Equilibrium and pre-equilibrium fluorescence studies on the interaction between protein L and kappa light chain.
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Bacterial Proteins↗
Biomedical subjects
M G Gore
Publications and source records attributed to M G Gore.
Bovine inositol monophosphatase: metal-binding interactions at two different sites.
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Animals↗
Refolding and reassociation of glycerol dehydrogenase from Bacillus stearothermophilus in the absence and presence of GroEL.
The refolding of the tetrameric, metalloenzyme glycerol dehydrogenase (GDH) from Bacillus stearothermophilus has been investigated using stopped-flow fluorescence and circular dichroism spectroscopy. The effects of metal ions on the refolding of the native enzyme and the refolding of a monomeric mutant ([A208]GDH) have also been studied. The refolding process of the wild-type enzyme is at least biphasic; 70% of the respective signal changes occur in the first 2 ms followed by a slower process with a half-life of 3 s. The presence of the metal ion does not affect the slowest biphasic refolding rate, which is virtually the same for all three versions of the enzyme. The presence of GroEL slows down the first phase of refolding. The reassociation of subunits was examined by measuring the regain in catalytic activity and the enhancement in the fluorescence emission from NADH on binding to the oligomeric form of the enzyme. The rate and extent of reassociation is dependent on enzyme concentration and the extent of reactivation is dependent on the presence of the metal ion. The reassociation process was more efficient in the presence of NADH particularly for the metal-depleted enzyme (apo-GDH). The presence of GroEL or GroEL plus ATP leads to a higher yield of reassociation and therefore catalytically active enzyme. The additional presence of Mg-ATP does not affect the extent of reassociation, but has a small positive effect on the rate of reassociation. These data suggest that GDH is bound weakly to GroEL and that GroES is not required for release of the protein.
Adenosine Triphosphate↗
Reversible denaturation of myo-inositol monophosphatase. The stability of the metal-binding loop.
The unfolding of bovine brain myo-inositol monophosphatase by guanidine. HCl (Gdn. HCl) has been investigated. The recovery of circular dichroism, emission spectra, and catalytic activity after dilution of Gdn.HCl-treated samples indicate that the overall process is reversible. The steepness of the spectroscopic changes between 3 M and 5 M Gdn.HCl, and the lack of any discernible plateau suggest that unfolding of the protein is a cooperative process. The sensitized luminescence of bound Tb(III) was used as a probe of conformational changes of the metal-binding loop. Denaturation of the enzyme by Gdn.HCl does not abolish sensitized luminescence. A 50% decrease in sensitized luminescence was observed in 5 M Gdn.HCl. Under this set of experimental conditions, the protein binds terbium with an association constant of 1 x 10(6)M-1. It is suggested that a residual structure of denatured myo-inositol monophosphatase is responsible for the binding of terbium ions. The kinetics of unfolding and refolding as a function of Gdn.HCl concentration were monitored by protein fluorescence in a stopped-flow instrument. The monophosphatase unfolded in a single kinetic phase with rate constants in the range 80-65 s-1 at 25 degrees C. The refolding kinetics fit monoexponential functions with rate constants in the range 120-65 s-1 depending on the Gdn.HCl concentration. Substantial refolding of the protein occurs within the dead time of mixing.
Animals↗
Bovine inositol monophosphatase: enzyme-metal-ion interactions studied by pre-equilibrium fluorescence spectroscopy.
Stopped-flow fluorescence spectroscopy has been used to determine the on-rate (kass) and the off-rate (kdiss) for the equilibrium between inositol monophosphatase and Mg2+ ions. The dissociation constant (Kd) for the equilibrium calculated from these constants suggests that the ions interact at site 1 on the enzyme with a Kd typically around 450 microM, close to values determined by equilibrium studies (270-300 microM). The affinity of this site on the wild-type enzyme for Mg2+ ions increases as the pH is increased. This is mediated almost entirely by change in the rate kdiss. A slow increase occurs in the fluorescence intensity of the pyrene-labelled enzyme after the initial, fast, increase in fluorescence caused by the binding of the Mg2+ ion. The rate of this change is independent of the concentration of the metal ion, implying that it may be a structural change in the enzyme-Mg2+ complex. Neither the fast nor the slow change in fluorescence intensity occurs when enzyme subjected to limited proteolysis by trypsin, which removes the N-terminal 36 residues, is mixed with Mg2+ ions. The data suggest that interaction with Mg2+ ions at a high-affinity site leads to a structural change in inositol monophosphatase. The data further confirm the importance of the presence of two metal ions in the structure/function of this enzyme, and show that the binding of the metal ions is not competitive with that of H+ ions and that the variation in Kd with pH is mediated almost totally by changes in kdiss.
