Salt and temperature-dependent conformation changes in spectrin from human erythrocyte membranes.

ORD and CD measurements of spectrin, in both the dimer and tetramer association state, indicate a high proportion of alpha-helix in this protein. At temperatures below 27 degrees C and in 0.1 M NaCl, the tetramer has an apparent helix content of 73% and the dimer, 68%. The conformation of both states is dependent on salt concentration and temperature. Low ionic strength solutions of spectrin display lowered sedimentation coefficients and a decreased apparent helix content, indicating perhaps a slight refolding and expansion of the molecule. In addition, spectrin in low ionic strength solutions undergoes a broad temperature-dependent transition spread from 20 to 50 degrees C, while in the presence of salt the transition is sharp and centered on 49 degrees C. The temperature-dependent changes in low ionic strength solutions appear to parallel the dissociation of tetramer to dimer.     

Spectroscopic study of the self-association of myosin

Homodyne and heterodyne measurements have been made of the spectrum and intensity of laser light scattered from solutions of skeletal muscle myosin at high salt concentrations. The spectral broadening and intensity measurements are consistent with the hypothesis that a myosin monomer-dimer equilibrium exists in these solutions. Values have been calculated for molecular weight and radius of gyration of the myosin monomer, virial coefficients, and diffusion constants of both monomer and dimer, and equilibrium constant of the reaction to form dimer. Diffusion constant measurements yield an approximate length of 1481 Å for the monomer and 2121 Å for the dimer. The significance of these lengths is discussed in terms of the models of Huxley and Pepe for the structure of the myofilament.     

A study of the properties of hybrids of oxyhaemoglobin and deoxyhaemoglobin with two porphyringlobin species

The fluorescence of porphyringlobin is quenched on adding haemoglobin to its solutions. It is suggested that this result indicates the formation of hybrids (comprising a dimer of porphyringlobin and a dimer of haemoglobin) in which quenching occurs by energy transfer from the porphyrin to the haem groups of the protein. From an analysis of fluorescence quenching, dissociation constants were calculated for the hybrids of oxy- and deoxyhaemoglobin with the fast- and slow-moving porphyringlobin species isolated by chromatography on CM-Sephadex (Treffry & Ainsworth, 1974). The values obtained are: deoxyhaemoglobin-fast-moving porphyringlobin, 0.8x10(-9)m; deoxyhaemoglobin-slow-moving porphyringlobin, 5x10(-10)m; oxyhaemoglobin-fast-moving porphyringlobin, 0.8x10(-6)m; oxyhaemoglobin-slow-moving porphyringlobin, 1.2x10(-7)m. The rates of reactions of solutions of haemoglobin and porphyringlobin, containing hybrids, with the thiol reagent 4,4'-dithiodipyridine showed that the thiol groups of the hybrids deoxyhaemoglobin-fast-moving porphyringlobin and oxyhaemoglobin-slow-moving porphyringlobin react more slowly than expected on the basis of composition alone: this result indicates that the deoxy and slow-moving conformations are the more stable, imposing themselves partially on to the fast-moving or oxy dimer of the hybrid. Also the rate of the reaction of CO with deoxyhaemoglobin is decreased when slow-moving porphyringlobin is added to its solutions: this is reflected in a movement of the oxygen equilibrium curve of such a mixture to higher oxygen partial pressures. Similar experiments with deoxyhaemoglobin solutions containing fast-moving porphyringlobin, showed an initial increase in the rate of CO uptake. Correspondingly, the oxygen equilibrium curve of the mixture showed an increased affinity for oxygen. Approximate calculations to determine the oxygen equilibria of the hybrids indicate that a functional dimer retains co-operative characteristics even when the dimer accompanying it within the tetramer has the reacted conformation.     

