Self-association and oxygen-binding characteristics of the isolated subunits of Limulus polyphemus hemocyanin

The hemocyanin of the horseshoe crab Limulus polyphemus is characteristic of arthropod hemocyanins in that it is a high-molecular-weight oligomer composed of functionally and structurally distinct subunits. The protein forms a 48-subunit complex, the largest form of arthropod hemocyanin, whose oxygen-binding characteristics are modulated by subunit interaction within the oligomer. It has previously been shown that a number of electrophoretic isozymes, which are identical immunochemically, are present in dissociated Limulus hemocyanin. In this study it is demonstrated that the electrophoretic differences in the antigenically identical subunits are not reflected in their oxygen-binding and self-assembly properties or in the roles they play in reassembly and function of the 48-subunit native molecule. The chloride-dependent modulation of the oxygen-binding properties of those Limulus subunits which do not self-assemble, as documented here, illustrates that this allosteric effect may be operable at the tertiary level. For each of the purified subunits the effects of pH and calcium ions on oxygen-binding characteristics and self-assembly reactions are reported, and the roles of specific subunits in reassembly of distinct aggregation states are further documented.     

A new theoretical approach to the investigation of the symmetry of protein oligomers with bifunctional reagents

The use of bifunctional reagents to form cross-links between subunits in protein oligomers and subsequent disruption of noncovalent interactions with SDS allows comment upon the number of subunits and the symmetry in the original assembly. In existing treatments the number of equations needed to describe theoretically the proportions of all the cross-linked species that can be formed as a function of time in this way makes the analysis of the system unmanageable for proteins with more than four subunits. A method is presented that allows the required equations for any oligomer to be formulated as an algorithm suitable for solution by computer. Its application is illustrated with reference to experimental results obtained with two protein hexamers, Jasus hemocyanin and alpha-urease from jack bean.     

Electron microscopy of metal-shadowed and negatively stained microtubule protein. Structure of the 30 S oligomer

Microtubule protein purified from porcine brain was fixed at low protein concentration with glutaraldehyde under conditions which maximize the relative concentration of the ring-shaped 30 S oligomer. Fixed oligomer was separated from glutaraldehyde and other protein species by column chromatography. The fixed, isolated oligomer was deposited on electron microscopy grids, dehydrated, and then critical point-dried before shadow-coating with carbon/platinum alloy at a fixed angle. Analysis of the shadow lengths observed by electron microscopy revealed that the height of the 30 S oligomer is 15 nm. Microtubule protein deposited on electron microscope grids at high protein concentrations was examined by the negative stain technique and found to contain apparent stacks of oligomer from which the number of tubulin dimers per turn of the ring and the distance between turns could be determined. The number of subunits per turn was determined as 13.8. The distance between turns was found to be 7.4 nm, indicating that the 15 nm high, shadowed oligomers consisted of two turns. Additional information from the literature is considered and a model is presented for the oligomer. The model is a helix of 29 tubulin dimers and five high molecular weight protein molecules arranged so as to preserve intersubunit bonding patterns found in microtubules.     

Structure and electrophysiological properties of the YscC secretin from the type III secretion system of Yersinia enterocolitica

YscC is the integral outer membrane component of the type III protein secretion machinery of Yersinia enterocolitica and belongs to the family of secretins. This group of proteins forms stable ring-like oligomers in the outer membrane, which are thought to function as transport channels for macromolecules. The YscC oligomer was purified after solubilization from the membrane with a nonionic detergent. Sodium dodecyl sulfate did not dissociate the oligomer, but it caused a change in electrophoretic mobility and an increase in protease susceptibility, indicating partial denaturation of the subunits within the oligomer. The mass of the homo-oligomer, as determined by scanning transmission electron microscopy, was approximately 1 MDa. Analysis of the angular power spectrum from averaged top views of negatively stained YscC oligomers revealed a 13-fold angular order, suggesting that the oligomer consists of 13 subunits. Reconstituted in planar lipid bilayers, the YscC oligomer displayed a constant voltage-independent conductance of approximately 3 nS, thus forming a stable pore. However, in vivo, the expression of YscC did not lead to an increased permeability of the outer membrane. Electron microscopy revealed that the YscC oligomer is composed of three domains, two stacked rings attached to a conical domain. This structure is consistent with the notion that the secretin forms the upper part of the basal body of the needle structure of the type III secreton.     

