Association Of Protein Subunits Research Articles

Design of Tunable Protein Interfaces Controlled by Post-Translational Modifications.

The design of protein interaction interfaces is a cornerstone of synthetic biology, where they can be used to promote the association of protein subunits into active molecular complexes or into protein nanostructures. In nature, protein interactions can be modulated by post-translational modifications (PTMs) that modify the protein interfaces with the addition and removal of various chemical groups. PTMs thus represent a means to gain control over protein interactions, yet they have seldom been considered in the design of synthetic proteins. Here, we explore the potential of a reversible PTM, serine phosphorylation, to modulate the interactions between peptides. We designed a series of interacting peptide pairs, including heterodimeric coiled coils, that contained one or more protein kinase A (PKA) recognition motifs. Our set of peptide pairs comprised interactions ranging from nanomolar to micromolar affinities. Mass spectrometry analyses showed that all peptides were excellent phosphorylation substrates of PKA, and subsequent phosphate removal could be catalyzed by lambda protein phosphatase. Binding kinetics measurements performed before and after treatment of the peptides with PKA revealed that phosphorylation of the target serines affected both the association and dissociation rates of the interacting peptides. We observed both the strengthening of interactions (up to an 11-fold decrease in Kd) and the weakening of interactions (up to a 180-fold increase in Kd). De novo-designed PTM-modulated interfaces will be useful to control the association of proteins in biological systems using protein-modifying enzymes, expanding the paradigm of self-assembly to encompass controlled assembly of engineerable protein complexes.

Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly.

The binding of metal ions at the interface of protein complexes presents a unique and poorly understood mechanism of molecular assembly. A remarkable example is the Rad50 zinc hook domain, which is highly conserved and facilitates the Zn2+-mediated homodimerization of Rad50 proteins. Here, we present a detailed analysis of the structural and thermodynamic effects governing the formation and stability (logK12 = 20.74) of this evolutionarily conserved protein assembly. We have dissected the determinants of the stability contributed by the small β-hairpin of the domain surrounding the zinc binding motif and the coiled-coiled regions using peptides of various lengths from 4 to 45 amino acid residues, alanine substitutions and peptide bond-to-ester perturbations. In the studied series of peptides, an >650 000-fold increase of the formation constant of the dimeric complex arises from favorable enthalpy because of the increased acidity of the cysteine thiols in metal-free form and the structural properties of the dimer. The dependence of the enthalpy on the domain fragment length is partially compensated by the entropic penalty of domain folding, indicating enthalpy-entropy compensation. This study facilitates understanding of the metal-mediated protein-protein interactions in which the metal ion is critical for the tight association of protein subunits.

Determining the role of metal binding in protein cage assembly.

Assembly of protein cages may require structural metal ions to nucleate or stabilize association of protein subunits. We describe here an approach to establishing the role of metal ions in protein cage assembly and stability, focusing on detecting the presence of structural metal ions as well as establishing oligomeric state. A colorimetric assay for detection of metal is described, along with a gel filtration assay to assess protein cage assembly and a fluorescence-based method for determining protein stability.

Detection and characterization of hemoglobin dissociation and aggregation using microcalorimetry

Using micro-differential scanning calorimetry (μDSC) to probe hemoglobin structural changes, previously unreported large exothermic events have been repeatedly detected at temperatures below the tertiary-to-secondary structural transition temperature (ca. 72 °C), which could be important in understanding protein subunit association. Given the importance of protein–protein interactions and protein solution stability, this event was characterized with respect to concentration and scan rate. This detailed analysis revealed the formation of aggregates that differed in size, appearance, and settling time. While repeat μDSC scans confirmed that this aggregation event was irreversible when samples were heated beyond 55 °C, it was also found that aggregation could be prevented through the addition 0.02 M urea. All evidence points to a significant occurrence of a kinetic pathway that leads to an intermediate aggregation event that is believed to be formed from dissociated hemoglobin subunits.