Small-angle x-ray scattering studies of metastable intermediates of beta-lactoglobulin isolated after heat-induced aggregation

Small-angle x-ray scattering was used for studying intermediate species, isolated after heat-induced aggregation of the A variant of bovine beta-lactoglobulin. The intermediates were separated in two fractions, the heated metastable dimer and heated metastable oligomers larger than the dimer. The pair distance distribution functions for the two intermediate fractions as well as for the native protein have been obtained by indirect Fourier transformation. In addition, the scattering intensity data for samples of the native protein at different concentrations were fitted using a combination of monomer and dimer form factors, which provides an estimate of the amount of monomer in solutions. By subtracting the contribution from the monomer, the scattering intensity from the dimer of the native protein can be determined and compared with the results for the metastable dimer. An ellipsoidal model was used to fit the data for the metastable dimer, and for comparison the same analysis was performed on the dimer of the native protein. The results show that the metastable dimer is more elongated than the dimer of the native protein and it occupies a volume 1.4-fold larger, in agreement with a more loose, partially unfolded conformation. The same ellipsoidal model was used to analyze the data for the fraction of larger metastable oligomers. In this case, an even more elongated ellipsoid was obtained, suggesting a linear association of monomers in the oligomers.     

A new crystal form of the complex between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus that diffracts to 2.8 A resolution.

Two distinct complexes between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus have been crystallized using ammonium sulphate as a precipitant. Form III crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA. They are of monoclinic space group C2 with unit cell dimensions a = 211.6 A, b = 126.8 A, c = 197.1 A, beta = 132.4 degrees and diffract to about 3.5 A. Preliminary crystallographic results show that the crystallographic asymmetric unit contains two synthetase dimers. Form IV crystals grow from solutions containing a 1:1.5 stoichiometry of synthetase dimer to tRNA. They are of orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 124.5 A, b = 128.9 A, c = 121.2 A and diffract to 2.8 A resolution. Preliminary crystallographic results show that these crystals contain only one tRNA molecule bound to a synthetase dimer.     

Gating and Permeation in Ion Channels Formed by Gramicidin A and Its Dioxolane-linked Dimer in Na+ and Cs+ Solutions

The association of two gramicidin A (gA) peptides via H-bonds in lipid bilayers causes the formation of an ion channel that is selective for monovalent cations only. In this study, two gAs were covalently linked with a dioxolane group (SS dimer). Some functional properties of natural gA channels were compared to that synthetic dimer in Na⁺- or Cs⁺-containing solutions. The SS dimer remained in the open configuration most of the time, while natural gA channels had a relatively brief mean open time. Single channel conductances to Na⁺ (g _Na ) or Cs⁺ (g _Cs ) in the SS dimer were smaller than in natural gA. However, g _Na was considerably more attenuated than g _Cs . This probably results from a tight solvation of Na⁺ by the dioxolane linker in the SS channel. In Cs⁺ solutions, the SS had frequent closures. By contrast, in Na⁺ solutions the synthetic dimer remained essentially in the open state. The mean open times of SS channels in different solutions (T _open,Na > T _open,Cs > T _open,H ) were inversely proportional to the single channel conductances (g _H > g _Cs > g _Na ). This suggests that ion occupancy inside the pore stabilizes the open configuration of the gA dimer. The mean closed time of the SS dimer was longer in Cs⁺ than in H⁺ solutions. Possible mechanisms for these effects are discussed. Received: 17 September 1999/Revised: 21 December 1999     

Hidden self-association of proteins

Sedimentation equilibrium measurements were carried out on solutions of bovine serum albumin, aldolase, and ovalbumin in phosphate-buffered saline, pH 7.2, at 10 degrees C. The data obtained for each protein were analyzed to yield the dependence of apparent weight-average molecular weight upon protein concentration, over a concentration range of ca 1-200 g/L. Using the approximate theory of Chatelier and Minton [1987) Biopolymers 26, 507-524), models are formulated for the dependence of apparent weight-average molecular weight upon concentration in non-ideal solutions containing proteins which may self-associate according to a monomer/n-mer or a monomer/dimer/tetramer scheme. The concentration dependence data for serum albumin may be accounted for, assuming either no self-association or weak monomer/dimer association. The data for aldolase may be accounted for assuming either weak monomer/dimer or weak monomer/trimer association. The data for ovalbumin may be accounted for assuming either weak monomer/trimer or weak monomer/dimer/tetramer association. The associations do not approach saturation at the highest concentrations studied, and the standard-state free energy changes accompanying self-association amount to less than 4 kcal/mol of intermolecular contacts, suggesting that non-specific clustering of protein molecules at high concentration rather than the formation of specific complexes is being observed.     

Construction of the simplest model to explain complex receptor activation kinetics

We study the mathematical solutions to the kinetic equations arising from various simple ligand-receptor [corrected] models. Focusing on the prediction of the various models for the activity vs. concentration curve, we find that solutions to the kinetic equations arising from the so-called dimer model exhibit features observed in some experiments, most noticeably a distinct maximum in the activity curve.     