The relationship between molecular weight and quaternary structure of globular proteins

The relationship between the molecular weight and the number of subunits in oligomeric globular proteins consisting of identical subunits has been analyzed. It has been shown that the molecular weights of the subunits are distributed about a mean value of 48,000 and consequently that the molecular weights of the native oligomeric proteins are distributed in clearly distinguishable molecular weight regions. This observation allows the probability of a particular oligomeric structure to be predicted from a measurement of the oligomer molecular weight alone, which is useful in a number of types of study of protein structure, particularly comparative studies. Calculations have been performed which suggest that there is no thermodynamic limitation, in terms of the subunit interactions themselves, to the size of an oligomeric protein with a given number of subunits. Rather, an individual polypeptide chain itself has inherent size limitations, which consequently limits the molecular weight of the corresponding oligomer.     

Effect of Substitution for Arginine Residues near Position 146 of the A Subunit of Escherichia coli Heat-Labile Enterotoxin on the Holotoxin Assembly

Escherichia coli heat-labile enterotoxin (LT) is a holotoxin which consists of one A and five B subunits. Although B subunit monomers released into periplasm can associate into pentameric structures in the absence of the A subunit, the A subunit accelerates the assembly. To express the function, A subunit constructs the proper spatial structure. However, the regions involved in the construction are unknown. To identify the regions, we substituted arginine residues near position 146 of the A subunit with glycine by oligonucleotide-directed site-specific mutagenesis and obtained the mutants expressing LT(R141G), LT(R143G), LT(R146G), LT(R143G, R146G), LT(R141G, R143G, R146G) and LT(R143G, R146G, R148G). We purified these mutant LTs by using an immobilized d -galactose column and analyzed the purified mutant LTs by SDS-PAGE to examine the amount of A subunit associated with B-subunit oligomer. The substitution of an arginine residue at any position did not induce a significant alteration in the amount of A subunit associated with B-subunit oligomer. However, the substitution of more than two arginine residues induced a significant decrease in the amount of A subunits associated with the B-subunit oligomer. Subsequently, we measured the level of the intracellular B-subunit oligomer of these mutant strains. The measurement revealed that the amount of B-subunit oligomer in cells decreased as the number of substituted arginine residues increased. These results show that all arginine residues near position 146 are important for the construction of the functional A subunit, and thus for holotoxin formation, although each individual arginine residue is not an absolute requirement.     

Structural interpretations of F(0) rotary function in the Escherichia coli F(1)F(0) ATP synthase

F(1)F(0) ATP synthases are known to synthesize ATP by rotary catalysis in the F(1) sector of the enzyme. Proton translocation through the F(0) membrane sector is now proposed to drive rotation of an oligomer of c subunits, which in turn drives rotation of subunit gamma in F(1). The primary emphasis of this review will be on recent work from our laboratory on the structural organization of F(0), which proves to be consistent with the concept of a c(12) oligomeric rotor. From the NMR structure of subunit c and cross-linking studies, we can now suggest a detailed model for the organization of the c(12) oligomer in F(0) and some of the transmembrane interactions with subunits a and b. The structural model indicates that the H(+)-carrying carboxyl of subunit c is located between subunits of the c(12) oligomer and that two c subunits pack in a front-to-back manner to form the proton (cation) binding site. The proton carrying Asp61 side chain is occluded between subunits and access to it, for protonation and deprotonation via alternate entrance and exit half-channels, requires a swiveled opening of the packed c subunits and stepwise association with different transmembrane helices of subunit a. We suggest how some of the structural information can be incorporated into models of rotary movement of the c(12) oligomer during coupled synthesis of ATP in the F(1) portion of the molecule.     

