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.     

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.     

Differential detergent solubility investigation of thermally induced transitions in cytochrome c oxidase

The thermal denaturation of membrane-reconstituted cytochrome c oxidase (EC 1.9.3.1) occurs at approximately 63 degrees C as determined by high-sensitivity differential scanning calorimetry. The heat capacity profile associated with this process is characterized by the presence of two well-defined peaks, indicating that all the enzyme subunits do not have the same thermal stability. This thermal denaturation of the enzyme complex is coupled to a change in its solubility properties. This change in solubility allows separation of the native and denatured protein fractions by detergent solubilization followed by centrifugation under conditions in which only the native fraction is solubilized. Using this principle, it has been possible to study the denaturation of membrane-reconstituted cytochrome c oxidase and quantitatively identify the protein subunits undergoing thermal denaturation using computer-assisted gel electrophoresis analysis. This technique allows calculation of single-subunit thermal denaturation profiles within the intact enzyme complex, and as such, it can be used to obtain transition temperatures, molecular populations, and van't Hoff enthalpy changes for individual protein subunits, thus complementing results obtained by high-sensitivity differential scanning calorimetry.     

Particle weights and protein composition of the ribosomal subunits of the extremely thermoacidophilic archaebacterium Caldariella acidophila.

1. The ribosomal subunits of one thermoacidophilic archaebacterium (Caldariella acidophila) and of two reference eubacterial species (Bacillus acidocaldarius, Escherichia coli) were compared with respect to ribosome mass and protein composition by (i) equilibrium-density sedimentation of the particles in CsCl and (ii) gel-electrophoretic estimations of the molecular weights of the protein and the rRNA. 2. By either procedure, it is estimated that synthetically active archaebacterial 30S subunits (52% protein by wt.) are appreciably richer in protein than the corresponding eubacterial particles (31% protein by wt.) 3. The greater protein content of the archaebacterial 30S subunits is accounted for by both a larger number and a greater average molecular weight of the subunit proteins; specifically, C. acidophila 30S subunits yield 28 proteins whose combined mass is 0.6 X 10(6) Da, compared with 20 proteins totalling 0.35 X 10(6) Da mass for eubacterial 30S subunits. 4. No differences in protein number are detected among the large subunits, but C. acidophila 50S subunits exhibit a greater number-average molecular weight of their protein components than do eubacterial 50S particles. 5. Particle weights estimated by either buoyant-density data, or molecular weights of rRNA plus protein, agree to within less than 2%. By either procedure C. acidophila 30S subunits 1.15 X 10(6) Da mass) are estimated to be about 300 000 Da heavier than their eubacterial counterparts (0.87 X 10(6) Da mass); a smaller difference. 0.15 X 10(6) Da, exists between the archaebacterial and the eubacterial 50S subunits (respectively 1.8 X 10(6) and 1.65 X 10(6) Da). It is concluded that the heavier-than-eubacterial mass of the C. acidophila ribosomes resides principally in their smaller subunits.     

Creating hetero-11-mers composed of wild-type and mutant subunits to study RNA binding to TRAP.

TRAP (trp RNA-binding attenuation protein) is an RNA-binding protein that regulates expression of the tryptophan biosynthetic genes in Bacillus subtilis by binding to RNA targets that contain multiple GAG and UAG repeats. TRAP is composed of 11 identical subunits arranged symmetrically in a ring. The secondary structure of the protein consists entirely of antiparallel beta-sheets, beta-turns, and loops. We show here that the TRAP 11-mer can be reversibly denatured into unfolded monomers by guanidine hydrochloride. Removing the denaturant allows the protein to spontaneously renature into fully functional 11-mers. Based on this finding, we developed a subunit mixing method to hybridize wild-type and mutant subunits into heteromeric 11-mers by denaturation followed by subunit mixing renaturation. This method allows the study of subunit cooperativity in protein-ligand interaction such as RNA binding. Our data further support and extend the previously proposed two-step model for RNA binding to TRAP by showing that the initiation of binding requires at least one fully active subunit in the protein combined with one fully functional repeat in the RNA. The initiation complex tethers the RNA on the protein, thus allowing cooperative interaction with the remainder of the repeats.     

