Characterization of heterologous protein-protein interactions using analytical ultracentrifugation.

Methods for quantitative characterization of heterologous protein-protein interactions by means of analytical ultracentrifugation (AUC) include sedimentation equilibrium, tracer sedimentation equilibrium, sedimentation velocity, and analytical band sedimentation. Fundamental principles governing the behavior of macromolecules in a centrifugal field are summarized, and the application of these principles to the interpretation of data obtained from each type of experiment is reviewed. Instrumentation and software for the acquisition and analysis of data obtained from different types of AUC experiments are described.     

Determination of the sedimentation coefficient of vaccinia virus DNA by analytical ultracentrifugation

By releasing intact molecules of vaccinia virus DNA, it has been possible to determine the sedimentation coefficient by analytical ultracentrifugation. The value found was 92S, which corresponds to a molecular weight of 161×10⁶.     

Characterization of recombinant murine leukemia virus integrase.

Retroviral integration involves two DNA substrates that play different roles. The viral DNA substrate is recognized by virtue of specific nucleotide sequences near the end of a double-stranded DNA molecule. The target DNA substrate is recognized at internal sites with little sequence preference; nucleosomal DNA appears to be preferred for this role. Despite this apparent asymmetry in the sequence, structure, and roles of the DNA substrates in the integration reaction, the existence of distinct binding sites for viral and target DNA substrates has been controversial. In this report, we describe the expression in Escherichia coli and purification of Moloney murine leukemia virus integrase as a fusion protein with glutathione S-transferase, characterization of its activity by using several model DNA substrates, and the initial kinetic characterization of its interactions with a model viral DNA substrate. We provide evidence for functionally and kinetically distinct binding sites for viral and target DNA substrates and describe a cross-linking assay for DNA binding at a site whose specificity is consistent with the target DNA binding site.     

Role of DNA end distortion in catalysis by avian sarcoma virus integrase.

Retroviral integrase (IN) recognizes linear viral DNA ends and introduces nicks adjacent to a highly conserved CA dinucleotide usually located two base pairs from the 3'-ends of viral DNA (the "processing" reaction). In a second step, the same IN active site catalyzes the insertion of these ends into host DNA (the "joining" reaction). Both DNA sequence and DNA structure contribute to specific recognition of viral DNA ends by IN. Here we used potassium permanganate modification to show that the avian sarcoma virus IN catalytic domain is able to distort viral DNA ends in vitro. This distortion activity is consistent with both unpairing and unstacking of the three terminal base pairs, including the processing site adjacent to the conserved CA. Furthermore, the introduction of mismatch mutations that destabilize the viral DNA ends were found to stimulate the IN processing reaction as well as IN-mediated distortion. End-distortion activity was also observed with mutant or heterologous DNA substrates. However, further analyses showed that using Mn(2+) as a cofactor, processing site specificity of these substrates was also maintained. Our results support a model whereby unpairing and unstacking of the terminal base pairs is a required step in the processing reaction. Furthermore, these results are consistent with our previous observations indicating that unpairing of target DNA promotes the joining reaction.     

Computer aided study of ligand binding with catalytic domain of Avian sarcoma virus integrase and its ligand binding loops.

We have carried out 1 nanosecond (ns) Molecular Dynamics (MD) simulations of the drug Y3 (4-acetylamino-5-hydroxynaphthalene- 2, 7-disulfonic acid) complexed with catalytic domain of Avian sarcoma virus Integrase (ASV-IN), both in vacuum and in the presence of explicit solvent. Starting models were obtained on the basis of PDB co- ordinates (1A5X) of ASV-IN-Y3 complex. Mn(2+) cation was present in the active site. To neutralize the positive charge in the presence of explicit solvent, eight Cl(-)anions were added. Energy Minimization (EM) and MD simulations, for both the systems, were carried out using Sander's module of AMBER5.0 with all atom force field. We also carried out 1 ns MD simulation on two flexible loops--L1 (Gly54-Gln62) and L2 (Trp138-Met155) playing crucial role in interaction of IN with the drug, under differing environmental conditions (vacuum, aqueous and organic solvent methanol). Comparison of the conformational changes in the loops, monomer and dimer is presented in the paper. Our results showed that the conformation of the loop region was closest to crystallographic data in case of monomer and constrained loops in aqueous environment. However, the dimer in vacuum was more stable than monomer. The beta sheet structure of the monomer in aqueous environment was unstable. Latter also took long time for equilibration. The box formed by loops L1 and L2 from two sub units (IINA and INB) of the dimer satisfies prerequisites for ligand recognition site and seems to be the functional biological unit.     

