Tryptophan pyrrolase in haem regulation. The mechanism of the opposite effects of tryptophan on rat liver 5-aminolaevulinate synthase activity and the haem saturation of tryptophan pyrrolase.

The self-assembly of myosin monomer into thick filament occurs via a two-step mechanism. At first a pair of myosin monomers reacts to form a parallel dimer; the dimer in turn adds to the filament ends at a rate that is independent of filament length. The rate of the dissociation reaction on the other hand is length-dependent. The 'off' rate constant has been shown to increase exponentially by a factor of 500 as the filament grows from the bare-zone out to its full length. The length of the filament is thus kinetically controlled; myosin is added to the filament at a fixed rate, whereas the dissociation rate increases to a point where equilibrium is established and the filament ceases to grow. The structural implications implicit in the mechanism are discussed.     

Dynamic Exchange of Myosin VI on Endocytic Structures

The actin-based molecular motor myosin VI functions in the endocytic uptake pathway, both during the early stages of clathrin-mediated uptake and in later transport to/from early endosomes. This study uses fluorescence recovery after photobleaching (FRAP) to examine the turnover rate of myosin VI during endocytosis. The results demonstrate that myosin VI turns over dynamically on endocytic structures with a characteristic half-life common to both the large insert isoform of myosin VI on clathrin-coated structures and the no-insert isoform on early endosomes. This half-life is shared by the myosin VI-binding partner Dab2 and is identical for full-length myosin VI and the cargo-binding tail region. The 4-fold slower half-life of an artificially dimerized construct of myosin VI on clathrin-coated structures suggests that wild type myosin VI does not function as a stable dimer, but either as a monomer or in a monomer/dimer equilibrium. Taken together, these FRAP results offer insight into both the basic turnover dynamics and the monomer/dimer nature of myosin VI.     

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.     

Non-specific termination of simian virus 40 DNA replication.

Axial X-ray diffraction patterns have been studied from relaxed, contracted and rigor vertebrate striated muscles at different sarcomere lengths to determine which features of the patterns depend on the interaction of actin and myosin. The intensity of the myosin layer lines in a live, relaxed muscle is sometimes less in a stretched muscle than in the muscle at rest-length; the intensity depends not only on the sarcomere length but on the time that has elapsed since dissection of the muscle. The movement of cross-bridges giving rise to these intensity changes are not caused solely by the withdrawal of actin from the A-band.When a muscle contracts or passes into rigor many changes occur that are independent of the sarcomere length: the myosin layer lines decrease in intensity to about 30% of their initial value when the muscle contracts, and disappear completely when the muscle passes into rigor. Both in contracting and rigor muscles at all sarcomere lengths the spacings of the meridional reflections at 143 Å and 72 Å are 1% greater than from a live relaxed muscle at rest-length. It is deduced that the initial movement of cross-bridges from their positions in resting muscle does not depend on the interaction of each cross-bridge with actin, but on a conformational change in the backbone of the myosin filament: occurring as a result of activation. The possibility is discussed that the conformational change occurs because the myosin filament, like the actin filament, has an activation control mechanism. Finally, all the X-ray diffraction patterns are interpreted on a model in which the myosin filament can exist in one of two possible states: a relaxed state which gives a diffraction pattern with strong myosin layer lines and an axial spacing of 143.4 Å, and an activated state which gives no layer lines but a meridional spacing of 144.8 Å.     

Time-resolved intrinsic fluorescence of Enzyme I. The monomer/dimer transition.

Enzyme I of the bacterial phosphotransferase system can exist in a monomer/dimer equilibrium which may have functional significance. Each monomer contains two tryptophan residues. It is demonstrated that the decay of both the monomer and the dimer can be described by a biexponential. The decay times depend on the temperature and at 6 degrees C the decay times are tau 1 = 0.4 ns and tau 2 = 3.2 ns for the monomer and tau 3 = 3.2 ns and tau 4 = 7.2 ns for the dimer form of the enzyme. The changes in the fluorescence decay parameters can be utilized to measure the equilibrium constant for the monomer/dimer transition.     