Long-Lived Conformational Isomerism of Protein Dimers: The Role of the Free Energy of Subunit Association

The association of protein subunits to form N-mers (N≥3) does not follow the dependence on the law of mass action predicted by the classical thermodynamic description used for the equilibrium of association of small molecules. For those anomalous cases, a so-called deterministic model has been previously proposed. The latter model was based on the empirical observation that the dynamics of subunit exchange between protein oligomers can be very slow, leading to the existence of long-lived conformational isomers and to a persistently heterogeneous ensemble of oligomers in solution. Contrary to the expectation for a protein dimer, we have recently shown that the subunit association of triosephosphate isomerase (TIM) could also be described as a deterministic process and that long-lived conformational isomers of TIM could be isolated in solution. Here we show that a), observation of hysteresis in pressure dissociation curves is an additional indicator of deterministic behavior; b), the extent of deviation from the classical thermodynamic behavior correlates with the free-energy change of subunit association; and c), experimental manipulation of the free energy of subunit association through the addition of a subdenaturing concentration of a chaotropic agent restores the concentration dependence of subunit association of TIM. A model that explains these features and its biological relevance is discussed.

Crystal Structure of Dps-1, a Functionally Distinct Dps Protein from Deinococcus radiodurans

DNA protection during starvation (Dps) proteins play an important role in protecting cellular macromolecules from damage by reactive oxygen species (ROS). Unlike most orthologs that protect DNA by a combination of DNA binding and prevention of hydroxyl radical formation by ferroxidation and sequestration of iron, Dps-1 from the radiation-resistant Deinococcus radiodurans fails to protect DNA from hydroxyl radical-mediated cleavage through a mechanism inferred to involve continuous release of iron from the protein core. To address the structural basis for this unusual release of Fe 2+, the crystal structure of D. radiodurans Dps-1 was determined to 2.0 Å resolution. Two of four strong anomalous signals per protein subunit correspond to metal-binding sites within an iron-uptake channel and a ferroxidase site, common features related to the canonical functions of Dps homologs. Similar to Lactobacillus lactis Dps, a metal-binding site is found at the N-terminal region. Unlike other metal sites, this site is located at the base of an N-terminal coil on the outer surface of the dodecameric protein sphere and does not involve symmetric association of protein subunits. Intriguingly, a unique channel-like structure is seen featuring a fourth metal coordination site that results from 3-fold symmetrical association of protein subunits through α2 helices. The presence of this metal-binding site suggests that it may define an iron-exit channel responsible for the continuous release of iron from the protein core. This interpretation is supported by substitution of residues involved in this ion coordination and the observation that the resultant mutant protein exhibits significantly attenuated iron release. Therefore, we propose that D. radiodurans Dps-1 has a distinct iron-exit channel.

Differential association of protein subunits with the human RNase MRP and RNase P complexes

RNase MRP is a eukaryotic endoribonuclease involved in nucleolar and mitochondrial RNA processing events. RNase MRP is a ribonucleoprotein particle, which is structurally related to RNase P, an endoribonuclease involved in pre-tRNA processing. Most of the protein components of RNase MRP have been reported to be associated with RNase P as well. In this study we determined the association of these protein subunits with the human RNase MRP and RNase P particles by glycerol gradient sedimentation and coimmunoprecipitation. In agreement with previous studies, RNase MRP sedimented at 12S and 60-80S. In contrast, only a single major peak was observed for RNase P at 12S. The analysis of individual protein subunits revealed that hPop4 (also known as Rpp29), Rpp21, Rpp20, and Rpp25 only sedimented in 12S fractions, whereas hPop1, Rpp40, Rpp38, and Rpp30 were also found in 60-80S fractions. In agreement with their cosedimentation with RNase P RNA in the 12S peak, coimmunoprecipitation with VSV-epitope-tagged protein subunits revealed that hPop4, Rpp21, and in addition Rpp14 preferentially associate with RNase P. These data show that hPop4, Rpp21, and Rpp14 may not be associated with RNase MRP. Furthermore, Rpp20 and Rpp25 appear to be associated with only a subset of RNase MRP particles, in contrast to hPop1, Rpp40, Rpp38, and Rpp30 (and possibly also hPop5), which are probably associated with all RNase MRP complexes. Our data are consistent with a transient association of Rpp20 and Rpp25 with RNase MRP, which may be inversely correlated to its involvement in pre-rRNA processing.

