Aldolase Tetramers Research Articles

Precise Fabrication of De Novo Nanoparticle Lattices on Dynamic 2D Protein Crystalline Lattices.

The science of protein self-assembly has experienced significant development, from discrete building blocks of self-assembled nanoarchitectures to advanced nanostructures with adaptive functionalities. Despite the prominent achievements in the field, the desire of designing de novo protein-nanoparticle (NP) complexes and constructing dynamic NP systems remains highly challenging. In previous works, l-rhamnulose-1-phosphate aldolase (C98RhuA) tetramers were self-assembled into two-dimensional (2D) lattices via disulfide bond interactions. These interactions provided 2D lattices with high structural quality and a sophisticated assembly mode. In this study, we devised a rational design for RhuA building blocks to fabricate 2D functionalized protein lattices. More importantly, the lattices were used to direct the precise assembly of NPs into highly ordered and diverse nanoarchitectures. These structures can be employed as an excellent tool to adequately verify the self-assembly mode and structural quality of the designed RhuA crystals. The subsequent redesign of RhuA building blocks enabled us to predictably produce a novel protein lattice whose conformational dynamics can be controllably regulated. Thus, a dynamic system of AuNP lattices was achieved. Transmission electron microscopy and small-angle X-ray scattering indicated the presence of these diverse NP lattices. This contribution enables the fabrication of future NP structures in a more programmable manner with more expected properties for potential applications in nanoelectronics and other fields.

Ion Mobility-Mass Spectrometry Reveals Highly-Compact Intermediates in the Collision Induced Dissociation of Charge-Reduced Protein Complexes.

Protocols that aim to construct complete models of multiprotein complexes based on ion mobility and mass spectrometry data are becoming an important element of integrative structural biology efforts. However, the usefulness of such data is predicated, in part, on an ability to measure individual subunits removed from the complex while maintaining a compact/folded state. Gas-phase dissociation of intact complexes using collision induced dissociation is a potentially promising pathway for acquiring such protein monomer size information, but most product ions produced are possessed of high charge states and elongated/string-like conformations that are not useful in protein complex modeling. It has previously been demonstrated that the collision induced dissociation of charge-reduced protein complexes can produce compact subunit product ions; however, their formation mechanism is not well understood. Here, we present new experimental evidence for the avidin (64 kDa) and aldolase (157 kDa) tetramers that demonstrates significant complex remodeling during the dissociation of charge-reduced assemblies. Detailed analysis and modeling indicates that highly compact intermediates are accessed during the dissociation process by both complexes. Here, we present putative pathways that describe the formation of such ions, as well as discuss the broader significance of such data for structural biology applications moving forward.

Effect of acrylamide on aldolase structure. II. Characterization of aldolase unfolding intermediates

Molecules of muscle aldolase A exposed to acrylamide change their conformation via I 1, T, I 2, D intermediates [1] and undergo a slow irreversible chemical modification of thiol groups. There is no direct correlation between activity loss and thiol groups modification. In the native enzyme two classes of Trp residues of 1.8 ns and 4.9 ns fluorescence lifetime have been found. Acrylamide (0.2–0.5 M) increases lifetime of longer-lived component, yet the transfer of aldolase molecules even from higher (1.0 M) perturbant concentration to a buffer, allows regain original Trp fluorescence lifetime. I 1, detected at about 0.2 M acrylamide, represents low populated tetramers of preserved enzyme activity. T, of maximum population at about 0.7–1.0 M acrylamide, consists of meta-stable tetramers of partial enzymatic activity. These molecules are able to exchange their subunits with aldolase C in opposition to the native molecules. At transition point for I 2 appearance (1.8 M acrylamide), aldolase becomes highly unstable: part of molecules dissociate into subunits which in the absence of perturbant are able to reassociate into active tetramers, the remaining part undergoes irreversible denaturation and aggregation. Some expansion of aldolase tetramers takes place prior to dissociation. D, observed above 3.0 M acrylamide, consists of irreversibly denatured enzyme molecules.

Associations between erythrocyte band 3 protein and aldolase in detergent solution. Determining their stoichiometry by analytical ultracentrifugation.

The cytoplasmic domain of band 3, the predominant polypeptide of the erythrocyte membrane, represents a binding site for certain glycolytic enzymes. We have studied the association between human band 3 protein and aldolase, in order to clarify the role of the different band 3 oligomers as ligand binding sites. The experiments were performed on mixtures of solubilized band 3 and aldolase in solutions of a nonionic detergent, nonaethyleneglycol lauryl ether. The main technique applied was sedimentation equilibrium analysis in an analytical ultracentrifuge. In addition, nonequilibrium centrifugation techniques were used. To facilitate the evaluations, the aldolase was labelled with a dye. The following results were obtained. (1) With unmodified band 3, aldolase is bound exclusively or at least predominantly to the band 3 tetramer (but not to monomers or dimers). (2) The band 3 tetramer can bind up to four aldolase tetramers. (3) The band 3 tetramer/aldolase complex is unstable on the time scale of the techniques used. (4) Stable band 3 dimers (stabilized either covalently or noncovalently) can also associate with aldolase and can bind up to two aldolase tetramers. The results described, together with those reported previously, point at a prominent role of the band 3 tetramer in ligand binding.

Preliminary crystallographic investigation of rabbit liver aldolase.