Nature of protamine-DNA complexes. A special type of ligand binding co-operativity

The mode of protamine binding to DNA double helices has been analyzed for the example of clupein Z from herring and DNA samples from bacteriophages lambda and PM2 by measurements of light-scattering intensities, ultracentrifugation and kinetics. The light-scattering intensity of DNA increases co-operatively at a threshold clupein concentration suggesting co-operative binding of clupein to double helices. These data are first analyzed in terms of a model with a transition at a threshold degree of binding. The parameters resulting from this analysis appear to be reasonable, but are shown to be in contrast with data on the absolute degree of clupein binding to DNA obtained by centrifugation experiments. An analysis of the kinetics associated with clupein binding to DNA by measurements of the time-dependence of light-scattering intensities in the time range of seconds demonstrates directly that clupein-induced intermolecular interactions of DNA molecules are essential. The rate constants of DNA association increase co-operatively at threshold clupein concentrations, which correspond to those observed in the equilibrium titrations. Above the threshold, the rate constants arrive at a level that is almost constant, but shows some decrease with increasing clupein concentrations. These results are described by a model with a monomer and a dimer state of DNA, which bind ligands with different affinities according to an excluded-site binding scheme. When the ligand binding constant is larger for the dimer than for the monomer state, as should be expected, binding of ligands drives the DNA from the monomer to the dimer state, even if the dimerization equilibrium in the absence of ligands is far in favor of the monomer. The transition from the monomer to the dimer state proves to be strongly co-operative. When the ligand concentration is increased to higher values, the dimers may be converted back to monomers due to an increased extent of ligand binding to the monomer state. The model is consistent with the available experimental data. The analysis of the data by the model indicates the existence of a reaction unit much below the DNA chain length, corresponding to about 80 nucleotide residues. The present model describes ligand driven intermolecular association; an analogous model is applicable to ligand driven intramolecular association. In summary, the co-operativity of clupein binding to DNA double helices is not due to nearest neighbor interactions, but results from thermodynamic coupling of clupein binding with clupein-induced DNA association.     

Cyanide-linked dimer-monomer equilibrium of Chromatium vinosum ferric cytochrome c′

Cyanide binding to Chromatium vinosum ferricytochrome c′ has been studied to further investigate possible allosteric interactions between the subunits of this dimeric protein. Cyanide binding to C. vinosum cytochrome c′ appears to be cooperative. However, the cyanide binding reaction is unusual in that the overall affinity of cyanide increases as the concentration of cytochrome c′ decreases and that cyanide binding causes the ligated dimer to dissociate to monomers as shown by gel-filtration chromatography. Therefore, the cyanide binding properties of C. vinosum ferricytochrome c′ are complicated by a cyanide-linked dimer to monomer dissociation equilibrium of the complexed protein. The dimer to monomer dissociation constant is 20-fold smaller than that for CO linked dissociation constant of ferrocytochrome c′. Furthermore, the pH dependence of both the intrinsic equilibrium binding constant and the dimer to monomer equilibrium dissociation constant was investigated over the pH range of 7.0 to 9.2 to examine the effect of any ionizable groups. The equilibrium constants did not exhibit a significant pH dependence over this pH range.     

Self-association of β-lactoglobulin A in acid solution. I. Translational diffusion coefficients

We report measurements of the diffusion coefficient of β-lactoglobulin A (βLG-A) at pH = 5.60 and 4.58 in 0.10 ionic strength acetate buffer by the techniques of analog photocurrent signal correlation and digital single-clipped photon correlation. At a concentration of 21 mg/ml and a pH of 4.58, the self-association of β-lactoglobulin can be represented by a simple dimer–octamer equilibrium model. We determined the translational diffusion coefficient of the dimer and that of the octamer using the scattering results of Kumosinski and Timasheff in a dimer–octamer mixture. Our analysis shows that the dimer βLG-A does not change its size if the pH is varied from 5.60 to 4.58 and both species remain constant in size for temperature changes from 3.5° to 25°C Hydrodynamically, the octamers behave like closed-packed spheres with an effective radius of about 45 Å according to the Stokes-Einstein relation.     