Subunit Assembly for DNA Cleavage by Restriction Endonuclease SgrAI

The SgrAI endonuclease usually cleaves DNA with two recognition sites more rapidly than DNA with one site, often converting the former directly to the products cut at both sites. In this respect, SgrAI acts like the tetrameric restriction enzymes that bind two copies of their target sites before cleaving both sites concertedly. However, by analytical ultracentrifugation, SgrAI is a dimer in solution though it aggregates to high molecular mass species when bound to its specific DNA sequence. Its reaction kinetics indicate that it uses different mechanisms to cleave DNA with one and with two SgrAI sites. It cleaves the one-site DNA in the style of a dimeric restriction enzyme acting at an individual site, mediating neither interactions in trans, as seen with the tetrameric enzymes, nor subunit associations, as seen with the monomeric enzymes. In contrast, its optimal reaction on DNA with two sites involves an association of protein subunits: two dimers bound to sites in cis may associate to form a tetramer that has enhanced activity, which then cleaves both sites concurrently. The mode of action of SgrAI differs from all restriction enzymes characterised previously, so this study extends the range of mechanisms known for restriction endonucleases.

Mutations in the RNA Component of RNase MRP Cause a Pleiotropic Human Disease, Cartilage-Hair Hypoplasia

The recessively inherited developmental disorder, cartilage-hair hypoplasia (CHH) is highly pleiotropic with manifestations including short stature, defective cellular immunity, and predisposition to several cancers. The endoribonuclease RNase MRP consists of an RNA molecule bound to several proteins. It has at least two functions, namely, cleavage of RNA in mitochondrial DNA synthesis and nucleolar cleaving of pre-rRNA. We describe numerous mutations in the untranslated RMRP gene that cosegragate with the CHH phenotype. Insertion mutations immediately upstream of the coding sequence silence transcription while mutations in the transcribed region do not. The association of protein subunits with RNA appears unaltered. We conclude that mutations in RMRP cause CHH by disrupting a function of RNase MRP RNA that affects multiple organ systems.

Effect of hydrostatic pressure on the fluorescence of indole derivatives

Effects of hydrostatic pressure on the fluorescence emission of L-tryptophan, N-acetyl-L-trytophanamide and indole were investigated. An increase in pressure ranging from 1 bar to 2.4 kbar results in reversible red-shifts of the emission of the three fluorophores. The pressure-induced redshift amounts to about 170 cm(-1) at 2.4 kbar, and appears related to changes in Stokes shift of the fluorophores caused by pressure effects on the dielectric constant and/or refractive index of the medium. As the pressure range investigated here is the range commonly used in studies of protein subunit association and/or folding, these observations raise the need for caution in interpreting pressure-induced spectral shifts. The significance of these observations to pressure studies of proteins is illustrated by investigation of pressure effects on human Cu,Zn Superoxide dismutase (SOD) and azurin fromPseudomonas aeruginosa. A reversible 170 cm(-1) red-shift of the emission of SOD was observed upon pressurization to 2.4 kbar. This might be interpreted as pressure-induced conformational changes of the protein. However, further studies using SOD that had been fully unfolded by guanidine hydrochloride, and fluorescence anisotropy measurements indicated that the observed red-shift was likely due to a direct effect of pressure on the fluorescence of the single tryptophan residue of SOD. Similar pressure-induced red-shifts were also observed for the buried tryptophan residue of azurin or for azurin that had been previously denatured by guanidine hydrochloride. These observations further suggest that the effective dielectric constant of the protein matrix is affected by pressure similarly to water.