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.     

Reactivity of nicotinamide-adenine dinucleotide dimer in plant phenol oxidase systems

The enzymie system in mung bean seedlings which earlier workers characterized as a diaphorase capable of oxidizing the 1,2- and 1,6-dihydropyridine isomers of NADH is, in fact, a phenol oxidase. The NAD species whose oxidation was observed is actually the dimeric 1-electron product obtained in the electrolytic reduction of NAD⁺ solutions. The unidentified “cofactor” required along with the enzyme for the oxidation of the dimer is probably a naturally occurring phenol. Similar activity is found in a variety of plants, including other types of beans as well as corn, cotton, and wheat. The dimer is also reactive with respect to a commercial mushroom phenol oxidase preparation. It cannot be stated whether the NAD dimer is in any sense a natural reactant in such systems, but the supposedly unusual mung bean activity is clarified.     

Application of a genetic algorithm in the conformational analysis of methylene-acetal-linked thymine dimers in DNA: Comparison with distance geometry calculations

The three-dimensional spatial structure of a methylene-acetal-linked thymine dimer presentin a 10 base-pair (bp) sense–antisense DNA duplex was studied with a geneticalgorithm designed to interpret NOE distance restraints. Trial solutions were represented bytorsion angles. This means that bond angles for the dimer trial structures are kept fixed duringthe genetic algorithm optimization. Bond angle values were extracted from a 10 bpsense–antisense duplex model that was subjected to energy minimization by means ofa modified AMBER force field. A set of 63 proton–proton distance restraints definingthe methylene-acetal-linked thymine dimer was available. The genetic algorithm minimizesthe difference between distances in the trial structures and distance restraints. A largeconformational search space could be covered in the genetic algorithm optimization byallowing a wide range of torsion angles. The genetic algorithm optimization in all cases ledto one family of structures. This family of the methylene-acetal-linked thymine dimer in theduplex differs from the family that was suggested from distance geometry calculations. It isdemonstrated that the bond angle geometry around the methylene-acetal linkage plays animportant role in the optimization.     

EPR study of dimerization and conformational change of Cu(II)-cytochrome c and its undeca- and octapeptide

The EPR study of cytochrome c in which FE(III) ion is replaced with Cu(II) shows that there are two types of monomer (a: 4 less than pH less than 6, and b: 6 less than pH less than 11.5) and two types of dimer (A: pH less than 4 and B: pH less than 11.5) formed depending upon the pH value of the solution. Computer simulation of the EPR spectra of the dimers indicates that the structure of the dimer A has a larger lateral shift than in the dimer B. It is also shown that in monomer a, the imidazole nitrogen of 18-His is not bound to Cu(II), while it is bound in the monomer b. In the undeca- and octapeptide of Cu(II)-cytochrome c, polymers are formed in acidic solutions. As the pH is raised, depolymerization proceeds to yield the monomer and the dimer. The structure of the dimer in both peptides is found to be similar to that of the dimer B of Cu(II)-cytochrome c. In the monomer of the peptides, neither the imidazole of 18-His nor the imidazole added in excess is bound to Cu(II) in the entire pH range. It is also concluded that the dimerization in Cu(II)-porphyrins interferes with the apical coordination of basic ligand, or vice versa.     

Hydrogen peroxide triggers the formation of a disulfide dimer of muscle acylphosphatase and modifies some functional properties of the enzyme

Acylphosphatase is expressed in vertebrates as two molecular forms, the organ common and the muscle types. The former does not contain cysteine residues, whereas the latter contains a single conserved cysteine (Cys-21). We demonstrated that H(2)O(2) at micromolar levels induces, in vitro, the formation of a disulfide dimer of muscle acylphosphatase, which displays properties differing from those of the reduced enzyme. In particular, we observed changes in the kinetic behavior of its intrinsic ATPase activity, whereas the kinetic behavior of its benzoyl phosphatase activity does not change. Moreover, the disulfide dimer is capable of interacting with some polynucleotides such as poly(G), poly(C), and poly(T) but not with poly(A), whereas the reduced enzyme does not bind polynucleotides. Experiments performed with H(2)O(2) in the presence of increasing SDS concentrations demonstrated that disulfide dimer formation is prevented by SDS concentrations higher than 300 microm, suggesting that a non-covalently-linked dimer is present in non-denaturing solvents. Light-induced cross-linking experiments performed on the Cys-21 --> Ser mutant in the pH range 3.8-9.0 have demonstrated that a non-covalently-linked dimer is in fact present in non-denaturing solutions and that an enzyme group with a pK(a) of 6.4 influences the monomer-dimer equilibrium.     