Functional oligomeric state of avian sarcoma virus integrase

Retroviral integrase, one of only three enzymes encoded by the virus, catalyzes the essential step of inserting a DNA copy of the viral genome into the host during infection. Using the avian sarcoma virus integrase, we demonstrate that the enzyme functions as a tetramer. In presteady-state active site titrations, four integrase protomers were required for a single catalytic turnover. Volumetric determination of integrase-DNA complexes imaged by atomic force microscopy during the initial turnover additionally revealed substrate-induced assembly of a tetramer. These results suggest that tetramer formation may be a requisite step during catalysis with ramifications for antiviral design strategies targeting the structurally homologous human immunodeficiency virus, type 1 (HIV-1) integrase.     

Caldesmon. Molecular weight and subunit composition by analytical ultracentrifugation

A wide range of values has been reported for the subunit and molecular weights of smooth muscle caldesmon. There have also been conflicting reports concerning whether caldesmon is a monomer or dimer. We attempted to resolve these uncertainties by determining the molecular weight of chicken gizzard smooth muscle caldesmon using the technique of sedimentation equilibrium in the analytical ultracentrifuge. Unlike previous methods that have been used to estimate the molecular weight of caldesmon, the molecular weight determined by equilibrium sedimentation does not depend upon assumptions about the shape of the molecule. We concluded that caldesmon in solution is monomeric with a molecular mass of 93 +/- 4 kDa, a value that is much less than those previously reported in the literature. This new value, in conjunction with sedimentation velocity experiments, led to the conclusion that caldesmon is a highly asymmetric molecule with an apparent length of 740 A in solution. The mass of a cyanogen bromide fragment, with an apparent mass of 37 kDa from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was determined to be 25.1 +/- 0.6 kDa using sedimentation equilibrium. These results imply that the reported molecular weights of other fragment(s) of caldesmon have also been overestimated. We have determined an optical extinction coefficient for caldesmon (E1%(280 nm) = 3.3) by determining its concentration from its refractive index which was measured in the analytical ultracentrifuge. From the above values of the molecular weight and the extinction coefficient, we redetermined that the caldesmon molecule has two cysteines and recalculated the stoichiometric molar ratio of actin/tropomyosin/caldesmon in the smooth muscle thin filament to be 28:4:1.     

Monoclonal antibodies against Rous sarcoma virus integrase protein exert differential effects on integrase function in vitro.

We have prepared and characterized several monoclonal antibodies (MAbs) against the Rous sarcoma virus integrase protein (IN) with the aim of employing these specific reagents as tools for biochemical and biophysical studies. The interaction of IN with the purified MAbs and their Fab fragment derivatives was demonstrated by Western blot (immunoblot), enzyme-linked immunosorbent assay, and size exclusion chromatography. A series of truncated IN proteins was used to determine regions in the protein important for recognition by the antibodies. The MAbs described here recognize epitopes that lie within the catalytic core region of IN (amino acids 50 to 207) and are likely to be conformational. A detailed functional analysis was carried out by investigating the effects of Fab fragments as well as of intact MAbs on the activities of IN in vitro. These studies revealed differential effects which fall into three categories. (i) One of the antibodies completely neutralized the processing as well as the joining activity and also reduced the DNA binding capacity as determined by a nitrocellulose filter binding assay. On the other hand, this MAb did not abolish the cleavage-ligation reaction on a disintegration substrate and the nonspecific cleavage of DNA by IN. The cleavage pattern generated by the IN-MAb complex on various DNA substrates closely resembled that produced by mutant IN proteins which show a deficiency in multimerization. Preincubation of IN with substrate protected the enzyme from inhibition by this antibody. (ii) Two other antibodies showed a general inhibition of all IN activities tested. (iii) In contrast, a fourth MAb stimulated the in vitro joining activity of IN. Size exclusion chromatography demonstrated that IN-Fab complexes from representatives of the three categories of MAbs exhibit different stoichiometric compositions that suggest possible explanations for their contrasting effects and may provide clues to the relationship between the structure and function of IN.     