Animals↗
Spectroscopic studies of myo-inositol monophosphatase with a novel fluorescent substrate.
myo-Inositol monophosphatase catalyzes dephosphorylation of the synthetic substrate anthraniloyl-2'-AMP. Binding of this fluorescent substrate to Tb(III)-monophosphatase was monitored by luminescence spectroscopy. The anthraniloyl chromophore excited at 330 nm sensitizes the long lived luminescence of enzyme bound Tb(III) at 490, 545, 585 and 620 nm. Assuming a mechanism of radiationless energy transfer, the actual distance of separation between the donor anthraniloyl moiety and the acceptor Tb(III) was calculated to be R = 10 angstroms. The binding studies support the earlier observation of Bone et al. (Proc. Natl. Acad. Sci. USA 89 (1992) 10031-10035) that the substrate and the lanthanide Gd(III) interact with a common binding domain of the protein. The catalytic activity of the monophosphatase is completely dependent upon Mg(II) ions which elicit changes in the secondary structure of the protein as revealed by circular dichroism measurements. Binding of Mg(II) ions tend to stabilize the secondary structure of the phosphatase against guanidinium-HCl denaturation.
Adenosine Monophosphate↗
Folding and conformational studies on SCR1-3 domains of human complement receptor 1.
Short consensus repeats SCR3 and SCR1-3 are soluble recombinant proteins, consisting of the third and first three N-terminal domains of complement receptor 1, respectively, which retain some anti-complement activity. The conformational stabilities and folding/unfolding of SCR3 and SCR1-3 have been studied using circular dichroism and equilibrium and pre-equilibrium fluorescence spectroscopy. Denaturation by guanidinium hydrochloride (GdnHCl) is rapid and completely reversible. Reduction of disulphide bridges in the folded proteins by beta-mercaptoethanol leads to an increase in fluorescence intensity. The fluorescence intensity of the folded proteins is approximately 7.5% of that of the respective unfolded proteins. The data can be approximated to a two-state transition between native and denatured forms of the proteins. SCR3 has a conformational stability in water of 12-13 kJ/mol whereas that of SCR1-3 is 19.5-19.9 kJ/mol depending upon the technique utilized. The heat capacity change associated with the unfolding of SCR1-3 was obtained by a series of GdnHCl unfolding experiments over a range of temperatures and was found to be 6.6 kJ/K.mol or 33.8 J/K.mol(residue). The refolding process of SCR3 was found to be simple, described by a single exponential equation, whereas that of SCR1-3 was found to be complex and could be fitted to a double exponential equation indicating the presence of folding intermediates.
Circular Dichroism↗
Crystallization of glycerol dehydrogenase from Bacillus stearothermophilus.
The NAD(+)-linked glycerol dehydrogenase from Bacillus stearothermophilus is a member of the so-called 'iron- containing' class of polyol dehydrogenases. This enzyme has been crystallized in three different forms in the presence of a range of divalent cations and glycerol or NAD+ using the hanging-drop method of vapour diffusion with ammonium sulfate as the precipitant. X-ray photographs have established that the crystals grown in the presence of zinc and glycerol (form A) most likely belong to space group I4(1)22 with cell parameters a = b = 102 and c = 728 A. The crystals grown with zlnc and NAD(+) (form B) belong to the tetragonal system and probably belong to the space group P42(1)2 with cell parameters a = b = 150 and c = 220 A. The crystals grown with lead and glycerol (form C) belong to a primitive orthorhombic system with cell parameters a = 127, b = 178 and c = 173 A. Experiments using the synchrotron radiation source at the DRAL Daresbury laboratory have shown all three crystal types diffract to at least 3 A resolution. Elucidation of the three-dimensional structure of this enzyme will provide a structural framework for this class of polyol dehydrogenases, which are not represented in the database at present, and enable comparisons to be made with enzymes belonging to the other classes.
Journal Article↗
Equilibrium and pre-equilibrium fluorescence spectroscopic studies of the binding of a single-immunoglobulin-binding domain derived from protein G to the Fc fragment from human IgG1.
