Tubulin dimer dissociation detected by fluorescence anisotropy

We have demonstrated a concentration-dependent dissociation of bovine brain tubulin dimer covalently labeled with 5-[(4,6-dichlorotriazin-2-yl)amino]fluorescein (DTAF) or with fluorescein isothiocyanate (FITC) by fluorescence anisotropy and size-exclusion HPLC. The fluorescence anisotropy values decreased to a limiting value upon dilution of tubulin from 10(-5) to 8 x 10(-8) M. A dissociation constant in 0.1 M Pipes, pH 6.9, 1 mM EGTA, and 1 mM MgSO4 at 20 degrees C was estimated to be (8.4 x 10(-7) +/- (0.4 x 10(-7) M. Control experiments using monomeric and other dimeric proteins, urea-denatured tubulin, and DTAF-tubulin diluted into solutions of bovine serum albumin or unlabeled tubulin were consistent with the finding that the changes in anisotropy upon dilution are due to protein dissociation. These results were supported by size-exclusion HPLC data where an increase in the elution volume of DTAF-tubulin and FITC-tubulin was observed with decreasing protein concentrations. Reversibility of the dissociation process and the lack of denaturation at high dilution were shown by the ability of reconcentrated protein to assemble into microtubules to about the same extent as undiluted protein. Fluorescent lifetimes and limiting anisotropy values were found to be approximately identical at different tubulin concentrations, indicating that the anisotropy changes reflect changes in size or rotational correlation time of the protein. Studies on the effects of tubulin ligands and promoters or inhibitors of assembly demonstrated that their effects on tubulin dimer-monomer equilibria are small but reproducible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tubulin folding cofactors: half a dozen for a dimer

Tubulin folding cofactors control the availability of tubulin subunits and microtubule stability in eukaryotic cells. Recent work on Arabidopsis mutants has provided a new experimental system for understanding the cellular functions of tubulin folding cofactors.     

Equilibrium dissociation and unfolding of the Arc repressor dimer

The equilibrium unfolding reaction of Arc repressor, a dimeric DNA binding protein encoded by bacteriophage P22, can be monitored by fluorescence or circular dichroism changes. The stability of Arc is concentration dependent, and the unfolding reaction is well described as a two-state transition from folded dimer to unfolded monomer. The stability of the protein is decreased at low pH and increased by high salt concentration. The salt dependence suggests that two ions bind preferentially to the folded protein. In 10 mM potassium phosphate (pH 7.3) and 100 mM KCl, the unfolding free energy reaches a maximum near room temperature. The results suggest that at the low protein concentrations where operator DNA binding is normally measured, Arc is predominantly monomeric and unfolded.     

Structural changes accompanying pH-induced dissociation of the beta-lactoglobulin dimer

We have used NMR spectroscopy to determine the three-dimensional (3D) structure, and to characterize the backbone dynamics, of a recombinant version of bovine beta-lactoglobulin (variant A) at pH 2. 6, where the protein is a monomer. The structure of this low-pH form of beta-lactoglobulin is very similar to that of a subunit within the dimer at pH 6.2. The root-mean-square deviation from the pH 6.2 (crystal) structure, calculated for backbone atoms of residues 6-160, is approximately 1.3 A. Differences arise from the orientation, with respect to the calyx, of the A-B and C-D loops, and of the flanking three-turn alpha-helix. The hydrophobic cavity within the calyx is retained at low pH. The E-F loop (residues 85-90), which moves to occlude the opening of the cavity over the pH range 7.2-6.2, is in the "closed" position at pH 2.6, and the side chain of Glu89 is buried. We also carried out measurements of (15)N T(1)s and T(2)s and (1)H-(15)N heteronuclear NOEs at pH 2.6 and 37 degrees C. Although the residues of the E-F loop (residues 86-89) have the highest crystallographic B-factors, the conformation of this loop is reasonably well defined by the NMR data, and its backbone is not especially mobile on the pico- to nanosecond time scale. Several residues (Ser21, Lys60, Ala67, Leu87, and Glu112) exhibit large ratios of T(1) to T(2), consistent with conformational exchange on a micro- to millisecond time scale. The positions of these residues in the 3D structure of beta-lactoglobulin are consistent with a role in modulating access to the hydrophobic cavity.     

