The dimerization of ferrihaems. II. Equilibrium and kinetic studies of mesoferrihaem dimerization.

Spectrophotometric data have been determined for mesoferrihaem at several pH values and over a range of concentration covering four orders of magnitude. The data reveal a dimerization process according to the equation 2 monomer in equilibrium dimer + H+, analogous to earlier findings for deuteroferrihaem and protoferrihaem. The value of K (defined as K = [dimer] [H+]/[monomer]2) was found to be 6.92.10(-2). This is close to the value for deuteroferrihaem but much less than that for protoferrihaem. This is interpreted in terms of possible additional bonding between the delocalized electron systems in protoferrihaem dimers relative to those of mesoferrihaem and deuteroferrihaem. Rate constants for dimerization were determined by temperature-jump spectrophotometry. The pH dependence of the rate constants is explained in terms of two distinct pathways for the dimerization process. These involve either direct reaction between two undissociated monomer molecules or alternatively an initial acid dissociation of a monomer molecule followed by reaction between an undissociated and dissociated molecule.     

Increased site 1 affinity improves biopotency of porcine growth hormone. Evidence against diffusion dependent receptor dimerization

Based on phage display optimization studies with human growth hormone (GH), it is thought that the biopotency of GH cannot be increased. This is proposed to be a result of the affinity of the first receptor for hormone far exceeding that which is required to trap the hormone long enough to allow diffusion of the second receptor to form the ternary complex, which initiates signaling. We report here that despite similar site 1 kinetics to the hGH/hGH receptor interaction, the potency of porcine GH for its receptor can be increased up to 5-fold by substituting hGH residues involved in site 1 binding into pGH. Based on extensive mutations and BIAcore studies, we show that the higher potency and site 1 affinity of hGH for the pGHR is primarily a result of a decreased off-rate associated with residues in the extended loop between helices 1 and 2 that interact with the two key tryptophans Trp104 and Trp169 in the receptor binding hot spot. Our mutagenic analysis has also identified a second determinant (Lys165), which in addition to His169, restricts the ability of non-primate hormones to activate hGH receptor. The increased biopotency of GH that we observe can be explained by a model for GH receptor activation where subunit alignment is critical for effective signaling.     

The dimerization of ferrihaems. III. Equilibrium and kinetic studies on the dimerization of coproferrihaem.

Spectrophotometric studies on the behaviour of coproferrihaem in aqueous solution showed that, in the pH range 6.66--8.04, a dimerization process occurs according to the equation 2 monomer K in equilibrium dimer + H+ The value of K, the pH-independent dimerization constant, was found to be 2.10 . 10(-3), signifying that coproferrihaem shows the least tendency to dimerize of any ferrihaem so far investigated. Forward and reverse rate constants for the dimerization process have been determined by the temperature-jump method. The results suggest that the cation-bridging between carboxyl residues, postulated for the dimers of the dicarboxylic ferrihaems, cannot occur between the additional carboxyl residues of coproferrihaem and that the increased negative charge may cause destabiliztion of the coproferrihaem dimer by repulsion effects.     

The dimerization of ferrihaems: II. Equilibrium and kinetic studies of mesoferrihaem dimerization

Spectrophotometric data have been determined for mesoferrihaem at several pH values and over a range of concentration covering four orders of magnitude. The data reveal a dimerization process according to the equation 2 monomer ? dimer + H⁺, analogous to earlier findings for deuteroferrihaem and protoferrihaem. The value of K (defined as $\text{K}\text{=}\text{[dimer][H}{}^{\mathrm{+}}\text{]}\text{[}\text{monomer}\text{])}{}^2\text{)}$ was found to be 6.92 · 10^?2. This is close to the value for deuteroferrihaem but much less than that for protoferrihaem. This is interpreted in terms of possible additional bonding between the delocalized electron systems in protoferrihaem dimers relative to those of mesoferrihaem and deuteroferrihaem.Rate constants for dimerization were determined by temperature-jump spectrophotometry. The pH dependence of the rate constants is explained in terms of two distinct pathways for the dimerization process. These involve either direct reaction between two undissociated monomer molecules or alternatively an initial acid dissociation of a monomer molecule followed by reaction between an undissociated and a dissociated molecule.     

Kinetics of alpha-chymotrypsin dimerization.

A method has been devised which permits the observation of the loss of active sites promoted by aggregation of alpha-chymotrypsin. When alpha-chymotrypsin in unbuffered solution at pH 7 is mixed with buffered proflavin by stopped flow instrumentation to give a final pH of 3.89, a decrease in active sites occurs, as measured by a decrease in enzyme-dye complex. The decrease in the rate of active sites shows a linear dependence on the square of the concentration of active sites remaining at equilibrium. The kinetic data of the reaction have been correlated with equilibrium measurements. Rate constants for formation and dissociation of dimer are 9.45 X 10(3) M(-1)S(-1) and 1.9 S(-1),, respectively. Calculation of Kdis for dimer from rate constants gives a value of 2.01 X 10(-4) M, while direct determination of Kdis gives a value of 1.44 X 10(-4) M.     