A single-immunoglobulin-binding protein based upon the C2 domain of Protein G from Streptococcus has been shown to bind tightly to the Fc fragment of IgG1. The binding interaction results in a decrease in the fluorescence intensity from the sole Trp residue (Trp-48) in this domain. This spectral change has been used to monitor the binding interactions between the two proteins using equilibrium and pre-equilibrium fluorescence spectroscopy. Comparison of the data from the two techniques suggests that a conformational change occurs after the initial formation of the complex. Mutagenesis studies have shown that the Trp residue is important for binding and that replacement by a Phe residue is important for binding and that replacement by a Phe residue leads to a 300-fold decrease in the affinity for Fc gamma 1. Determination of the rate constants kon and koff at different values of pH between 4.0 and 9.0 suggest that variations in Kd are mediated predominantly by changes in kon. Competition experiments between SpG1 and a single-IgG-binding domain from Protein A from Staphylococcus aureus have been used to determine the affinity of the latter for Fc gamma 1.
Bacterial Proteins↗
Elution of human IgG from affinity columns containing immobilised variants of protein A.
Immobilised analogues of protein A have been used for affinity chromatographic separation of human IgG. Truncation of the C-terminal region of an engineered IgG-binding domain based upon the B domain from protein A, in combination with site-directed mutagenesis, has led to the generation of a number of proteins with a decreased affinity for IgG. The elution of human IgG from these proteins when immobilised onto a solid support occurs over the pH range 3.2-5.0 with 0.5 M acetate buffer. These proteins may be effective alternatives to standard protein A columns when milder elution conditions are required.
Binding Sites↗
Crystallization and X-ray analysis of a single fab binding domain from protein L of Peptostreptococcus magnus.
Protein L is a multidomain cell wall constituent of certain strains of Peptostreptococcus magnus which binds to the variable domain of immunoglobulin kappa-light chains. A single immunoglobulin-binding domain of M(r) = 9000 from this protein has been isolated and crystallized. The crystals are of space group P4(2)2(1)2, with cell dimensions a = b = 66.9 A, c = 68.3 A, and diffract to at least 2.2 A resolution. The asymmetric unit of the crystal contains two molecules of the protein L domain, related by a noncrystallographic 2-fold axis, as revealed by a self-rotation function calculated with native diffraction data.
Bacterial Proteins↗
Cloning, expression and purification of Ppl-1, a kappa-chain binding protein, based upon protein L from Peptostreptococcus magnus.
Protein L is a multi-domain, cell wall constituent of certain strains of Peptostreptococcus magnus, which binds to the variable domain of the L-chains of Ig. A gene fragment which codes for a single Ig-binding domain of protein L (Ppl-1) was cloned into a modified pKK223-3 vector and over-expressed in E. coli JM103. A rapid protein purification protocol is described. In these studies, purified Ppl-1 was immobilised on to an agarose gel and tested against an array of Igs and Ig fragments. It was found that Ppl-1-bound Igs from a number of different sources via interactions with the L kappa-chain. An enzyme linked immunosorbant assay has been developed to assay the binding of Ppl-1 to IgG. The incubation of Ppl-1 with human serum does not produce an immunoprecipitate, thus suggesting one unique interaction per binding domain which has been confirmed by ELISA. These experiments demonstrate the potential value of Ppl-1 as an immunological tool and as an affinity chromatography ligand for the purification of Igs.
Amino Acid Sequence↗
Bovine inositol monophosphatase. Ligand binding to pyrene-maleimide-labelled enzyme.
Inositol monophosphatase can be modified at two sites by pyrene maleimide. These sites have been identified as Cys141 and Cys218. Stoichiometric addition of pyrene maleimide allows the sole modification of Cys218. The fluorescence of the pyrene moiety on the modified protein can be excited directly or by resonance energy transfer. The fluorescence properties of the pyrene group on Cys218 allows the interaction of ligands with the enzyme to be monitored. This feature has allowed dissociation constants for various metal ions to be determined and allowed the formation of various enzyme/ligand complexes to be observed. These studies have demonstrated that Mg2+ is required to support Pi binding and that Li+ interacts with a post-catalytic complex which is only formed in the forward reaction.
Amino Acid Sequence↗
The stability and unfolding of an IgG binding protein based upon the B domain of protein A from Staphylococcus aureus probed by tryptophan substitution and fluorescence spectroscopy.