A one-dimensional continuum elastic model for membrane-embedded gramicidin dimer dissociation

Membrane elastic properties, which are subject to alteration by compounds such as cholesterol, lipid metabolites and other amphiphiles, as well as pharmaceuticals, can have important effects on membrane proteins. A useful tool for measuring some of these effects is the gramicidin A channels, which are formed by transmembrane dimerization of non-conducting subunits that reside in each bilayer leaflet. The length of the conducting channels is less than the bilayer thickness, meaning that channel formation is associated with a local bilayer deformation. Electrophysiological studies have shown that the dimer becomes increasingly destabilized as the hydrophobic mismatch between the channel and the host bilayer increases. That is, the bilayer imposes a disjoining force on the channel, which grows larger with increasing hydrophobic mismatch. The energetic analysis of the channel-bilayer coupling is usually pursued assuming that each subunit, as well as the subunit-subunit interface, is rigid. Here we relax the latter assumption and explore how the bilayer junction responds to changes in this disjoining force using a simple one-dimensional energetic model, which reproduces key features of the bilayer regulation of gramicidin channel lifetimes.     

Pressure dissociation and conformational drift of the beta dimer of tryptophan synthase

Micromolar solutions of tryptophan synthase beta 2 dimer dissociate into monomers in the pressure range of 800-1600 bars as shown by studies of the spectral shift of the intrinsic fluorescence and of the fluorescence polarization of dansyl conjugates. At 25 degrees C the standard change in volume on dissociation (dV0) of the holoprotein was -162 mL mol-1, and the dissociation constant at 1 bar was K0 = 3.7 10(-10) M. Pyridoxal-reduced holoprotein and apoprotein had, within 10%, the same dV0, but K0 was decreased in the reduced protein (6 X 10(-11) M) and increased in the apoprotein (3.6 X 10(-9) M). At 4 degrees C the free energy of association of the holoprotein was reduced by 1.4 kcal mol-1, but dV0 was unchanged. In all the protein forms the decompression curves differed from the respective compression curves, indicating the loss of some free energy of association following separation of the monomers. This hysteretic behavior was largest in the apoprotein and amounted to a loss of 2.6 kcal mol-1 in the free energy of association. When the pressure was rapidly raised to 2.2 kbars, half-dissociation of the reduced pyridoxal beta 2 dimer took approximately 12 min. Upon return to atmospheric pressure reassociation was complete in 2-3 min and half of the enzyme activity was regained in 10 min; pyridoxal fluorescence recovered more slowly with a biphasic course. The independent return of these properties and the hysteretic behavior indicate that subunit separation is followed by a conformational drift like that observed in lactate dehydrogenase dissociated by either pressure or temperature or in enolase dissociated by dilution.     

Ionization of His 55 at the dimer interface of dynein light-chain LC8 is coupled to dimer dissociation

LC8 is a highly conserved light-chain subunit of cytoplasmic dynein that interacts with a wide variety of cellular proteins and is presumed to play a fundamental role in dynein assembly and cargo recruitment and in the assembly of protein complexes unrelated to dynein. LC8 is a dimer at physiological pH but dissociates to a folded monomer at pH < 4.8. We have suggested that acid-induced dimer dissociation is due to protonation of His 55, which is stacked against His 55' and completely buried in the dimer interface. In this work, we show that the pH-induced dissociation is reversible and indeed governed by the ionization state of His 55. Mutagenesis of His 55 to Lys results in a monomer in the pH range of 3-8, while the mutation to Ala results in a dimer in the same pH range. Mutations that disrupt intermolecular hydrogen bonds between Tyr 65 and Lys 44' and His 55 and Thr 67' do not change the association state of the dimer. Titration curves for His 55 and the two other histidines, His 72 and 68, were determined by (13)C-(1)H NMR for H55K and for WT-LC8 in the monomeric and dimeric states. The pK(a) values of His 72 and His 68 are 6 in the WT dimer and 6.2-6.5 in monomeric H55K, while the pK(a) of His 55 is about 4.5 in the WT dimer. These results indicate that deprotonation of His 55 is linked to dimer formation and that mutation of His 55 to a small neutral residue or to a positively charged residue uncouples the protonation and dissociation processes.     

Kinetics of the decamer - dimer dissociation of arginine decarboxylase.

Aurintricarboxylic acid inhibited replicative DNA synthesis in nucleotide-permeable mouse ascites sarcoma cells. DNA polymerase activity assayed with activated DNA template and DNA polymerase purified partially from sarcoma cells was also inhibited by aurintricarboxylic acid. The inhibition of DNA polymerase activity was probably due to the inhibitory interaction of aurintricarboxylic acid with DNA polymerase. The replicative DNA synthesis might be inhibited by aurintricarboxylic acid interacting with some essential protein component(s), such as DNA polymerase of the replication machinery.     

Reversible dimer dissociation of tubulin S and tubulin detected by fluorescence anisotropy.

