, including secondary structure, lack of ordered tryptophan, exposed hydrophobic binding sites, and a Stokes' radius between that of the folded and unfolded states.     

Isolation of Cry1Ab protein mutants of Bacillus thuringiensis by a highly efficient PCR site-directed mutagenesis system

A site-directed mutagenesis method was designed and used to create Cry1Ab mutant proteins in two of the five highly conserved blocks present in the Cry protein family. Region 1 comprises the central alpha-helix 5 of domain I and has been implicated in the pore formation activity of the toxin. Substitution of arginine by serine at position 173 (R173S) affects neither structural integrity nor toxicity. Region 2 comprises the major part of the domain I/domain II interface, characterized by the presence of numerous hydrogen bonds and electrostatic interactions. Mutations in the salt bridge formed by aspartic acid 242 and arginine 265 (D242N, D242C, R265C, and D242C/R265C) resulted in structurally unstable mutant proteins as is shown by their increased protease sensitivity and lack of biological activity.