Molecular size of nerve growth factor in dilute solution.

Nerve growth factor (NGF) is a protein composed of two identical chains of mass 13,259. An analysis of the sedimentation equilibrium, sedimentation velocity, and gel filtration behavior of dilute solutions of NGF indicates the existence of a rapidly reversible monomer in equilibrium dimer equilibrium and that the association constant K for the reaction at neutral pH is 9.4 X 10(6)M-1. Reaction mixtures consist of equal concentrations of monomer and dimer at a total protein concentration as high as 1.4 mug/ml, and at 1 ng/ml, monomer accounts for greater than 99% of the total. The latter concentration is 20 to 30 times that required for the biological activity of NGF. Several lines of evidence suggest that the dimerization reaction is highly stereospecific, although its biological significance is not known.     

Specific Optical Rotations and the Horeau Effect

The observation of nonequivalence of optical and enantiomeric purities, referred to as the Horeau effect, is thought to arise from molecular aggregation in liquid solutions. Although this effect was first observed in 1969, the conditions under which this effect may, or may not, be observable are not established. Considering the formation of dimers as the simplest form of aggregation, the expressions for specific optical rotations in the presence of homochiral and heterochiral monomer–dimer equilibria are presented. Analysis of these equations indicates that the Horeau effect will not be observable even in the presence of aggregation under either of the following two situations: 1) The specific optical rotation of the monomeric species is equal to that of the dimeric species; 2) The heterochiral equilibrium constant is twice that of the homochiral equilibrium constant. Chirality 28:181–185, 2016. © 2015 Wiley Periodicals, Inc.     

Cryo-EM structure of monomeric photosystem II at 2.78 Å resolution reveals factors important for the formation of dimer

Photosystem II (PSII) functions mainly as a dimer to catalyze the light energy conversion and water oxidation reactions. However, monomeric PSII also exists and functions in vivo in some cases. The crystal structure of monomeric PSII has been solved at 3.6 Å resolution, but it is still not clear which factors contribute to the formation of the dimer. Here, we solved the structure of PSII monomer at a resolution of 2.78 Å using cryo-electron microscopy (cryo-EM). From our cryo-EM density map, we observed apparent differences in pigments and lipids in the monomer-monomer interface between the PSII monomer and dimer. One β-carotene and two sulfoquinovosyl diacylglycerol (SQDG) molecules are found in the monomer-monomer interface of the dimer structure but not in the present monomer structure, although some SQDG and other lipid molecules are found in the analogous region of the low-resolution crystal structure of the monomer, or cryo-EM structure of an apo-PSII monomer lacking the extrinsic proteins from Synechocystis sp. PCC 6803. In the current monomer structure, a large part of the PsbO subunit was also found to be disordered. These results indicate the importance of the β-carotene, SQDG and PsbO in formation of the PSII dimer.     

Rabbit skeletal myosin heads in solution, as observed by ultracentrifugation and freeze-fracture electron microscopy: dimerization and maximum chord

The use of analytical ultracentrifugation and freeze-fracture electron microscopy in solution allowed us to observe the monomeric and dimeric forms of Mg.71. This subfragment of the myosin molecule contains the LC2 light chain and is comparable to a "native" myosin head. Sedimentation-diffusion equilibrium ultracentrifugation shows that it is necessary to use slightly different conditions in order to obtain a pure Mg.S1 dimer, as compared to the case of chymotryptic S1 (LC2-free S1). For example, in a buffer leading to a complete dimerization of chymotryptic S1, Mg.S1 is only in the form of a monomer-dimer mixture, with comparable proportions of monomer and dimer. The freeze-fracture technique, applied to solutions containing Mg.S1 or chymotryptic S1, revealed that the monomeric species both have the same maximum chord (about 120 A) and that both dimeric species also have the same maximum chord (about 250 A). The maximum chord of the monomer is comparable to the surface-to-surface spacing between the myosin and actin filaments, in a fiber at the slack length. In sharp contrast this chord is higher than this spacing in a stretched fiber. The consequences of this fact are discussed, with particular reference to the sarcomere length-tension relationship.     