Concentration-dependent dissociation of dimers of goat brain tubulin S and tubulin was studied by fluorescence anisotropy. Upon dilution, assembly-competent fluorescein 5'-maleimide labeled dimers of tubulin S and tubulin show a progressive decrease in fluorescence anisotropy. That this lowering of anisotropy results from the dissociation of tubulin S dimers into monomers was shown by dilution experiments with unlabeled homologous and heterologous proteins. A nonlinear least-squares fit of the data gave a dissociation constant of 7.1 x 10(-8) M for tubulin S compared to 7.2 x 10(-7) M for tubulin at 25 degrees C in 0.1 M PEM buffer, pH 7.0. van't Hoff plots of dimer-monomer dissociation of tubulin S and tubulin also show considerable differences in delta H and delta S. Effects of ionic strength and colchicine on the equilibrium constants are also substantially different for tubulin and tubulin S. The implications of these observations on the influence of C-terminal tails on tubulin structure are discussed.     

Formation, structure, and dissociation of the ribonuclease S three-dimensional domain-swapped dimer

Post-translational events, such as proteolysis, are believed to play essential roles in amyloid formation in vivo. Ribonuclease A forms oligomers by the three-dimensional domain-swapping mechanism. Here, we demonstrate the ability of ribonuclease S, a proteolytically cleaved form of ribonuclease A, to oligomerize efficiently. This unexpected capacity has been investigated to study the effect of proteolysis on oligomerization and amyloid formation. The yield of the RNase S dimer was found to be significantly higher than that of RNase A dimers, which suggests that proteolysis can activate oligomerization via the three-dimensional domain-swapping mechanism. Characterization by chromatography, enzymatic assays, and NMR spectroscopy indicate that the structure of the RNase S dimer is similar to that of the RNase A C-dimer. The RNase S dimer dissociates much more readily than the RNase A C-dimer does. By measuring the dissociation rate as a function of temperature, the activation enthalpy and entropy for RNase S dimer dissociation were found to resemble those for the release of the small fragment (S-peptide) from monomeric RNase S. Excess S-peptide strongly slows RNase S dimer dissociation. These results strongly suggest that S-peptide release is the rate-limiting step of RNase S dimer dissociation.     

Inactivation of creatine kinase by high pressure may precede dimer dissociation.

The effects of hydrostatic pressure on creatine kinase activity and conformation were investigated using either the high-pressure stopped-flow method in the pressure range 0.1-200 MPa for the activity determination, or the conventional activity measurement and fluorescence spectroscopy up to 650 MPa. The changes in creatine kinase activity and intrinsic fluorescence show a total or partial reversibility after releasing pressure, depending on both the initial value of the high pressure applied and on the presence or absence of guanidine hydrochloride. The study on 8-anilinonaphthalene-1-sulfonate binding to creatine kinase under high pressure indicates that the hydrophobic core of creatine kinase was progressively exposed to the solvent at pressures above 300 MPa. This data shows that creatine kinase is inactivated at low pressure, preceding both the enzyme dissociation and the unfolding of the hydrophobic core occurring at higher pressure. Moreover, in agreement with the recently published structure of the dimer, it can be postulated that the multistate transitions of creatine kinase induced both by pressure and guanidine denaturation are in direct relationship with the existence of hydrogen bonds which maintain the dimeric structure of the enzyme.     

Mapping protein surface accessibility via an electron transfer dissociation selectively cleavable hydrazone probe

A protein's surface influences its role in protein-protein interactions and protein-ligand binding. Mass spectrometry can be used to give low resolution structural information about protein surfaces and conformations when used in combination with derivatization methods that target surface accessible amino acid residues. However, pinpointing the resulting modified peptides upon enzymatic digestion of the surface-modified protein is challenging because of the complexity of the peptide mixture and low abundance of modified peptides. Here a novel hydrazone reagent (NN) is presented that allows facile identification of all modified surface residues through a preferential cleavage upon activation by electron transfer dissociation coupled with a collision activation scan to pinpoint the modified residue in the peptide sequence. Using this approach, the correlation between percent reactivity and surface accessibility is demonstrated for two biologically active proteins, wheat eIF4E and PARP-1 Domain C.     