Expression of Torpedo nicotinic acetylcholine receptor subunits in yeast is enhanced by use of yeast signal sequences

We have produced the four subunits of the nicotinic acetylcholine receptor of Torpedo californica, an integral membrane protein, in the yeast Saccharomyces cerevisiae. Two of the subunits (alpha and delta) were readily produced from their cDNAs after simply subcloning them into a yeast shuttle vector adjacent to a yeast promoter. The other two protein subunits (beta and gamma) were not produced by this strategy, although the amounts of mRNA produced from these expression constructs are similar to those for alpha and delta. Replacing the DNA coding for the normal N-terminal signal sequences for the beta and gamma subunits with DNA coding for the signal sequence of yeast invertase results in successful protein synthesis. The yeast signal sequence allows these subunits to be translocated across the membrane of the endoplasmic reticulum and to be glycosylated. The appropriate final size of the subunit proteins suggests that the yeast signal sequence has been properly cleaved after translocation.     

Estimating friction coefficients of mixed globular/chain molecules, such as protein/DNA complexes.

Existing methods for predicting translational friction properties of complex molecules start by explicitly building up their three-dimensional shape with spherical subunits. This treatment has been used especially for two types of systems: rigid assemblies and flexible chain molecules. However, many protein/DNA complexes such as chromatin consist of a small number of globular, relatively rigid, bound protein interspersed by long stretches of flexible DNA chain. I present a higher level of treatment of such macromolecules that avoids explicit subunit modeling as much as possible. An existing analytical formulation of the hydrodynamics equations is shown to be accurate when used with the present treatment. Thus the approach is fast and can be applied to hydrodynamic studies of highly degenerate multiple equilibria, such as those encountered in problems of the regulation of chromatin structure. I demonstrate the approach by predicting the effect of a hypothetical unwinding process in dinucleosomes and by simulating the distribution of sedimentation coefficients for cooperative and random models for a chromatin saturation process.     

Salt bridges at the inter-ring interface regulate the thermostat of GroEL

The chaperonin GroEL consists of a double-ring structure made of identical subunits and displays unusual allosteric properties caused by the interaction between its constituent subunits. Cooperative binding of ATP to a protein ring allows binding of GroES to that ring, and at the same time negative inter-ring cooperativity discharges the ligands from the opposite ring, thus driving the protein-folding cycle. Biochemical and electron microscopy analysis of wild type GroEL, a single-ring mutant (SR1), and two mutants with one inter-ring salt bridge of the chaperonin disrupted (E461K and E434K) indicate that these ion pairs form part of the interactions that allow the inter-ring allosteric signal to be transmitted. The wild type-like activities of the ion pair mutants at 25 degrees C are in contrast with their lack of inter-ring communication and folding activity at physiological temperatures. These salt bridges stabilize the inter-ring interface and maintain the inter-ring spacing so that functional communication between protein heptamers takes place. The characterization of GroEL hybrids containing different amounts of wild type and mutant subunits also indicates that as the number of inter-ring salt bridges increases the functional properties of the hybrids recover. Taken together, these results strongly suggest that inter-ring salt bridges form a stabilizing ring-shaped, ionic zipper that ensures inter-ring communication at the contact sites and therefore a functional protein-folding cycle. Furthermore, they regulate the chaperonin thermostat, allowing GroEL to distinguish physiological (37 degrees C) from stress temperatures (42 degrees C).     

Packaging of up to 240 subunits of a 17 kDa nuclease into the interior of recombinant hepatitis B virus capsids

The icosahedral nucleocapsid of hepatitis B virus (HBV) consists of multiple subunits of a single 183 amino acids (aa) core protein encasing the viral genome. However, recombinant core protein alone also forms capsid-like particles. We have recently shown that a 238 aa protein centrally inserted into the core protein can be displayed on the particle surface. Here we demonstrate that replacement of the C-terminal basic domain by the 17 kDa Staphylococcus aureus nuclease also yields particles but that in these the foreign domains are located in the interior. The packaged nuclease is enzymatically active, and the chimeric protein forms mosaic particles with the wild-type core protein. Hence the HBV capsid is useful as a molecular platform which, dependent on the fusion site, allows foreign protein domains to either be packaged into or be exposed on the exterior of the particle. These results are of relevance for the use of the HBV capsid as a vaccine carrier, and as a target for antiviral therapy.     

3D complex: a structural classification of protein complexes

Most of the proteins in a cell assemble into complexes to carry out their function. It is therefore crucial to understand the physicochemical properties as well as the evolution of interactions between proteins. The Protein Data Bank represents an important source of information for such studies, because more than half of the structures are homo- or heteromeric protein complexes. Here we propose the first hierarchical classification of whole protein complexes of known 3-D structure, based on representing their fundamental structural features as a graph. This classification provides the first overview of all the complexes in the Protein Data Bank and allows nonredundant sets to be derived at different levels of detail. This reveals that between one-half and two-thirds of known structures are multimeric, depending on the level of redundancy accepted. We also analyse the structures in terms of the topological arrangement of their subunits and find that they form a small number of arrangements compared with all theoretically possible ones. This is because most complexes contain four subunits or less, and the large majority are homomeric. In addition, there is a strong tendency for symmetry in complexes, even for heteromeric complexes. Finally, through comparison of Biological Units in the Protein Data Bank with the Protein Quaternary Structure database, we identified many possible errors in quaternary structure assignments. Our classification, available as a database and Web server at http://www.3Dcomplex.org, will be a starting point for future work aimed at understanding the structure and evolution of protein complexes.     