Rotation of the c subunit oligomer in EF(0)EF(1) mutant cD61N

ATP synthases (F(0)F(1)-ATPases) mechanically couple ion flow through the membrane-intrinsic portion, F(0), to ATP synthesis within the peripheral portion, F(1). The coupling most probably occurs through the rotation of a central rotor (subunits c(10)epsilon gamma) relative to the stator (subunits ab(2)delta(alpha beta)(3)). The translocation of protons is conceived to involve the rotation of the ring of c subunits (the c oligomer) containing the essential acidic residue cD61 against subunits ab(2). In line with this notion, the mutants cD61N and cD61G have been previously reported to lack proton translocation. However, it has been surprising that the membrane-bound mutated holoenzyme hydrolyzed ATP but without translocating protons. Using detergent-solubilized and immobilized EF(0)F(1) and by application of the microvideographic assay for rotation, we found that the c oligomer, which carried a fluorescent actin filament, rotates in the presence of ATP in the mutant cD61N just as in the wild type enzyme. This observation excluded slippage among subunit gamma, the central rotary shaft, and the c oligomer and suggested free rotation without proton pumping between the oligomer and subunit a in the membrane-bound enzyme.     

Depolymerization of phospholamban in the presence of calcium pump: a fluorescence energy transfer study

Phospholamban (PLB), a 52-amino acid protein, regulates the Ca-ATPase (calcium pump) in cardiac sarcoplasmic reticulum (SR) through PLB phosphorylation mediated by beta-adrenergic stimulation. The mobility of PLB on SDS-PAGE indicates a homopentamer, and it has been proposed that the pentameric structure of PLB is important for its regulatory function. However, the oligomeric structure of PLB must be determined in its native milieu, a lipid bilayer containing the Ca-ATPase. Here we have used fluorescence energy transfer (FET) to study the oligomeric structure of PLB in SDS and dioleoylphosphatidylcholine (DOPC) lipid bilayers reconstituted in the absence and presence of Ca-ATPase. PLB was labeled, specifically at Lys 3 in the cytoplasmic domain, with amine-reactive fluorescent donor/acceptor pairs. FET between donor- and acceptor-labeled subunits of PLB in SDS solution and DOPC lipid bilayers indicated the presence of PLB oligomers. The dependence of FET efficiency on the fraction of acceptor-labeled PLB in DOPC bilayers indicated that it is predominantly an oligomer having 9-11 subunits, with approximately 10% of the PLB as monomer, and the distance between dyes on adjacent PLB subunits is 0.9 +/- 0.1 nm. When labeled PLB was reconstituted with purified Ca-ATPase, FET indicated the depolymerization of PLB into smaller oligomers having an average of 5 subunits, with a concomitant increase in the fraction of monomer to 30-40% and a doubling of the intersubunit distance. We conclude that PLB exists primarily as an oligomer in membranes, and the Ca-ATPase affects the structure of this oligomer, but the Ca-ATPase binds preferentially to the monomer and/or small oligomers. These results suggest that the active inhibitory species of PLB is a monomer or an oligomer having fewer than 5 subunits.     

Region 1112–1123 in the central domain of 18S rRNA in 40S subunits of plant ribosomes: Accessibility for complementary interactions and the functional role

The binding of the 18S rRNA of the 40S subunits of wheat germ ribosomes to an oligodeoxyribonucleotide complementary to the 1112–1123 region of the central domain of this RNA molecule has been studied. The selective binding of this oligomer to the complementary RNA fragment and the inhibition of the translation of uncapped chimeric RNA containing enhancer sequences in the 5′-untranslated region upstream of the reporter sequence coding for β-glucuronidase has been shown in a cell-free protein-synthesizing system. The use of a derivative of the aforementioned oligomer containing an alkylating group at the 5′ end allowed for the demonstration that the 1112–1123 region of 18S RNA can form a heteroduplex with the complementary sequence of the oligomer. The data obtained show that the 1112–1123 region in loop 27 of the central domain of 18S RNA of 40S ribosomal subunits is exposed on the subunit surface and probably participates in the cap-independent binding of the subunits to mRNA due to the complementary interaction with the enhancer sequences.     