Dimer-tetramer association equilibria of human adult hemoglobin and its mutants as observed by analytical ultracentrifugation

Dimer-tetramer equilibrium of human adult hemoglobin in CO form (COHb A) and its mutants were measured by sedimentation velocity and sedimentation equilibrium. In sedimentation velocity, the association constants were estimated by measuring the concentration dependence of the weight average sedimentation coefficients at pH 6 and 7 and fitting the data to the theoretical binding isotherms with association constants as a parameter. Association constants of wild type Hb A and three mutant Hbs, Hb Hirose(βW37S), recombinant (r)Hb(βW37H) and rHb(αY42S), in which an amino acid was replaced at the α(1)β(2) interface, were measured in the presence and absence of inositol hexaphosphate (IHP). All the three mutations lowered the value of association constants, but the presence of IHP shifted the equilibrium toward tetramer. Although the association constant between dimer and tetramer of rHb(βW37H) and rHb(αY42S) were similar, sedimentation coefficient distribution function, c(s), analysis indicated that the association and dissociation rate constants of the former is higher than the latter.     

Characterization of protein detergent complexes by NMR, light scattering, and analytical ultracentrifugation

Abstract  Bottlenecks in expression, solubilization, purification and crystallization hamper the structural study of integral membrane proteins (IMPs). Successful crystallization is critically dependent on the purity, stability and oligomeric homogeneity of an IMP sample. These characteristics are in turn strongly influenced by the type and concentration of the detergents used in IMP preparation. By utilizing the techniques and analytical tools we earlier developed for the characterization of protein-detergent complexes (PDCs) [21], we demonstrate that for successful protein extraction from E. coli membrane fractions, the solubilizing detergent associates preferentially to IMPs rather than to membrane lipids. Notably, this result is contrary to the generally accepted mechanism of detergent-mediated IMP solubilization. We find that for one particular member of the family of proteins studied (E. coli receptor kinases, which is purified in mixed multimeric states and oligomerizes through its transmembrane region), the protein oligomeric composition is largely unaffected by a 10-fold increase in protein concentration, by alteration of micelle properties through addition of other detergents to the PDC sample, or by a 20-fold variation in the detergent concentration used for solubilization of the IMP from the membrane. We observed that the conditions used for expression of the IMP, which impact protein density in the membrane, has the greatest influence on the IMP oligomeric structure. Finally, we argue that for concentrating PDCs smaller than 30 kDa, stirred concentration cells are less prone to over-concentration of detergent and are therefore more effective than centrifugal ultrafiltration devices.     

Mutational analysis of the energetics of the GrpE.DnaK binding interface: equilibrium association constants by sedimentation velocity analytical ultracentrifugation

DnaK, the prokaryotic Hsp70 molecular chaperone, requires the nucleotide exchange factor and heat shock protein GrpE to release ADP. GrpE and DnaK are tightly associated molecules with an extensive protein-protein interface, and in the absence of ADP, the dissociation constant for GrpE and DnaK is in the low nanomolar range. GrpE reduces the affinity of DnaK for ADP, and the reciprocal linkage is also true: ADP reduces the affinity of DnaK for GrpE. The energetic contributions of GrpE side-chains to GrpE-DnaK binding were probed by alanine-scanning mutagenesis. Sedimentation velocity (SV) analytical ultracentrifugation (AUC) was used to measure the equilibrium constants (Keq) for GrpE binding to the ATPase domain of DnaK in the presence of ADP. ADP-bound DnaK is the natural target of GrpE, and the addition of ADP (final concentration of 5 microM) to the preformed GrpE-DnaK(ATPase) complexes allowed the equilibrium association constants to be brought into an experimentally accessible range. Under these experimental conditions, the substitution of one single GrpE amino acid residue, arginine 183 with alanine, resulted in a GrpE-DnaK(ATPase) complex that was weakly associated (Keq =9.4 x 10(4) M). This residue has been previously shown to be part of a thermodynamic linkage between two structural domains of GrpE: the thermosensing long helices and the C-terminal beta-domains. Several other GrpE side-chains were found to have a significant change in the free energy of binding (DeltaDeltaG approximately 1.5 to 1.7 kcal mol(-1)), compared to wild-type GrpE.DnaK(ATPase) in the same experimental conditions. Overall, the strong interactions between GrpE and DnaK appear to be dominated by electrostatics, not unlike barnase and barstar, another well-characterized protein-protein interaction. GrpE, an inherent thermosensor, exhibits non-Arrhenius behavior with respect to its nucleotide exchange function at bacterial heat shock temperatures, and mutation of several solvent-exposed side-chains located along the thermosensing indicated that these residues are indeed important for GrpE-DnaK interactions.     