A dynamic quaternary structure of bovine alpha-crystallin as indicated from intermolecular exchange of subunits

The structural bovine eye lens protein alpha-crystallin was dissociated in 7 M urea and its four subunits, A1, A2, B1, and B2, were separated by means of ion-exchange chromatography. Homopolymeric reaggregates of these subunits were prepared by removal of the denaturant via dialysis. It was found that subunits were exchanged upon incubation of mixtures of two homopolymers under native conditions. New hybrid species were formed within 24 h as demonstrated by isoelectric focusing. Moreover, native alpha-crystallin molecules also exchanged subunits when incubated with homopolymeric aggregates of B2 subunits. Subunit exchange between native alpha-crystallin molecules is postulated, and a "dynamic quaternary structure" is presented that allows the polydisperse protein to adapt to changes in cytoplasmic conditions upon aging of the lens tissue.     

Stability and self-assembly of the S-layer protein of the cell wall of Bacillus stearothermophilus

The surface layer of the cell envelope of Bacillus stearothermophilus consists of a regular array of protein subunits. As shown by dodecyl sulfate polyacrylamide gel-electrophoresis and ultracentrifugation, the fully solubilized S-layer protein represents a homogeneous entity with a subunit molecular mass of 115 +/- 5 kDa. Solubilization of the protein may be accomplished at acid pH, or using high concentrations of urea or guanidine X HCl. It is accompanied by (partial) denaturation, thus interfering with the characterization of the protein in its unperturbed native state. Removal of the solubilizing agent by dialysis or dilution allows the S-layer to be reassembled into two-dimensional crystalline lattices identical to those observed in intact cells. To determine the kinetics of association, optimum conditions are found to be rapid mixing with 0.1 M sodium phosphate pH 7.0, 20 degrees C, final protein concentration greater than 10 micrograms/ml. If the time course of the self-assembly is monitored by light scattering, as well as by chemical cross-linking with glutardialdehyde, multiphasic kinetics with a rapid initial phase and slow consecutive processes of higher than second-order are observed. The rapid phase may be attributed to the formation of oligomeric precursors (Mr greater than 10(6) ). Concentration-dependent light scattering measurements give evidence for a "critical concentration" of association, suggesting that patches of 12-16 protein subunits fuse and recrystallize into the final (native) S-layer structure. Recrystallization tends to be complete.     

Elucidation of the stator organization in the V-ATPase of Neurospora crassa

V-ATPases are membrane protein complexes that pump protons in the lumen of various subcellular compartments at the expense of ATP. Proton pumping is done by a rotary mechanism that requires a static connection between the membrane pumping domain (V(0)) and the extrinsic catalytic head (V(1)). This static connection is composed of several known subunits of the V-ATPase, but their location and topological relationships are still a matter of controversy. Here, we propose a model for the V-ATPase of Neurospora crassa on the basis of single-particle analysis by electron microscopy. Comparison of the resulting map to that of the A-ATPase from Thermus thermophilus allows the positioning of two subunits in the static connecting region that are unique to eukaryotic V-ATPases (C and H). These two subunits seem to be located on opposite sides of a semicircular arrangement of the peripheral connecting elements, suggesting a role in stabilizing the stator in V-ATPases.     

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.     

Analysis of the interaction mode between hyperthermophilic archaeal group II chaperonin and prefoldin using a platform of chaperonin oligomers of various subunit arrangements

Prefoldin is a co-chaperone that captures an unfolded protein substrate and transfers it to the group II chaperonin for completion of protein folding. Group II chaperonin of a hyperthermophilic archaeon, Thermococcus strain KS-1, interacts and cooperates with archaeal prefoldins. Although the interaction sites within chaperonin and prefoldin have been analyzed, the binding mode between jellyfish-like hexameric prefoldin and the double octameric ring group II chaperonin remains unclear. As prefoldin binds the chaperonin β subunit more strongly than the α subunit, we analyzed the binding mode between prefoldin and chaperonin in the context of Thermococcus group II chaperonin complexes of various subunit compositions and arrangements. The oligomers exhibited various affinities for prefoldins according to the number and order of subunits. Binding affinity increased with the number of Cpnβ subunits. Interestingly, chaperonin complexes containing two β subunits adjacently exhibited stronger affinities than other chaperonin complexes containing the same number of β subunits. The result suggests that all four β tentacles of prefoldin interact with the helical protrusions of CPN in the PFD–CPN complex as the previously proposed model that two adjacent PFD β subunits seem to interact with two CPN adjacent subunits.     