Answer to the Comments on G. Weber's "van't Hoff Revisited: Enthalpy of Association of Protein Subunits" by A. Holtzer and by R. Ragone, G. Colonna, and L. Ambrosone

Answer to the Comments on G. Weber's "van't Hoff Revisited: Enthalpy of Association of Protein Subunits" by A. Holtzer and by R. Ragone, G. Colonna, and L. Ambrosone

Comment on "van't Hoff Revisited: Enthalpy of Association of Protein Subunits"

Comment on "van't Hoff Revisited: Enthalpy of Association of Protein Subunits"

Effect of high pressure on activity of rabbit reticulocyte cell-free protein-synthesizing system

Studies so far indicate that many processes in the interactions between biomacromole-cules are accompanied by a change in occupied volume. For instance, the association ofprotein subunits into oligomer and the formation of protein-nucleic acid complex give anincrease in volume. The increase in volume in these processes mainly results from: (i) theformation of the dead space between the subunits of the oligomeric protein or the com-plexes in the processes mentioned above; (ii) the formation of salt bonds and (iii) theinteractions between hydrophobic groups. It is known from the Lechatelier principle that ifhigh pressure is applied to an equilibrium system mentioned above, the original equilibrium

Van't Hoff revisited: enthalpy of association of protein subunits

van't Hoff revisited: enthalpy of association of protein subunits

Rapid kinetics of G protein subunit association: A rate-limiting conformational change?

G protein subunit association and dissociation are thought to play an important role in signal transduction. We measured αβγ heterocomplex formation using resonance energy transfer. Fluorescein-labelled α(F-α) emission was quenched ∼ 10% on mixing with eosin-labelled βγ(E-βγ). Unlabelled βγ did not quench F-α fluorescence. Stopped-flow kinetics showed a t 1 2 ranging from 2.5 s to 0.25 s for 50 nM to 1200 nM E-βγ. The rate saturated at high E-βγ concentrations consistent with a two-step mechanism. We report the first rapid-mix studies of G protein subunit association kinetics which suggest that α and βγ combine by a two-step process with a maximal rate of 4.1 ± 0.4 s −1.

Interaction of Indoleacrylic Acid with Trp Aporepressor from Escherichia coli

Trans-β-Indoleacrylic acid (IAA) binds with moderately high affinity to the dimeric protein, trp aporepressor from Escherichia coli. IAA is itself nonfluorescent, and, upon binding, IAA quenches ∼95% of the intrinsic tryptophan fluorescence of the protein. Since there is an overlap between the absorbance of IAA and the emission of tryptophan, this quenching appears to be due to resonance energy transfer. To adequately fit binding isotherms, it was necessary to use a model in which the binding of IAA to one subunit is able to quench fluorescence from both subunits of the protein. This model also assumes that binding to the two subunits occurs independently. Binding data were obtained as a function of the concentration of trp aporepressor, over the range of 1 × 10 −8 to 5 × 10 −5 M, in order to test the possibility that protein subunit association or dissociation occurs. When analyzed in terms of the above model, no significant protein concentration dependence is seen for the IAA association constant, indicating that either (i) the protein does not undergo changes in oligomerization over this concentration range or that (ii) different states of aggregation bind IAA with similar affinity. The affinity of IAA was found to increase at higher ionic strength and at lower pH. Also, evidence is presented for a photoinduced change in the configuration of IAA.

Labeling of the beta gamma subunit complex of transducin with an environmentally sensitive cysteine reagent. Use of fluorescence spectroscopy to monitor transducin subunit interactions

In this study, we have examined the interactions of the beta gamma subunit complex of the retinal GTP-binding protein transducin (beta gamma T) with its alpha subunit (alpha T) using fluorescence spectroscopic approaches. The beta gamma T subunit complex was covalently labeled with 2-(4'-maleimidylanilino)napthalene-6-sulfonic acid (MIANS), an environmentally sensitive fluorescent cysteine reagent. The formation of the MIANS beta gamma T complexes (two to five MIANS adducts per beta gamma T) resulted in 2-3-fold enhancements in the MIANS fluorescence, and 20-25-nm blue shifts in the fluorescence emission maxima, relative to the emission for identical concentrations of MIANS-labeled MIANS complexes. The addition of alpha T.GDP to these MIANS beta gamma T complexes resulted in an additional enhancement in the MIANS fluorescence (typically ranging from 20 to 40%) and a 5-10-nm blue shift in the wavelength for maximum emission. These fluorescence changes were specifically elicited by the GDP-bound form of alpha T and were not observed upon the addition of purified alpha T.guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) complexes to the MIANS beta gamma T species. Conditions which resulted in the activation of the alpha T.GDP subunit (i.e. the addition of AlF4- or the addition of rhodopsin-containing vesicles and GTP gamma S) resulted in a reversal of the alpha T.GDP-induced enhancement of the MIANS beta gamma T fluorescence. Thus the MIANS beta gamma T fluorescence provided a spectroscopic monitor for transducin-subunit association and transducin-activation. Based on the results from studies using this spectroscopic read-out, it appears that the association of the alpha T.GDP species with the beta gamma T subunit complex to form the holotransducin molecule is rapid and does not limit the rate of the rhodopsin-stimulated activation of holotransducin. However, either the dissociation of the activated alpha T subunit from the beta gamma T complex, or a conformational change in beta gamma T which occurs as a result of the subunit dissociation event, appears to be slow relative to the G protein-subunit association event.