Chain length characterization of oligodeoxyribonucleotides by analytical ultracentrifugation

Analytical ultracentrifugation was used to determine chain length of oligomers in the deoxyribonucleotide series. Uncertainties with respect to partial specific volume of the solute and questions of charge compel a semiempirical approach: variations of these parameters introduce fluctuations well within the experimental error of the method. Measurements of the sedimentation equilibria for three homooligomer series—d(pT)_n, d(pA)_n and d(pC)_n—as well as a number of oligomers of varying base composition generated straight-line plots passing through the origin.     

Self-association of adrenodoxin studied by using analytical ultracentrifugation

The mitochondrial steroid hydroxylase system of vertebrates utilizes adrenodoxin (Adx), a small iron–sulfur cluster protein of about 14 kDa as an electron carrier between a reductase and cytochrome P450. Although the crystal structure of this protein has been elucidated, the solution structure of Adx was discussed contrary in the literature [I.A. Pikuleva, K. Tesh, M.R. Waterman, Y. Kim, The tertiary structure of full-length bovine adrenodoxin suggests functional dimers, Arch. Biochem. Biophys. 373 (2000) 44–55; D. Beilke, R. Weiss, F. Löhr, P. Pristovsek, F. Hannemann, R. Bernhardt, H. Rüterjans, A new electron mechanism in mitochondrial steroid hydroxylase systems based on structural changes upon the reduction of adrenodoxin, Biochemistry 41 (2002) 7969–7978]. Therefore, it was necessary to study the self-association of this protein by using analytical ultracentrifugation over a larger concentration range. As could be demonstrated in sedimentation velocity experiments, as well as sedimentation equilibrium runs with explicit consideration of thermodynamic non-ideality, the full-length protein (residues 1–128) in the oxidized state resulted in a monomer–dimer equilibrium (K_a ~ 3 × 10² M^− 1). For truncated Adx (1–108), as well as the reduced Adx, the association behavior was strongly reduced. The consequences of this behavior are discussed with respect to the physiological meaning for the Adx system.     

Analysis of interacting biopolymer systems by analytical ultracentrifugation

Many of the functions of biological macromolecules are based on specific interactions. Extended concentration dependent studies of sedimentation coefficients or molecular masses of biopolymers are highly useful for describing the different kinds of association phenomena. These studies allow one to determine the partial concentrations of monomers and associates or reactants and complexes in self-associating systems or heterologous associations, respectively. Furthermore, in combination with corresponding measurements of biological activity these data allow one to estimate the individual activity parameters of components involved in equilibrium processes. The study of self-association and heterologous association using analytical ultracentrifugation, some recent developments therein, and its application to different examples are outlined here. Accepted: 18 October 1996     

Avian sarcoma virus-transformed quail clones defective in the production of focus-forming virus.

Quail embryo fibroblasts were infected at low multiplicity with avian sarcoma virus, and transformed cells were selected by their ability to form colonies in agar. Five clones that failed to produce focus-forming virus were examined for (i) intactness of the integrated proviral DNA, (ii) intracellular viral RNA production, (iii) intracellular viral antigen production, (iv) production of virus particles, and (v) rescue of a functional src gene and of parental host range determinants by superinfection with Rous-associated virus-60, an avian leukosis virus of subgroup E. Deletions in the integrated viral DNA were apparent in three of the five nonproducer clones. In one clone producing focus-forming virus, analysis of the integrated viral DNA revealed an insertion in the region of the genome that codes for src.     