Glycosaminoglycan disaccharide alters the dimer dissociation constant of the chemokine MIP-1 beta

Chemokines are immune system proteins that recruit and activate leukocytes to sites of infection. This recruitment is believed to involve the establishment of a chemokine concentration gradient by the binding of chemokines to glycosaminoglycans (GAGs). In previous studies, we elucidated the GAG binding site of the chemokine MIP-1beta and implicated the involvement of the chemokine dimer in GAG binding through residues across the dimer interface. In the present studies, nuclear magnetic resonance spectroscopy was used to investigate the effect of GAG binding on MIP-1beta dimerization. Using several dimerization-impaired variants of MIP-1beta (F13Y, F13L, L34W, and L34K), these studies indicate that the addition of disaccharide to the mutants increases their dimerization affinities. For MIP-1beta F13Y, the presence of the disaccharide increases the chemokine dimerization affinity about 9-fold as evidenced by a decrease in the dimer dissociation constant from 610 to 66 microM. Even more dramatically, the dimerization affinity of MIP-1beta L34W also increases upon addition of disaccharide, with the dimer dissociation constant decreasing from 97 to 6.5 microM. After this effect for the mutants of MIP-1beta was shown, similar experiments were conducted with the CC chemokine RANTES, and it was demonstrated that the presence of disaccharide increases its dimerization affinity by almost 7-fold. These findings provide further evidence of the importance of the dimer in chemokine function and provide the first quantitative investigation of the role of GAGs in the manipulation of the MIP-1beta quaternary structure.     

Cysteine accessibility probes timing and extent of NBD separation along the dimer interface in gating CFTR channels

Cystic fibrosis transmembrane conductance regulator (CFTR) channel opening and closing are driven by cycles of adenosine triphosphate (ATP) binding–induced formation and hydrolysis-triggered disruption of a heterodimer of its cytoplasmic nucleotide-binding domains (NBDs). Although both composite sites enclosed within the heterodimer interface contain ATP in an open CFTR channel, ATP hydrolysis in the sole catalytically competent site causes channel closure. Opening of the NBD interface at that site then allows ADP–ATP exchange. But how frequently, and how far, the NBD surfaces separate at the other, inactive composite site remains unclear. We assessed separation at each composite site by monitoring access of nucleotide-sized hydrophilic, thiol-specific methanothiosulfonate (MTS) reagents to interfacial target cysteines introduced into either LSGGQ-like ATP-binding cassette signature sequence (replacing equivalent conserved serines: S549 and S1347). Covalent MTS-dependent modification of either cysteine while channels were kept closed by the absence of ATP impaired subsequent opening upon ATP readdition. Modification while channels were opening and closing in the presence of ATP caused macroscopic CFTR current to decline at the same speed as when the unmodified channels shut upon sudden ATP withdrawal. These results suggest that the target cysteines can be modified only in closed channels; that after modification the attached MTS adduct interferes with ATP-mediated opening; and that modification in the presence of ATP occurs rapidly once channels close, before they can reopen. This interpretation was corroborated by the finding that, for either cysteine target, the addition of the hydrolysis-impairing mutation K1250R (catalytic site Walker A Lys) similarly slowed, by an order of magnitude, channel closing on ATP removal and the speed of modification by MTS reagent in ATP. We conclude that, in every CFTR channel gating cycle, the NBD dimer interface separates simultaneously at both composite sites sufficiently to allow MTS reagents to access both signature-sequence serines. Relatively rapid modification of S1347C channels by larger reagents—MTS-glucose, MTS-biotin, and MTS-rhodamine—demonstrates that, at the noncatalytic composite site, this separation must exceed 8 Å.     

Studies on the dissociation and urea-induced unfolding of FtsZ support the dimer nucleus polymerization mechanism

FtsZ is a major protein in bacterial cytokinesis that polymerizes into single filaments. A dimer has been proposed to be the nucleating species in FtsZ polymerization. To investigate the influence of the self-assembly of FtsZ on its unfolding pathway, we characterized its oligomerization and unfolding thermodynamics. We studied the assembly using size-exclusion chromatography and fluorescence spectroscopy, and the unfolding using circular dichroism and two-photon fluorescence correlation spectroscopy. The chromatographic analysis demonstrated the presence of monomers, dimers, and tetramers with populations dependent on protein concentration. Dilution experiments using fluorescent conjugates revealed dimer-to-monomer and tetramer-to-dimer dissociation constants in the micromolar range. Measurements of fluorescence lifetimes and rotational correlation times of the conjugates supported the presence of tetramers at high protein concentrations and monomers at low protein concentrations. The unfolding study demonstrated that the three-state unfolding of FtsZ was due to the mainly dimeric state of the protein, and that the monomer unfolds through a two-state mechanism. The monomer-to-dimer equilibrium characterized here (K(d) = 9 μM) indicates a significant fraction (~10%) of stable dimers at the critical concentration for polymerization, supporting a role of the dimeric species in the first steps of FtsZ polymerization.