The dimerization of ferrihaems. I. The effect of buffer ions and specific cations on deuteroferrihaem dimerization.

The dimerization of dueteroferrihaem in aqueous solution has been investigated using a parameter, named the dimerization index (Robs). This is defined as the ratio of extinction coefficients at wavelengths corresponding to Soret band maxima for the monomeric and dimeric species, respectively. For solutions containing mainly monomeric species, Robs greater than 2, whereas for solutions containing mainly dimeric species Robs less than 1. A computer programme has been applied to determine values of the dimerization constant, K, defined as: K = [dimer] [H+]/[monomer]2. Phosphate buffer anions and Tris . HCl buffer enhanced dimerization. Monovalent and divalent cations also increased dimerization, but in a specific manner. The magnitudes of their effects increased in the order K+ less than Na+ less than Li+ less than Sr2+ less than Mg2+ approximately or equal to Ca2+. Values of K were determined for several concentrations of Na+ and Sr2+. These data are interpreted in terms of a stabilization of the ferrihaem dimer by the formation of ion triplets with the added cation 'sandwiched' between carboxyl residues of the adjacent ferrihaem monomeric units. General guidelines are recommended for the choice of conditions which minimize dimerization.     

Biologically-generated primer for PCR: PCR primer of unknown sequence.

We describe a method for producing specific PCR primers directly from PCR product, bypassing the usual need to know the primer sequence. Lack of abundance of primers derived from a PCR product is compensated for by the incorporation of an arbitrary 5'TAG sequence which acts as a surrogate template target for the bulk amplification phase. We use the technique to amplify clonospecific rearranged immunoglobulin genes, which have applications as markers of lymphoid neoplasms for tracing the success of therapy. The principle may have wider application wherever conserved and variable regions of DNA are juxtaposed.     

The dimerization of ferrihaems. IV. Studies on haematoferrihaem and a general appraisal of the nature and implications of dimerization.

The dimerization of haematoferrihaem was studied in phosphate buffer in the pH range 7.02--8.14. The absorbance of dilute solutions decreased over a period of several hours due to adsorption of haematoferrihaem to glass vessels. This problem was overcome by using dilute solutions within 10 min of preparation. Spectrophotometric data were consistent with a dimerization process according to the equation 2 monomer in equilibrium dimer + H+ as found earlier for other ferrihaems studied. The value of K, defined as K = [dimer] [H+]/[monomer]2, was found to be 1.00 . 10(-2). Rate constants for the forward and reverse steps in dimerization were determined at pH values of 6.63, 7.01 and 7.44, using the temperature-jump technique. The reaction pathway for dimerization was found to be similar to those of deuteroferrihaem and mesoferrihaem, but different from that of coproferrihaem. A general appraisal of the factors influencing dimerization is attempted in the light of accumulated data on various ferrihaems. With the exception of protoferrihaem, it is suggested that dimerzation increases with the hydrophobicity of the 2,4 substituents. The additional stability of the protoferrihaem dimer is explained in terms of increased interaction due to conjugation of the vinyl groups with the porphyrin macrocycle.     

Random diffusion can account for topA-dependent suppression of partition defects in low-copy-number plasmids.

The maintenance of partition-defective (Par-) mini-P1 and mini-F plasmids was studied in topA strains of Escherichia coli, which are defective in topoisomerase I activity. The partition defects were substantially but not completely suppressed in broth-grown cultures. This suppression was not due to a large increase in copy number. However, the absolute number of copies of Par- mini-P1 plasmids per average dividing cell is sufficiently high to account for the modest stability observed if a random distribution of the copies to daughter cells is assumed. The similar number of Par- plasmid copies in wild-type cells are distributed in a considerably worse-than-random fashion. Thus, it is unnecessary to propose, as was suggested previously, that an active, par-independent pathway operates in topA strains to ensure proper segregation of the plasmids to daughter cells. Rather, it seems likely that the lack of topoisomerase I activity aids the random distribution of the partition-defective plasmids, perhaps by facilitating their separation after replication. The results of studies carried out at reduced growth rates were consistent with this view; when topA cells containing Par- mini-P1 plasmids were cultured in minimal medium, in which the copy number of the plasmids per average cell is sharply reduced, very little suppression of the partition defect was observed.     

Evidence of noncovalent dimerization of calmodulin.