Thermodynamics of information transfer between subunits in oligomeric enzymes and kinetic cooperativity. 1. Thermodynamics of subunit interactions, partition functions and enzyme reaction rate

The strength of quaternary constraints between two subunits of a polymeric enzyme depends upon the number of neighboring subunits and upon whether these subunits are liganded or not. These quaternary constraints between two subunits of a complex polymeric enzyme may be expressed, however, in terms of quaternary constraints that exist within ideal dimers. The influence of quaternary constraints on the reaction rate of a complex polymeric enzyme may thus be expressed in terms of the intersubunit strain that exists within dimers. This conclusion, that was far from evident, appears to be the consequence of the postulates of structural kinetics, and derive as well from usual thermodynamic principles. The structural steady-state equations may be expressed in terms of partition and sub-partition functions. As applied to structural kinetic models, a partition function expresses how, during the steady state, the energy of a population of enzyme molecules is distributed over n states. Similarly a sub-partition function describes how, during the steady state, the energy of these enzyme molecules is partitioned among only n-k of these states. Although the concept of partition function was initially formulated for equilibrium processes, it may be extended without any loss of generality to non-equilibrium processes. Moreover it is reminiscent of the concept of binding polynomial presented some years ago by Wyman for the equilibrium binding of a ligand to a protein. With this formalism derived from statistical mechanics, a structural rate equation may be derived from the ratio of a sub-partition function of degree n-1 and of a partition function of degree n. Again these properties are the consequence of the postulates of structural kinetics associated with simple ideas derived from statistical thermodynamics.     

Localization of nuclear subunits of cyclic AMP-dependent protein kinase by the immunocolloidal gold method

An immunocolloidal gold electron microscopy method is described allowing the ultrastructural localization and quantitation of the regulatory subunits RI and RII and the catalytic subunit C of cAMP-dependent protein kinase. Using a postembedding indirect immunogold labeling procedure that employs specific antisera, the catalytic and regulatory subunits were localized in electron-dense regions of the nucleus and in cytoplasmic areas with a minimum of nonspecific staining. Antigenic domains were localized in regions of the heterochromatin, nucleolus, interchromatin granules, and in the endoplasmic reticulum of different cell types, such as rat hepatocytes, ovarian granulosa cells, and spermatogonia, as well as cultured H4IIE hepatoma cells. Morphometric quantitation of the relative staining density of nuclear antigens indicated a marked modulation of the number of subunits per unit area under various physiologic conditions. For instance, following partial hepatectomy in rats, the staining density of the nuclear RI and C subunits was markedly increased 16 h after surgery. Glucagon treatment of rats increased the staining density of only the nuclear catalytic subunit. Dibutyryl cAMP treatment of H4IIE hepatoma cells led to a marked increase in the nuclear staining density of all three subunits of cAMP-dependent protein kinase. These studies demonstrate that specific antisera against cAMP-dependent protein kinase subunits may be used in combination with immunogold electron microscopy to identify the ultrastructural location of the subunits and to provide a semi-quantitative estimate of their relative cellular density.     

Different packaging of DNA in the filamentous viruses Pf1 and Xf

Xf Virus DNA, like Pf1 DNA, is a single-stranded circular molecule and contains, within experimental error, the same number of nucleotides, 7400. This was unexpected since Pf1 virus is 2 μm long while Xf virus is only 1 μm long. The ratio of nucleotides to major coat protein subunits has been found to be nearly unity in Pf1 and nearly two in Xf, but it is not certain that the ratios have exactly integer values. Calculations give the average axial internucleotide separation in Pf1virus as 5.3 Å whereas in Xf virus, the calculated separation is only 2.6 Å. The protein subunits in both Pf1 and Xf have calculated axial separations close to 2.6 Å. The results provide a solution to a problem encountered in the interpretation of X-ray diffraction patterns of these viruses concerning the number of protein subunits per helical turn.     

The Enzyme Activity Allosteric Regulation Model Based on the Composite Nature of Catalytic and Regulatory Sites Concept

Abstract

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.