Showing papers on "Cooperative binding published in 1999"
TL;DR: The authors' working model for the structure of the PrP complex shows the histidine residues in successive octarepeats bridged between two copper ions, with both the Nepsilon2 and Ndelta1 imidazole nitrogen of each histidine residue coordinated and the remaining coordination sites occupied by a backbone amide nitrogen and a water molecule.
Abstract: Evidence is growing to support a functional role for the prion protein (PrP) in copper metabolism. Copper ions appear to bind to the protein in a highly conserved octapeptide repeat region (sequence PHGGGWGQ) near the N terminus. To delineate the site and mode of binding of Cu(II) to the PrP, the copper-binding properties of peptides of varying lengths corresponding to 2-, 3-, and 4-octarepeat sequences have been probed by using various spectroscopic techniques. A two-octarepeat peptide binds a single Cu(II) ion with Kd ≈ 6 μM whereas a four-octarepeat peptide cooperatively binds four Cu(II) ions. Circular dichroism spectra indicate a distinctive structuring of the octarepeat region on Cu(II) binding. Visible absorption, visible circular dichroism, and electron spin resonance spectra suggest that the coordination sphere of the copper is identical for 2, 3, or 4 octarepeats, consisting of a square-planar geometry with three nitrogen ligands and one oxygen ligand. Consistent with the pH dependence of Cu(II) binding, proton NMR spectroscopy indicates that the histidine residues in each octarepeat are coordinated to the Cu(II) ion. Our working model for the structure of the complex shows the histidine residues in successive octarepeats bridged between two copper ions, with both the Nɛ2 and Nδ1 imidazole nitrogen of each histidine residue coordinated and the remaining coordination sites occupied by a backbone amide nitrogen and a water molecule. This arrangement accounts for the cooperative nature of complex formation and for the apparent evolutionary requirement for four octarepeats in the PrP.
514 citations
01 Jan 1999
TL;DR: In this article, the copper-binding properties of peptides of varying lengths corresponding to 2-, 3-, and 4-octarepeat sequences have been probed by using various spectroscopic techniques.
Abstract: Evidence is growing to support a functional role for the prion protein (PrP) in copper metabolism. Copper ions appear to bind to the protein in a highly conserved octapeptide repeat region (sequence PHGGGWGQ) near the N terminus. To delineate the site and mode of binding of Cu(II) to the PrP, the copper-binding properties of peptides of varying lengths corresponding to 2-, 3-, and 4-octarepeat sequences have been probed by using various spectroscopic techniques. A two-octarepeat peptide binds a single Cu(II) ion with Kd ' 6 mM whereas a four-octarepeat peptide coopera- tively binds four Cu(II) ions. Circular dichroism spectra indicate a distinctive structuring of the octarepeat region on Cu(II) binding. Visible absorption, visible circular dichroism, and electron spin resonance spectra suggest that the coordi- nation sphere of the copper is identical for 2, 3, or 4 octare- peats, consisting of a square-planar geometry with three nitrogen ligands and one oxygen ligand. Consistent with the pH dependence of Cu(II) binding, proton NMR spectroscopy indicates that the histidine residues in each octarepeat are coordinated to the Cu(II) ion. Our working model for the structure of the complex shows the histidine residues in successive octarepeats bridged between two copper ions, with both the N«2 and Nd1 imidazole nitrogen of each histidine residue coordinated and the remaining coordination sites occupied by a backbone amide nitrogen and a water molecule. This arrangement accounts for the cooperative nature of complex formation and for the apparent evolutionary require- ment for four octarepeats in the PrP.
497 citations
TL;DR: Evidence that the TCR and/or peptide-MHC have flexible binding surfaces that are stabilized upon binding is reported, which is likely to contribute to cross-reactivity in antigen recognition.
313 citations
TL;DR: It is demonstrated that a dimeric but tetramerization-deficient Stat5 protein can activate only a subset of target sites, and for functional activity on a wider range of potential recognition sites, N-domain-mediated oligomerization is essential.
Abstract: Stat5a and Stat5b are rapidly activated by a wide range of cytokines and growth factors, including interleukin-2 (IL-2). We have previously shown that these signal transducers and activators of transcription (STAT proteins) are key regulatory proteins that bind to two tandem gamma interferon-activated site (GAS) motifs within an IL-2 response element (positive regulatory region III [PRRIII]) in the human IL-2Ralpha promoter. In this study, we demonstrate cooperative binding of Stat5 to PRRIII and explore the molecular basis underlying this cooperativity. We demonstrate that formation of a tetrameric Stat5 complex is essential for the IL-2-inducible activation of PRRIII. Stable tetramer formation of Stat5 is mediated through protein-protein interactions involving a tryptophan residue conserved in all STATs and a lysine residue in the Stat5 N-terminal domain (N domain). The functional importance of tetramer formation is shown by the decreased levels of transcriptional activation associated with mutations in these residues. Moreover, the requirement for STAT protein-protein interactions for gene activation from a promoter with tandemly linked GAS motifs can be relieved by strengthening the avidity of protein-DNA interactions for the individual binding sites. Taken together, these studies demonstrate that a dimeric but tetramerization-deficient Stat5 protein can activate only a subset of target sites. For functional activity on a wider range of potential recognition sites, N-domain-mediated oligomerization is essential.