Calcium-binding proteins, such as S-100, dimerize readily, and this phenomenon plays an important role in their regulation of target enzymes [Krebs, J., Quadroni, M. & Van Eldik, L.J. (1995) Nat. Struct. Biol. 2, 711-714; Kilby, P.M., Van Eldik, L.J. & Roberts, G. C. (1996) Structure 4, 1041-1052]. We have investigated by Fourier-transform ion cyclotron resonance (FTICR) MS the conformational states of the calcium-binding protein calmodulin, and present clear evidence for a calmodulin dimer formed as a result of noncovalent interactions between folded monomers. Ultra-high-resolution electrospray ionization (ESI) mass spectra for calmodulin, obtained with a 9.4 T FTICR mass spectrometer, are presented. With the use of denaturing solutions (1 : 1 acetonitrile/water + 1% formic acid), relatively high charge states (20 < z < 10) of monomeric calmodulin ions were detected, whereas when calmodulin was electrosprayed from buffer, monomers ions with only 5-10 charges were detected. CD measurements for calmodulin in buffered solution revealed that its alpha-helical content was significantly higher than that for calmodulin in acetonitrile/water solutions, consistent with a proposition that changes in charge state distributions observed in the MS experiments reflect differing states of calmodulin folding. Under buffered conditions, noncovalently bound calmodulin dimers were observed by ESI FTICR MS. Analytical ultracentrifugation experiments carried out in the same solution conditions as those used in the MS experiments were consistent with the proposed calmodulin dimer-monomer equilibrium. The ultra-high mass resolution achieved with the 9.4 T FTICR mass spectrometer allowed unequivocal identification of the noncovalent, as opposed to covalent, character of the calmodulin dimer.     

Disulfide-bonded dimerization of fibronectin in vitro.

Human plasma fibronectin was denatured with 8 M urea and reduced with dithiothreitol. Dialysis or dilution of the solution led to formation of fibronectin dimers which migrated in non-reducing SDS/PAGE similarly to untreated control protein. When the redimerized fibronectin was reduced and re-electrophoresed it formed a doublet of alpha and beta chains of equal intensity indicating that it was a heterodimer. Low concentrations (less than 1 mM) of Fe3+ enhanced the redimerization of fibronectin, suggesting that metal ions may mediate oxidative reactions in the formation of the disulfides. Consequently, redimerization of fibronectin was completely prevented by deferoxamine, an iron chelator. Dimerization of fibronectin took place most effectively at pH greater than or equal to 8.8 but decreased strongly at lower pH, representing more unfavourable conditions for the action of the thiolate anion in the thiol/disulfide exchange reaction. Redimerized fibronectin, however, lost many of its binding properties to macromolecular ligands, suggesting that the disulfide bonding did not entirely regenerate the proper conformation of the protein. Pulse/chase experiments of fibroblast cultures showed that the initially monomeric fibronectin was rapidly and quantitatively dimerized under conditions representing natural pH and environment. SDS/PAGE analysis of the dialyzed urea-denatured/reduced thrombin and plasmin digests of fibronectin revealed that the NH2-terminal 30-kDa fragment and other fragments that contained intrachain disulfides quantitatively regained their non-reduced electrophoretic mobility. The results show that the dimerization and formation of intrachain disulfides of fibronectin may occur, in part, spontaneously, based on the amino acid sequence information of the protein. However, complete disulfide formation may also need other factors, present only in living cells, as suggested by pulse/chase experiments in fibroblasts.     

The dimerization of ferrihaems: I. The effect of buffer ions and specific cations on deuteroferrihaem dimerization

The dimerization of deuteroferrihaem in aqueous solution has been investigated using a parameter, named the dimerization index (R_obs). This is defined as the ratio of extinction coefficients at wavelengths corresponding to Soret band maxima for the monomeric and dimeric species, respectively. For solutions containing mainly monomeric species, R_obs > 2, whereas for solutions containing mainly dimeric species R_obs < 1.A computer programme has been applied to determine values of the dimerization constant, K, defined as: $\text{K =}\text{[}\text{dimer}\text{][}\text{H}{}^{\mathrm{+}}\text{]}\text{[}\text{monomer}\text{]}{}^2$.Phosphate buffer anions and Tris · HCl buffer enhanced dimerization. Monovalent and divalent cations also increased dimerization, but in a specific manner. The magnitudes of their effects increased in the order $\text{K}{}^{\mathrm{+}}\text{< Na}{}^{\mathrm{+}}\text{< Li}{}^{\mathrm{+}}\text{<}\text{Sr}{}^{\mathrm{2+}}\text{< Mg}{}^{\mathrm{2+}}\text{? Ca}{}^{\mathrm{2+}}$. Values of K were determined for several concentrations of Na⁺ and Sr²⁺. These data are interpreted in terms of a stabilization of the ferrihaem dimer by the formation of ion triplets with the added cation ‘sandwiched’ between carboxyl residues of the adjacent ferrihaem monomeric units. General guidelines are recommended for the choice of conditions which minimize dimerization.