The stability and unfolding of an immunoglobulin (Ig) G binding protein based upon the B domain of protein A (SpAB) from Staphylococcus aureus were studied by substituting tryptophan residues at strategic locations within each of the three alpha-helical regions (alpha 1-alpha 3) of the domain. The role of the C-terminal helix, alpha 3, was investigated by generating two protein constructs, one corresponding to the complete SpAB, the other lacking a part of alpha 3; the Trp substitutions were made in both one- and two-domain versions of each of these constructs. The fluorescence properties of each of the single-tryptophan mutants were studied in the native state and as a function of guanidine-HCl-mediated unfolding, and their IgG binding activities were determined by a competitive enzyme-linked immunosorbent assay. The free energies of folding and of binding to IgG for each mutant were compared with those for the native domains. The effect of each substitution upon the overall structure and upon the IgG binding interface was modelled by molecular graphics and energy minimization. These studies indicate that (i) alpha 3 contributes to the overall stability of the domain and to the formation of the IgG binding site in alpha 1 and alpha 2, and (ii) alpha 1 unfolds first, followed by alpha 2 and alpha 3 together.
Base Sequence↗
Mammalian inositol monophosphatase: the identification of residues important for the binding of Mg2+ and Li+ ions using fluorescence spectroscopy and site-directed mutagenesis.
The fluorescence properties of residue Trp-219 in inositol monophosphatase are sensitive to the ionization of neighbouring groups. The pH-dependent changes in the fluorescence emission intensity and wavelength of maximum emission appear to arise as the result of two separate ionizations in the proximity of Trp-219, namely due to the ionization of His-217 and Cys-218. By studying the curve of fluorescence intensity against pH, given by the mutants Cys-218-->Ala or His-217-->Gln, the pK of His-217 was determined to be 7.54 and the pK of Cys-218 was estimated to be about 8.2. These mutants have altered kinetic parameters for catalytic Mg2+ ions and inhibitory Mg2+ and Li+ ions. The Cys-218-->Ala mutant enzyme is not subject to inhibition by concentrations of Mg2+ ions up to 400 mM and has a specific activity of 156% of the maximum obtainable activity of the native enzyme. The His-217-->Gln mutant enzyme shows reduced sensitivity to inhibition by Mg2+ and Li+ ions, and has a specific activity of 110% of that obtainable for the native enzyme.
Base Sequence↗
The identification of a lysine residue reactive to pyridoxal-5-phosphate in the glycerol dehydrogenase from the thermophile Bacillus stearothermophilus.
The glycerol dehydrogenase (GDH) from Bacillus stearothermophilus is inactivated by incubation with pyridoxal-5-phosphate (PALP). The complex formed between the two can be trapped by reduction with sodium borohydride to yield a protein with an absorbance band at 325 nm and a fluorescence emission band at 430 nm, typical of trapped pyridoxal-5-phosphate moieties. Total loss of catalytic activity of the enzyme is associated with the modification of approximately one equivalent of the reagent; the incorporation of the reagent and the loss of activity can be prevented by the additional presence of the oxidised or reduced coenzyme. Peptides derived from the labelled protein have been sequenced and have identified Lys-97 as the reactive residue. Site-directed mutagenesis had been used to replace Lys-97 by a His residue. This mutated enzyme has no catalytic activity and fluorescence spectroscopy studies suggest that it is unable to bind NADH.
Amino Acid Sequence↗
Bovine inositol monophosphatase. Studies on the binding interactions with magnesium, lithium and phosphate ions.
Rapid equilibrium dialysis has been used to show that recombinant bovine brain inositol monophosphatase binds one equivalent of Pi per subunit of enzyme. Pi is only bound in the presence of Mg2+ ions. The dissociation constant for the equilibrium is approximately 50 microM. This value of Kd is independent of the concentration of the Mg2+ ions and of the presence or absence of Li+ ions. Lithium ions which inhibit the enzyme uncompetitively are not able to support the binding of the Pi to the enzyme. The observation that Pi only binds in the presence of Mg2+ ions supports similar conclusions made in experiments which studied the protection of the enzyme from proteolytic degradation and chemical modification.
Animals↗
Labeling the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum at Glu-439 with 5-(bromomethyl)fluorescein.
The (Ca(2+)-Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum was labeled with 5-(bromomethyl)fluorescein. A stoichiometry of one label per ATPase molecule was found, which was unaffected by the presence of ATP. Labeling resulted in a 60% decrease in ATPase activity. Sequencing identified the labeled residue as Glu-439. The fluorescence emission spectrum of the labeled ATPase was unaffected by the addition of Ca2+ or vanadate or by phosphorylation with either Pi or ATP. Measurement of the pK of the bound fluorescein and observation of quenching by KI were consistent with a relatively exposed location for the fluorophore. Measurements of fluorescence energy transfer located the position of Glu-439 relative to Lys-515 and Cys-344 and relative to the membrane surface. None of these distances changed in binding Ca2+ or vanadate.
Amino Acid Sequence↗