Coordination of Substrate Binding and ATP Hydrolysis in Vps4-Mediated ESCRT-III Disassembly

ESCRT-III undergoes dynamic assembly and disassembly to facilitate membrane exvagination processes including multivesicular body (MVB) formation, enveloped virus budding, and membrane abscission during cytokinesis. The AAA-ATPase Vps4 is required for ESCRT-III disassembly, however the coordination of Vps4 ATP hydrolysis with ESCRT-III binding and disassembly is not understood. Vps4 ATP hydrolysis has been proposed to execute ESCRT-III disassembly as either a stable oligomer or an unstable oligomer whose dissociation drives ESCRT-III disassembly. An in vitro ESCRT-III disassembly assay was developed to analyze Vps4 function during this process. The studies presented here support a model in which Vps4 acts as a stable oligomer during ATP hydrolysis and ESCRT-III disassembly. Moreover, Vps4 oligomer binding to ESCRT-III induces coordination of ATP hydrolysis at the level of individual Vps4 subunits. These results suggest that Vps4 functions as a stable oligomer that acts upon individual ESCRT-III subunits to facilitate ESCRT-III disassembly.     

StHsp14.0, a small heat shock protein of Sulfolobus tokodaii strain 7, protects denatured proteins from aggregation in the partially dissociated conformation

The small heat shock protein (sHsp), categorized into a class of molecular chaperones, binds and stabilizes denatured proteins for the purpose of preventing aggregation. The sHsps undergo transition between different oligomeric states to control their nature. We have been studying the function of sHsp of Sulfolobus tokodaii, StHsp14.0. StHsp14.0 exists as 24meric oligomer, and exhibits oligomer dissociation and molecular chaperone activity over 80°C. We constructed and characterized StHsp14.0 mutants with replacement of the C-terminal IKI to WKW, IKF, FKI and FKF. All mutant complexes dissociated into dimers at 50°C. Among them, StHsp14.0FKF is almost completely dissociated, probably to dimers. All mutants protected citrate synthase (CS) from thermal aggregation at 50°C. But, the activity of StHsp14.0FKF was the lowest. Then, we examined the complexes of StHsp14.0 mutants with denatured CS by SAXS. StHsp14.0WKW protects denatured CS by forming the globular complexes of 24 subunits and a substrate. StHsp14.0FKF also formed similar complex but the number of subunits in the complex is a little smaller. These results suggest that the dimer itself exhibits low chaperone activity, and a partially dissociated oligomer of StHsp14.0 protects a denatured protein from interacting with other molecules by surrounding it.     

Molecular weight determination of phospholamban oligomer in the presence of sodium dodecyl sulfate: application of low-angle laser light scattering photometry

The number of polypeptides constituting the oligomeric structure of canine phospholamban (a putative regulator of Ca(2+)-ATPase of cardiac sarcoplasmic reticulum) stable even in the presence of sodium dodecyl sulfate was estimated through determination of the molecular weight of the oligomer. Owing to the small molecular size, the low UV-absorptivity and the limited availability, the molecular weight determination required very sophisticated application of the following technique, used as the only recourse: low-angle laser light scattering measurement combined with high-performance gel chromatography. The molecular weight of phospholamban oligomer was found to be 30,400 and the number of subunits was concluded to be five after correction for the dependence of the apparent molecular weights on the protein concentration.     