Macromolecular size-and-shape distributions by sedimentation velocity analytical ultracentrifugation

Sedimentation velocity analytical ultracentrifugation is an important tool in the characterization of macromolecules and nanoparticles in solution. The sedimentation coefficient distribution c(s) of Lamm equation solutions is based on the approximation of a single, weight-average frictional coefficient of all particles, determined from the experimental data, which scales the diffusion coefficient to the sedimentation coefficient consistent with the traditional s approximately M(2/3) power law. It provides a high hydrodynamic resolution, where diffusional broadening of the sedimentation boundaries is deconvoluted from the sedimentation coefficient distribution. The approximation of a single weight-average frictional ratio is favored by several experimental factors, and usually gives good results for chemically not too dissimilar macromolecules, such as mixtures of folded proteins. In this communication, we examine an extension to a two-dimensional distribution of sedimentation coefficient and frictional ratio, c(s,f(r)), which is representative of a more general set of size-and-shape distributions, including mass-Stokes radius distributions, c(M,R(S)), and sedimentation coefficient-molar mass distributions c(s,M). We show that this can be used to determine average molar masses of macromolecules and characterize macromolecular distributions, without the approximation of any scaling relationship between hydrodynamic and thermodynamic parameters.     

The formation of actin oligomers studied by analytical ultracentrifugation

The small oligomers formed from Mg-G-actin under favorable conditions were studied by sedimentation velocity ultracentrifugation. The critical concentration of actin at pH 7.8 in the presence of 100 microM MgCl2 and 200 microM ATP was 12.5 +/- 2.8 microM. Under these conditions, about 15% of 7.5 microM Mg-actin was converted to oligomers of subunit size four to eight in 5 h at 20 degrees C. In 100 microM MgCl2 and no free ATP, the critical concentration was about 6.5 microM, and about 22% of 7.5 microM Mg-actin was converted to dimers in 80 min. There were no detectable higher oligomers or F-actin present in either case. As determined by the analysis of ATP hydrolysis, most, if not all, of the oligomer subunits contained ATP. When 28.5 microM actin was polymerized to steady state in 100 microM MgCl2 and 200 microM ATP, about 50% of the actin was present as F-actin, consistent with the critical concentration (approximately 12.5 microM), about 50% as oligomers as large as seven subunits, and only about 5% as monomers. When solutions containing oligomers were diluted the oligomers dissociated. Alternatively, when the MgCl2 concentration was raised to 1 mM, the solutions containing oligomers polymerized more rapidly than monomeric Mg-G-actin and to the same final steady state. These data are entirely consistent with the condensation-elongation model for helical polymerization proposed by Oosawa and Kasai (Oosawa, F., and Kasai, M. (1962) J. Mol. Biol. 4, 10-21) according to which, under certain conditions, substantial amounts of short linear and helical oligomers should be formed below the critical concentration and linear oligomers should coexist with monomers and F-actin at steady state.     

Rous sarcoma virus integrase protein: mapping functions for catalysis and substrate binding.

Rous sarcoma virus (RSV), like all retroviruses, encodes an integrase protein that is responsible for covalently joining the reverse-transcribed viral DNA to host DNA. We have probed the organization of functions within RSV integrase by constructing mutant derivatives and assaying their activities in vitro. We find that deletion derivatives lacking the amino-terminal 53 amino acids, which contain the conserved H-X(3-7)-H-X(23-32)-C-X(2)-C (HHCC) Zn(2+)-binding motif, are greatly impaired in their ability to carry out two reactions characteristic of integrase proteins: specific cleavage of the viral DNA termini and DNA strand transfer. Deletion mutants lacking the carboxyl-terminal 69 amino acids are also unable to carry out these reactions. However, all deletion mutants that retain the central domain are capable of carrying out disintegration, an in vitro reversal of the normal DNA strand transfer reaction, indicating that the catalytic center probably lies within this central region. Another conserved motif, D-X(39-58)-D-X(35)-E, is found in this central domain. These findings with RSV integrase closely parallel previous findings with human immunodeficiency virus integrase, indicating that a modular catalytic domain is a general feature of this family of proteins. Surprisingly, and unlike results obtained so far with human immunodeficiency virus integrase, efficient strand transfer activity can be restored to a mutant RSV integrase lacking the amino-terminal HHCC domain by fusion to various short peptides. Furthermore, these fusion proteins retain the substrate specificity of RSV integrase. These data support a model in which the integrase activities required for strand transfer in vitro, including substrate recognition, multimerization, and catalysis, all lie primarily outside the amino-terminal HHCC domain.