216 citations
TL;DR: A structure-based statistical thermodynamic approach is presented and used to predict the propagation of the stabilization effects triggered by the binding of the monoclonal antibody D1.3 to hen egg white lysozyme.
Abstract: The interaction of a ligand with a protein occurs at a local site (the binding site) and involves only a few residues; however, the effects of that interaction are often propagated to remote locations. The chain of events initiated by binding provides the basis for fundamental biological phenomena such as allosterism, signal transduction, and structural-stability modification. In this paper, a structure-based statistical thermodynamic approach is presented and used to predict the propagation of the stabilization effects triggered by the binding of the monoclonal antibody D1.3 to hen egg white lysozyme. Previously, Williams et al. [Williams, D. C., Benjamin, D. C., Poljak, R. J. & Rule, G. S. (1996) J. Mol. Biol. 257, 866-876] showed that the binding of this antibody affects the stability of hen egg white lysozyme and that the binding effects propagate to a selected number of residues at remote locations from the binding epitope. In this paper, we show that this phenomenon can be predicted from structure. The formalism presented here permits the identification of the structural path followed by cooperative interactions that originate at the binding site. It is shown that an important condition for the propagation of binding effects to distal regions is the presence of a significant fraction of residues with low structural stability in the uncomplexed binding site. A survey of protein structures indicates that many binding sites have a dual character and are defined by regions of high and low structural stabilities. The low-stability regions might be involved in the transmission of binding information to other regions in the protein.
181 citations
TL;DR: Direct biochemical evidence of cooperative interaction in nucleotide binding of the two NBFs of SUR1 suggests that glibenclamide both blocks this cooperative binding of ATP and MgADP and, in cooperation with the Mg ADP bound at NBF2, causes ATP to be released from NBF1.
Abstract: The ATP-sensitive potassium (KATP) channels in pancreatic beta cells are critical in the regulation of glucose-induced insulin secretion. Although electrophysiological studies provide clues to the complex control of KATP channels by ATP, MgADP, and pharmacological agents, the molecular mechanism of KATP-channel regulation remains unclear. The KATP channel is a heterooligomeric complex of SUR1 subunits of the ATP-binding-cassette superfamily with two nucleotide-binding folds (NBF1 and NBF2) and the pore-forming Kir6.2 subunits. Here, we report that MgATP and MgADP, but not the Mg salt of gamma-thio-ATP, stabilize the binding of prebound 8-azido-[alpha-32P]ATP to SUR1. Mutation in the Walker A and B motifs of NBF2 of SUR1 abolished this stabilizing effect of MgADP. These results suggest that SUR1 binds 8-azido-ATP strongly at NBF1 and that MgADP, either by direct binding to NBF2 or by hydrolysis of bound MgATP at NBF2, stabilizes prebound 8-azido-ATP binding at NBF1. The sulfonylurea glibenclamide caused release of prebound 8-azido-[alpha-32P]ATP from SUR1 in the presence of MgADP or MgATP in a concentration-dependent manner. This direct biochemical evidence of cooperative interaction in nucleotide binding of the two NBFs of SUR1 suggests that glibenclamide both blocks this cooperative binding of ATP and MgADP and, in cooperation with the MgADP bound at NBF2, causes ATP to be released from NBF1.
159 citations
TL;DR: The number of Smad-binding sites within the 94-base pair major TGFβ-responsive region and the mechanism ofSmad-mediated transactivation are mapped and it is shown that Smad4 and Smad3 bind cooperatively to two adjacent DNA elements in this region.
123 citations
TL;DR: NF-Y can be added to the list of transcription factors interacting with Sp1 by demonstrating by the GST pull-down assay that NF-YA interacts physically with Sp 1 in-vitro in the absence of DNA.
114 citations
TL;DR: In this article, the authors compared FGFR1 and FGFR3 binding domains to determine the mechanism regulating ligand binding affinity and specificity, and identified two regions that contribute to FGF binding.
101 citations
TL;DR: It is shown that there are three different types of cooperative interactions during the DNA binding of DnaA proteins from E. coli, Streptomyces lividans, and Thermus thermophilus: i) binding to distant binding sites; ii)binding to closely spaced binding Sites; and iii) bindingto non-canonical binding sites.
101 citations
TL;DR: A small 19-amino-acid synthetic peptide whose sequence corresponds to the putative RNA-binding domain of CarMV p7, at the basic central part of the protein, was synthesized, and it was demonstrated that it binds viral RNA probes.
TL;DR: The effects of the peptide inhibitor acetyl pepstatin on the structural stability of the HIV‐1 protease have been measured by high sensitivity calorimetric techniques and a structure‐based thermodynamic analysis of the binding process indicates that the stabilization effect is not distributed uniformly throughout the protease molecule.