Two-dimensional electrophoresis as a tool for structural and genetic studies of seed proteins from Poaceae and Fagaceae.

The application of two-dimensional electrophoretic procedures to structural and genetic studies of seed proteins from Poaceae (including the cultivated cereals) and Fagaceae is described. The following related problems have been considered: covalent and non-covalent association of protein subunits in multiple oligomeric structures; chromosomal locations of genes encoding seed proteins; quantitation of gene products in relation to gene expression and regulation; purification of protein components to study their homology relationships and in vitro activities; evolutionary and phylogenetic relationships; identification of genetic stocks. Isoelectric focusing, pore-gradient electrophoresis, electrophoresis at different pH's, are among the separation procedures used in the first dimension, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis and starch-gel electrophoresis at acid pH have been the preferred second-dimensional methods. Dissociating conditions (sodium dodecyl sulfate, Nonidet P-40, or urea) and reducing conditions (2-mercaptoethanol) have been used when required.

Phenomenological description of the association of protein subunits subjected to conformational drift. Effects of dilution and of hydrostatic pressure.

The native conformation of oligomers may be expected to undergo reversible changes when they separate upon dissociation of the original aggregate. When these changes are slow in comparison with the time of an association-dissociation (AD) cycle, they give rise to characteristic effects in the dependence of the dissociation: upon dilution, at constant pressure, and upon the applied pressure, at constant concentration. The phenomenological description of these effects is examined by comparing two possible models: The first model assumes a continuous loss in free energy of association with the extent of dissociation; the second supposes the existence of two or more distinct aggregates differing in subunit affinity and present in proportions that vary with the extent of dissociation. The latter model fits better the experimental data available, with regard to both the concentration and the pressure dependence of the association, and gives a particularly simple explanation of the hysteresis phenomena observed in several oligomeric proteins after application of pressure. The validity of the principle of detailed balance, often assumed in dealing with complex equilibria, is discussed in detail as it does not appear possible to reconcile it with some of the experimental observations or with the proposed model.

Conformational flexibility of neurophysin as investigated by local motions of fluorophores. Relationships with neurohypophyseal hormone binding

Flexibility of various structural domains of neurophysin and neurophysin-neurohypophyseal hormone complexes has been investigated through the fast rotational motion of fluorophores in highly viscous medium. Despite seven intrachain disulfide links, it is shown that some domains of neurophysin remain highly flexible. Dimerization of neurophysin does not affect the structural integrity of the individual subunits, each subdomain being conformationally equivalent within each protomer of the unliganded dimer. The absence of heterogeneous fluorescence anisotropy precludes the existence of a dimer tautomerization equilibrium. Binding of the hormonal ligands to neurophysin dimer promotes a large conformational change over the whole protein structure as assessed by differential alterations of the flexibility-rigidity and intrasegmental interaction properties of domains that do not participate directly to the dimerization/binding areas. The order of free-energy coupling between ligand binding and protein subunit association has been evaluated. Data are consistent with a model in which the first mole of bound ligand stabilizes the dimer by increasing the intersubunit contacts while the second mole of ligand induces most of the described conformational change. Accordingly, the positive cooperativity between the two dimeric binding sites is linked mainly to the binding of the second ligand. The induced structural change is perceived differently by each subunit as assessed by opposite local motions of Tyr49 in each liganded protomer and leads to the formation of a dimeric complex with a global pseudospherical symmetry although containing domains of local asymmetry.