Absorption and circular dichroic spectral studies on the self-association of daunorubicin

The self-association of daunorubicin in aqueous solution has been examined using visible absorption, fluorescence, and CD measurements. Spectral changes in the concentration range 10^?6 to 1.5 × 10^?3M have been interpreted in terms of a monomer–dimer equilibrium for daunorubicin. The data have been analyzed using a nonlinear curve-fitting technique. The results obtained in this study differ markedly from previously published results, and possible reasons for this difference are discussed. The effect of solvent, temperature, and ionic strength on the dimerization process is investigated.     

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 bound nucleotide of actin

The extent of actin polymerization has been studied for samples in which the bound nucleotide of the actin was ATP, ADP, or an analog of ATP that was not split (AMPPNP). The equilibrium constants for the addition of a monomer to a polymer end were determined from the concentration of monomer coexisting with the polymer. An analysis of these results concludes that the bound ATP on G-actin provides little energy to promote the polymerization of the actin. AMPPNP was incorporated into F-actin and the interaction of F-actin · AMPPNP with myosin was studied. F-actin · AMPPNP activated the ATPase of myosin to the same extent as did F-actin · ADP. However, the rate of superprecipitation was slower in the case of F-actin · AMPPNP than in the control.     

Regulation of UVR8 photoreceptor dimer/monomer photo‐equilibrium in Arabidopsis plants grown under photoperiodic conditions

The UV RESISTANCE LOCUS 8 (UVR8) photoreceptor specifically mediates photomorphogenic responses to UV‐B. Photoreception induces dissociation of dimeric UVR8 into monomers to initiate responses. However, the regulation of dimer/monomer status in plants growing under photoperiodic conditions has not been examined. Here we show that UVR8 establishes a dimer/monomer photo‐equilibrium in plants growing in diurnal photoperiods in both controlled environments and natural daylight. The photo‐equilibrium is determined by the relative rates of photoreception and dark‐reversion to the dimer. Experiments with mutants in REPRESSOR OF UV‐B PHOTOMORPHOGENESIS 1 (RUP1) and RUP2 show that these proteins are crucial in regulating the photo‐equilibrium because they promote reversion to the dimer. In plants growing in daylight, the UVR8 photo‐equilibrium is most strongly correlated with low ambient fluence rates of UV‐B (up to 1.5 μmol m^?2 s^?1), rather than higher fluence rates or the amount of photosynthetically active radiation. In addition, the rate of reversion of monomer to dimer is reduced at lower temperatures, promoting an increase in the relative level of monomer at approximately 8–10 °C. Thus, UVR8 does not behave like a simple UV‐B switch under photoperiodic growth conditions but establishes a dimer/monomer photo‐equilibrium that is regulated by UV‐B and also influenced by temperature.     

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.     

High resolution X-ray structure of yeast hexokinase,an allosteric protein exhibiting a non-symmetric arrangement of subunits

The structure determination of yeast hexokinase has been extended to 3.5 Å resolution for the dimer and to 2.7 Å resolution for the monomer using multiple isomorphous replacement. The electron density maps of both the monomer and dimer crystal forms have been substantially improved by an averaging procedure. From these maps the course of the polypeptide backbone and some aspects of the dimer interaction have been established.The hexokinase subunit arrangement is contrary to a major tenet of the Monod et al. (1965) theory of allosteric proteins which postulated that only symmetric or isologous interactions of subunits would occur in oligomeric proteins. One subunit of the dimer is related to the other by a 156 ° rotation about and a 13.8 Å translation along a molecular screw axis. In the hexokinase dimer the set of residues in one subunit that is interacting with the other subunit is different from the set of residues in the second subunit that is interacting with the first subunit. This heterologous or non-symmetric interaction of subunits is associated with some small differences in the structure of the two subunits, particularly at the subunit interface, and accounts for some of this enzyme's non-symmetric interactions with substrates and activators. Indeed, the non-symmetric subunit association may play an important role in the control of this enzyme's activity.The overall structure of hexokinase is considerably different than the known structures of the other enzymes in the glycolytic pathway. Although there is a striking similarity between the domain of hexokinase that binds AMP and the domain of lactate dehydrogenase that binds NAD, the former structure contains both antiparallel and parallel β-pleated strands, while the latter contains only parallel β-structure. In an attempt to assess the significance of this structural similarity, the structure of the nucleotide binding domains of hexokinase and lactate dehydrogenase are compared to a portion of carboxypeptidase A. The observed similarities among these structures suggests that a central β-pleated sheet flanked by α-helices is a common supersecondary structure that probably arose by convergent as well as divergent evolution. Thus, there appears to be no compelling evidence at this time to support the hypothesis that a part of hexokinase has evolved from the same gene as the dinucleotide binding domain of lactate dehydrogenase.     