Rabbit liver aldolase was purified by affinity elution on a CM52 ion exchanger. Crystals of rabbit liver aldolase suitable for X-ray diffraction experiments were grown from 45% saturated ammonium sulfate solution at 4 degrees C. The enzyme crystallizes in space group C222(1) having cell dimensions a = 377.02 A, b = 130.35 A, c = 80.04 A and diffracts to at least 3.5 A resolution. On the basis of a 55% solvent content there are eight aldolase tetramers in the unit cell. Rotational symmetry analysis to 6.7 A is consistent with the aldolase tetramers having a high degree of internal symmetry corresponding to point group 222. The crystallized enzyme is catalytically active.

Stability of quaternary structure and mode of dissociation of fructosediphosphate aldolase isoenzymes.

Using a highly sensitive "subunit exchange" assay, we have studied the relative strengths of interactions between different subunit types (A and C) of fructosediphosphate aldolase and have determined the mode of dissociation of aldolase tetramers in vitro. Interactions between C subunits within C4 tetramers were found to be considerably more resistant to disruption than were interactions between A subunits in A4 tetramers with regard to increasing concentrations of H+, OH-, or urea. Slight dissociation of A4 was also observed in 1.2 M magnesium chloride. These observations suggest that the quaternary structure of aldolase C4 is inherently more stable than that of aldolase A4. Also, the symmetrical heterotetramer A2C2 was found to be more resistant to urea-mediated dissociation than was the aldolase A4 homotetramer; this observation suggests that, even when in heteromeric combination, C subunits have a stabilizing influence on the quaternary structure of aldolase tetramers. In no case did we find evidence for a stable dimeric intermediate in the dissociation of aldolase tetramers to monomers. These observations are considered in terms of the tetrahedral arrangement of subunits in the aldolase tetramer. The general applicability of the subunit exchange assay described here for studying the subunit structure and mode of dissociation of oligomeric enzymes is discussed.

Mechanisms for the genesis of aldolase tetramers in cell-free protein synthesizing systems and in vivo.

Mechanisms for the genesis of aldolase tetramers in cell-free protein synthesizing systems and in vivo.

Physico-chemical evidence for the interaction between aldolase and glyceraldehyde-3-phosphate dehydrogenase.

Polarization of fluorescence measurements of aldolase and D-glyceraldehyde-3-phosphate dehydrogenase labeled with fluorescein isothiocyanate have been used to detect the possible formation of a soluble complex between the proteins. The results suggest an interaction between aldolase and D-glyceraldehyde-3-phosphate dehydrogenase with an apparent dissociation constant 3 X 10(-7) M and an apparent stoichiometry of two aldolase tetramers bound per tetramer of D-glyceraldehyde-3-phosphate dehydrogenase.

Synthesis of functional aldolase tetramers in a heterologous cell-free system.

The present report describes the complete synthesis of a functional oligomeric enzyme in a heterologous cell-free system. Polysomal RNA from chicken skeletal muscle was used to direct the production of functional aldolase tetramers in wheat germ extracts. The aldolase product was (a) specifically precipitated with monospecific antibodies raised against pure muscle aldolase, (b) had the same subunit molecular weight (40,000) as that of native aldolase (as determined by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate), (c) presumably contained a functional active site since it co-purified with authentic muscle aldolase upon substrate elution from phosphocellulose, and (d) had associated into tetrameric units (Mr=160,000) as shown by centrifugation in sucrose gradients. The present work suggests that, within the cell, post-translational processing of aldolase polypeptide chains is not involved in the formation of functional aldolase tetramers.

Evidence for the lack of subunit exchange between aldolase tetramers in vivo.

The results of a double isotope experiment using 3H- and 14C-labeled leucine as precursors of protein synthesis demonstrated that the aldolase C to A subunit transition which is associated with chick skeletal muscle development involves the preferential synthesis of different aldolase isoenzymes. This developmental system was used to test for subunit exchange between aldolase tetramers in vivo. In a second double isotope experiment, it was found that the 14C:3H ratios of A and C subunits derived from the same heterotetramer were essentially identical, while the isotope ratios of the same subunit type derived from different isoenzymes were considerably different. Had subunit exchange between the isoenzymes occurred, A subunits of a given heterotetramer would have been expected to have higher isotope ratios than the corresponding C subunits. Therefore, these data suggest that subunit exchange between aldolase tetramers does not occur in vivo, at least not in skeletal muscle to an appreciable extent. The results of the present study suggest that all aldolase tetramers are constructed at the time of the initial assembly of newly synthesized subunits, that is, "new" tetramers would not be generated by subunit exchange between already constructed tetramers. In addition, the present work suggests that the degradation of all four subunits of an aldolase tetramer are coupled inasmuch as the subunits would not be reincorporated into other tetramers. Thus, in contrast to some other proteins, it appears that the subunits of the aldolase tetramer turn over coordinately.

Catalytic and Immunochemical Properties of Homomeric and Heteromeric Combinations of Aldolase Subunits

Abstract The catalytic and immunochemical properties of homomeric and heteromeric rabbit fructose diphosphate aldolase tetramers are described. Heteromers composed of either A and C (A3C, A2C2, AC3) or B and C (B3C, B2C2, BC3) subunits have maximal velocities and Michaelis constants indistinguishable from those of an equivalent mixture of homomers. These results indicate the subunits act independently and also imply the existence of four catalytically active sites in the aldolase molecule (one active site per subunit). Specific noncross-reaction antibodies prepared against the homomeric aldolases A4, B4, and C4 react with heteromers containing one or more subunits to which the antibodies had been directed. This interaction results in essentially complete inhibition of the catalytic activity of the heteromer. The amount of antibody required is inversely proportional to the number of antigenic subunits in the heteromer. These results suggest that (a) interaction with antibody may alter subunit interactions that are required for catalytic activity, and (b) antigenic sites may be modified in homomeric and heteromeric combination.