The stoichiometry of the chloroplast ATP synthase oligomer III in Chlamydomonas reinhardtii is not affected by the metabolic state

The chloroplast H(+)-ATP synthase is a key component for the energy supply of higher plants and green algae. An oligomer of identical protein subunits III is responsible for the conversion of an electrochemical proton gradient into rotational motion. It is highly controversial if the oligomer III stoichiometry is affected by the metabolic state of any organism. Here, the intact oligomer III of the ATP synthase from Chlamydomonas reinhardtii has been isolated for the first time. Due to the importance of the subunit III stoichiometry for energy conversion, a gradient gel system was established to distinguish oligomers with different stoichiometries. With this methodology, a possible alterability of the stoichiometry in respect to the metabolic state of the cells was examined. Several growth parameters, i.e., light intensity, pH value, carbon source, and CO(2) concentration, were varied to determine their effects on the stoichiometry. Contrary to previous suggestions for E. coli, the oligomer III of the chloroplast H(+)-ATP synthase always consists of a constant number of monomers over a wide range of metabolic states. Furthermore, mass spectrometry indicates that subunit III from C. reinhardtii is not modified posttranslationally. Data suggest a subunit III stoichiometry of the algae ATP synthase divergent from higher plants.     

Theory and application of fluorescence homotransfer to melittin oligomerization.

Fluorescence homotransfer (electronic energy transfer between identical fluorophores) has the potential to quantitate the number of subunits in membrane protein oligomers. Homotransfer strongly depolarizes fluorescence emission as a result of intermolecular excitation energy exchange between an initially excited, oriented molecule and a randomly oriented neighbor. We have theoretically treated fluorescein labeled subunits in an oligomer as a cluster of molecules that can exchange excitation energy back and forth among the subunits within that group. We find that the larger the number of subunits, the more depolarized is the emission. The general equations to calculate the expected anisotropy for complexes composed of varying numbers of labeled subunits are presented. Self-quenching of fluorophores, orientation, and changes in lifetime are also discussed and/or considered. To test this theory, we have specifically labeled melittin on its N-terminal with fluorescein and monitored its monomer to tetramer equilibrium both in solution and in lipid bilayers. The calculated anisotropies are close to the experimental values when non-fluorescent fluorescein dimers are taken into account. Our results show that homotransfer may be a promising method to study membrane-protein oligomerization.     

The enzyme activity allosteric regulation model based on the composite nature of catalytic and regulatory sites concept

A new kinetic model of enzymatic catalysis is proposed, which postulates that enzyme solutions are equilibrium systems of oligomers differing in the number of subunits and in the mode of their assembly. It is suggested that the catalytic and regulatory sites of allosteric enzymes are of composite nature and appear as a result of subunits joining. Two possible joining modes are postulated at each oligomerization step. Catalytic site may arise on oligomer formed only by one of these modes. Effector acts by fastening together components of certain oligomeric form and increases the life time of this form. It leads to a shift of oligomer equilibrium and increases a proportion of effector-binding oligomers. Effectors-activators bind the oligomers carrying composite catalytic sites and effectors-inhibitors bind the oligomers, which do not carry active catalytic sites. Thus, catalytic activity control in such system is explained by effector-induced changes of a catalytic sites number, but not of a catalytic site activity caused by changes of subunit's tertiary structure. The postulates of the model do not contradict available experimental data and lead to a new type of general rate equation, which allows to describe and understand the specific kinetic behavior of allosteric enzymes as well as Michaelis type enzymes. All known rate equations of allosteric The equation was tested by modeling the kinetics of human erythrocyte phosphofructokinase. It enabled to reproduce quantitatively the 66 kinetic curves experimentally obtained for this enzyme under different reaction conditions.     

The subunit S1 is important for pertussis toxin secretion

Pertussis toxin is a protein containing five noncovalently linked subunits which are assembled into the monomer A (containing the subunit S1) and the oligomer B (containing subunits S2, S3, S4, and S5 in a 1:1:2:1 ratio). Each of the five subunits is synthesized as a precursor containing a secretory leader peptide and is secreted into the periplasm of Bordetella pertussis where the five subunits are assembled into the oligomeric structure and then released into the culture medium. In the absence of subunit S3 the remaining subunits are not secreted into the medium, thus suggesting that the assembled structure is necessary for the release of the toxin into the supernatant. In this study we describe four B. pertussis mutants which secrete into the medium low amounts of the B oligomer of pertussis toxin. These mutants have single or multiple changes in the gene encoding the S1 subunit and synthesize S1 proteins with altered conformation which are not assembled into the holotoxin and are apparently degraded in the periplasm. These data indicate that while the B oligomer alone has the structural information necessary for the extracellular export of pertussis toxin, the S1 subunit is required for its efficient release into the medium.     