Monomer concentrations and dimerization constants in crystallizing lysozyme solutions by dialysis kinetics.

Dialysis kinetics measurements have been made to study the effect of ionic strength on the dimerization of lysozyme in acidic solutions that lead to the growth of tetragonal lysozyme crystals. Using glutaraldehyde cross-linked dimers of lysozyme, we have determined that both monomers and dimers can escape from 25,000 molecular weight cutoff dialysis membranes with velocity constants of 5.1 x 10(-7) and 1.0 x 10(-7) s(-1) for the monomer and dimer species, respectively. The flux from 25K MWCO membranes has been measured for lysozyme in pH 4.0 buffered solutions of 1, 3, 4, 5, and 7% NaCl over a wide range of protein concentrations. Assuming that dimerization is the first step in crystallization, a simple monomer to dimer equilibrium was used to model the flux rates. Dimerization constants calculated at low protein concentrations were 265, 750, 1212, and 7879 M(-1) for 3, 4, 5, and 7% NaCl, respectively. These values indicate that dimerization increases with the ionic strength of the solution suggesting that aggregation is moderated by electrostatic interactions. At high protein concentrations and high supersaturation, the dimerization model does not describe the data well. However, the Li model that uses a pathway of monomer <-> dimer <-> tetramer <-> octamer <-> 16-mer fits the measured flux data remarkably well suggesting the presence of higher order aggregates in crystallizing solutions.     

The state of association of band 3 protein of the human erythrocyte membrane in solutions of nonionic detergents

Band 3 protein, the anion transport protein of the human erythrocyte membrane, was solubilized and purified in aqueous solutions of two nonionic detergents: Ammonyx-LO (dimethyl laurylamine oxide) and C12E9 (nonaethylene glycol lauryl ether). The state of association of the purified protein was studied by analytical ultracentrifugation. Band 3 protein solubilized and studied in solutions of Ammonyx-LO was found to be in a monomer/dimer/tetramer association equilibrium. Band 3 protein freshly prepared in C12 E9 showed the same behaviour; however, during aging the protein was converted into stable noncovalent dimers. The conversion was retarded by the presence of beta-mercaptoethanol or by treatment of the samples with iodoacetamide; it seems to be due to oxidation of the protein by degradation products of the detergent. It is concluded that a monomer/dimer/tetramer association equilibrium is the native state of association of band 3 protein solubilized by nonionic detergents. Since nonionic detergents are assumed not to interfere with protein-protein interactions among membrane proteins, the results strongly support the claim that, in the erythrocyte membrane, band 3 is in a monomer/dimer/tetramer association equilibrium (Dorst, H.-J. and Schubert, D. (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1605-1618).     

Structure of deoxyhemoglobin: ionizable groups and polyethylene glycol

X-ray studies on deoxy-hemoglobin have been reported on crystals grown under conditions of high (about 2.5 M) and low salt (about 0.1 M). The high-salt crystals were grown in water, whereas the low-salt crystals were prepared in polyethylene glycol solutions (m.w. 6000-8000 Da, 10-30% w/v). Oxygen-binding characteristics of hemoglobin in these two environments differ radically. In salt solutions, hemoglobin binds oxygen with a p50 value of about 5 torr of oxygen and in a cooperative manner characterized with an n value of Hill varying from two to three. In polyethylene glycol solutions, hemoglobin crystals are oxygenated with a p50 value of about 270 torr of oxygen, without exhibiting a cooperative effect. I report on a detailed study of the X-ray data defining the dimer interface (alpha1beta2) of these two forms of hemoglobin. The study reveals that the main difference between the two structures lies in the number and arrangement of the water molecules and in distances between ionizable side chains in the dimer interface. I propose that these differences lead to significant shifts in the pK values of the ionizable groups in the dimer interface.     