The Specific Monomer/Dimer Equilibrium of the Corticotropin-releasing Factor Receptor Type 1 Is Established in the Endoplasmic Reticulum

G protein-coupled receptors (GPCRs) represent the most important drug targets. Although the smallest functional unit of a GPCR is a monomer, it became clear in the past decades that the vast majority of the receptors form dimers. Only very recently, however, data were presented that some receptors may in fact be expressed as a mixture of monomers and dimers and that the interaction of the receptor protomers is dynamic. To date, equilibrium measurements were restricted to the plasma membrane due to experimental limitations. We have addressed the question as to where this equilibrium is established for the corticotropin-releasing factor receptor type 1. By developing a novel approach to analyze single molecule fluorescence cross-correlation spectroscopy data for intracellular membrane compartments, we show that the corticotropin-releasing factor receptor type 1 has a specific monomer/dimer equilibrium that is already established in the endoplasmic reticulum (ER). It remains constant at the plasma membrane even following receptor activation. Moreover, we demonstrate for seven additional GPCRs that they are expressed in specific but substantially different monomer/dimer ratios. Although it is well known that proteins may dimerize in the ER in principle, our data show that the ER is also able to establish the specific monomer/dimer ratios of GPCRs, which sheds new light on the functions of this compartment.     

Dimer dissociation and unfolding mechanism of coagulation factor XI apple 4 domain: spectroscopic and mutational analysis

The blood coagulation protein factor XI (FXI) consists of a pair of disulfide-linked chains each containing four apple domains and a catalytic domain. The apple 4 domain (A4; F272-E362) mediates non-covalent homodimer formation even when the cysteine involved in an intersubunit disulfide is mutated to serine (C321S). To understand the role of non-covalent interactions stabilizing the FXI dimer, equilibrium unfolding of wild-type A4 and its C321S variant was monitored by circular dichroism, intrinsic tyrosine fluorescence and dynamic light scattering measurements as a function of guanidine hydrochloride concentration. Global analysis of the unimolecular unfolding transition of wild-type A4 revealed a partially unfolded equilibrium intermediate at low to moderate denaturant concentrations. The optically detected equilibrium of C321S A4 also fits best to a three-state model in which the native dimer unfolds via a monomeric intermediate state. Dimer dissociation is characterized by a dissociation constant, K(d), of approximately 90 nM (in terms of monomer), which is in agreement with the dissociation constant measured independently using fluorescence anisotropy. The results imply that FXI folding occurs via a monomeric equilibrium intermediate. This observation sheds light on the effect of certain naturally occurring mutations, such as F283L, which lead to intracellular accumulation of non-native forms of FXI. To investigate the structural and energetic consequences of the F283L mutation, which perturbs a cluster of aromatic side-chains within the core of the A4 monomer, it was introduced into the dissociable dimer, C321S A4. NMR chemical shift analysis confirmed that the mutant can assume a native-like dimeric structure. However, equilibrium unfolding measurements show that the mutation causes a fourfold increase in the K(d) value for dissociation of the native dimer and a 1 kcal/mol stabilization of the monomer, resulting in a highly populated intermediate. Since the F283 side-chain does not directly participate in the dimer interface, we propose that the F283L mutation leads to increased dimer dissociation by stabilizing a monomeric state with altered side-chain packing that is unfavorable for homodimer formation.