The biological functions of low-frequency vibrations (phonons) 5. A phenomenological theory

Low-frequency internal motions of a biomacromolecule are thought to possess significant biological function from the dynamic point of view. In this paper, a general phenomenological theory is established by which it is clearly verified that low-frequency resonance plays a central role in the energy transmission required during the cooperative interaction between subunits in a protein oligomer. According to the present theory, it is found that the energy transmission between a pair of diagonal subunits in a protein oligomer with a polygon arrangement is the most efficient, so as to in a sense further predict that after a ligand is bound to a subunit by random collision, its diagonal subunit in the same protein oligomer will possess the greatest probability of binding with the next ligand. Furthermore, based on the concept of the 'resonance-controlled trigger' derived from the phenomenological theory, it is feasible to estimate the lower time limit of allosteric transition from one subunit to the other. Such a time limit depends on the dominant low-frequency mode of each subunit, the ratio of the coupling force constant to the corresponding inherent force constant, as well as the geometrical arrangement of subunits in a protein oligomer. So far none of the allosteric transitions observed in proteins has exceeded the time limit as defined here, indicating a logical consistency between our theory and the experiments.     

Reversible fast-dimerization of bovine serum albumin detected by fluorescence resonance energy transfer

Self-association of bovine serum albumin (BSA) was explored using fluorescence resonance energy transfer (FRET) between two populations of the protein labeled separately with either fluorescein-5'-isothiocyanate (FITC) or eosin-5'-isothiocyanate (EITC). The energy transfer reached the steady state after 5 s at 25 degrees C, indicating a fast exchange between oligomer subunits. The dependence of the energy transfer efficiency on the protein concentration and its reversion by unlabeled BSA demonstrate that association between BSA monomers occurs through a reversible path that involves specific interactions between the protein molecules. Because energy transfer took place even after blocking Cys 34 with iodoacetamide, this residue might not be involved in the reversible self-association process. The number of subunits forming the oligomer and its dissociation constant were determined from measurements of energy transfer as a function of the donor-acceptor ratio and of the total protein concentration. Analysis of these data indicated that BSA is in a monomer-dimer equilibrium with a dissociation constant of 10 +/- 2 microM at 25 degrees C in 10 mM MOPS-K (pH 5.8).     

Biochemical and genetic properties of oligomeric structures: a general approach

The oligomers constituted by association of different subunits can exist under multiple forms. In the case of the genetically variable proteins, such a multiplicity leads to numerous questions (i) on the enumerations: what is the number of active forms when a given subunit can make the oligomer inactive, or when the subunits are encoded by s alleles; (ii) on the subunit effects on biochemical properties: how to estimate these effects, are they equal, are there interactions between subunits, etc. Theoretical methods for the study of such oligomeric structures are developed, which mainly rely on linear model techniques. Peculiar properties examined are Vmax and Km, but also the quantities of the various oligomers, which depend on their association law. This approach is extended to the oligomers composed of different sets of subunits, as are for example some enzymes. These aspects are discussed from numerous bibliographic examples, with special reference to molecular interactions (protein complementation or molecular heterosis). Otherwise the genetic application of this theoretical approach is presented: it is possible to consider a genotype as an oligomer of alleles, and thus to study their effects and their interactions, in the one-locus case as well as in the several-loci case. The relevance of this generalization is discussed in connection with two other concepts, the "sequence space" used in molecular evolution and the regression of the genotypic values on the number of alleles used in quantitative genetics.