Mineral Catalysis of a Potentially Prebiotic Aldol Condensation

Minerals may have played a significant role in chemical evolution. In the course of investigating the chemistry of phosphonoacetaldehyde (PAL), an analogue of glycolaldehyde phosphate, we have observed a striking case of catalysis by the layered hydroxide mineral hydrotalcite ([Mg₂Al(OH)₆][Cl.nH₂O]). In neutral or moderately basic aqueous solutions, PAL is unreactive even at a concentration of 0.1 M. In the presence of a large excess of NaOH (2 M), the compound undergoes aldol condensation to produce a dimer containing a C3–C4 double-bond. In dilute neutral solutions and in the presence of the mineral, however, condensation takes place rapidly, to produce a dimer which is almost exclusively the C2–C3 unsaturated product. Received: 11 February 1998 / Accepted: 12 May 1998     

High resolution proton magnetic resonance spectroscopy of histones and histone--histone complexes in aqueous solution

Low molecular weight histone complexes of H2A (congruent to dimer), H2B (congruent to tetramer), H3--H4 (congruent to tetramer), H2A--H2B (congruent to dimer), and H2B--H4 (congruent to dimer) have been prepared in 2 M NaCl and neutral pH at 4 degrees C. These materials are free of nonspecific aggregate and are suitable for study by high resolution proton magnetic resonance spectroscopy. Such spectra have been recorded in aqueous solutions under conditions allowing a study of the exchangeable proton resonances of histone complexes for the first time and indicate that the structured regions are rich in hydrophobic amino acids, as well as arginine and some acidic amino acids. Most of the lysine and probably alanine residues remain in a motile, random coil-like state after formation of the complexes. It is suggested that arginine residues may be important in inter- and/or intra-subunit interactions in histone complexes.     

On the quaternary structure of native rabbit muscle phosphofructokinase.

Small angle X-ray scattering measurements on solutions of native rabbit muscle phosphofructokinase (EC 2.7.1.11; ATP; D-fructose-6-phosphate 1 phosphotransferase) show that the dimer has a radius of gyration of 32.5 Å and a molecular weight of 160,000, and that the biologically active tetramer has a radius of gyration of 51.5 Å and a molecular weight of 320.000. A possible model was calculated from scattering curves of the dimer and tetramer suggesting two hollow cylinders with cell dimensions for the dimer of a height of 78.0 Å and a long half axis of 38.0 Å, and for the tetramer of a height of 155.0 Å and an outer radius of 35.0 Å. The tetramer is formed along the 78.0 Å axis of the dimer by means of an end-to-end aggregation. The overall particle dimensions of the protomer of molecular weight 80,000 is calculated to be 35.0 × 30.0 × 55.0 Å, assuming an elliptical molecule. The distance between the centers of the two dimeric units within the tetramer is 104.5 ± 1.5 Å.     

The association−dissociation behavior of the ApoE proteins: kinetic and equilibrium studies

The apolipoprotein E family consists of three major protein isoforms: apolipoprotein E4 (ApoE4), ApoE3, and ApoE2. The isoforms, which contain 299 residues, differ only by single-amino acid changes, but of the three, only ApoE4 is a risk factor for Alzheimer’s disease. At micromolar concentrations, lipid-free ApoE exists predominantly as tetramers. In more dilute solutions, lower-molecular mass species predominate. Using fluorescence correlation spectroscopy (FCS), intermolecular fluorescence resonance energy transfer (FRET), and sedimentation methods, we found that the association?dissociation reaction of ApoE can be modeled with a monomer?dimer?tetramer process. Equilibrium constants have been determined from the sedimentation data, while the individual rate constants for association and dissociation were determined by measurement of the kinetics of dissociation of ApoE and are in agreement with the equilibrium constants. Dissociation kinetics as measured by intermolecular FRET show two phases reflecting the dissociation of tetramer to dimer and of dimer to monomer, with dissociation from tetramer to dimer being more rapid than the dissociation from dimer to monomer. The rate constants differ for the different ApoE isoforms, showing that the association?dissociation process is isoform specific. Strikingly, the association rate constants are almost 2 orders of magnitude slower than expected for a diffusion-controlled process. Dissociation kinetics were also monitored by tryptophan fluorescence in the presence of acrylamide and the data found to be consistent with the monomer?dimer?tetramer model. The approach combining multiple methods establishes the reaction scheme of ApoE self-association.