Abstract: The effects of the peptide inhibitor acetyl pepstatin on the structural stability of the HIV-1 protease have been measured by high sensitivity calorimetric techniques. At 25°C and pH 3.6, acetyl pepstatin binds to HIV-1 protease with an affinity of 1.6 × 107 M-1 and an enthalpy of 7.3 ± 0.5 kcal/mol, indicating that binding is not favored enthalpically and that the favorable Gibbs energy originates from a large positive entropy. Since the binding of acetyl pepstatin is associated with a negative change in heat capacity (−450 cal/K⋅mol) the association reaction becomes enthalpically favored at temperatures higher than 40°C. The presence of the inhibitor stabilizes the dimeric structure of the protease in a fashion that can be quantitatively described by a set of thermodynamic linkage equations. The combination of titration and differential scanning calorimetry provides an accurate way of determining binding constants for high affinity inhibitors that cannot be determined by titration calorimetry alone. A structure-based thermodynamic analysis of the binding process indicates that the stabilization effect is not distributed uniformly throughout the protease molecule. The binding of the inhibitor selectively stabilizes those conformational states in which the binding site is formed, triggering a redistribution of the state probabilities in the ensemble of conformations populated under native conditions. As a result, the stability constants for individual residues do not exhibit the same change in magnitude upon inhibitor binding. Residues in certain areas of the protein are affected significantly whereas residues in other areas are not affected at all. In particular, inhibitor binding has a significant effect on those regions that define the binding site, especially the flap region which becomes structurally stable as a result of the additional binding free energy. The induced stabilization propagates to regions not in direct contact with the inhibitor, particularly to the strand between residues Pro9 and Ala22 and the helix between Arg87 and Gly94. On the other hand, the stability of the strand between Asp60 and Leu76 is not significantly affected by inhibitor binding. The structural distribution of binding effects define cooperative pathways within the protease molecule. Proteins 1999;36:147–156. © 1999 Wiley-Liss, Inc.
TL;DR: It is reported that trypsin-fragmented mouse IP3R 1 (mIP3R1) retains significant inositol 1,4,5-trisphosphate (IP3) binding activity that is comparable to the intact receptor in affinity, capacity, and specificity and suggests that the IP3-binding pocket consists of two non-covalently but tightly associated structural domains each of which has a discrete function.
TL;DR: It is demonstrated that although the hRad51 and RecA proteins display many structural and functional similarities, they show profound inherent mechanistic differences.
TL;DR: It is found that for amphiphiles which are sufficiently hydrophobic, a charge neutralization, or even charge inversion of the complex is possible, which is of particular importance in applications to gene therapy, for which the functional delivery of specific base sequence into living cells remains an outstanding problem.
Abstract: We study complex formation between the DNA and cationic amphiphilic molecules. As the amphiphile is added to the solution containing DNA, a cooperative binding of surfactants to the DNA molecules is found. This binding transition occurs at a specific density of amphiphile, which is strongly dependent on the concentration of the salt and on the hydrophobicity of the surfactant molecules. We find that for amphiphiles which are sufficiently hydrophobic, a charge neutralization, or even charge inversion of the complex is possible. This is of particular importance in applications to gene therapy, for which the functional delivery of specific base sequence into living cells remains an outstanding problem. The charge inversion could, in principle, allow the DNA–surfactant complexes to approach the negatively charged cell membranes permitting the transfection to take place.
TL;DR: A simple mechanism for cooperativity, in the form of a tension-mediated interaction between proteins bound to DNA at two different locations, is proposed and properties of the cooperative interaction are discussed, along with experimental implications.
TL;DR: In this article, surface plasmon resonance was used to show that alphaIIbbeta3 contains two distinct ligand binding pockets, one binding to fibrinogen and a separate binding to Arg-Gly-Asp (RGD)-type ligands.
TL;DR: Newly designed constrained methionines are incorporated into the interactions between the crucial C-terminal methionine of substance P and these two binding sites to highlight the need of such constrained amino acids to probe subtle interactions in ligand-receptor complexes.
TL;DR: It is proposed that heparin binding to basic amino acids in IGFBP-5 between 201-218 may physically occlude subsequent interaction between IGF-I and Gly203/Gln209, and that this may explain previous work of others showing reduced affinity of ECM bound IGF BP-5 for IGF- I.
Abstract: The highly conserved N-and C-terminal domains of IGFBPs are believed to participate in IGF binding, but only recently have some of the critical residues in the IGFBP sequence involved in ligand binding been identified. Here we describe two highly conserved amino acids in the C-terminal domain of rat IGFBP-5 that are involved in binding IGF-I. Site-directed mutagenesis was used to produce two mutants, G203K and Q209A, of rIGFBP-5. Relative to wild-type rIGFBP-5, an 8-fold reduction in affinity for human IGF-I was found for recombinant G203K protein in both IGF-I ligand blots and solution phase ligand binding assays, and a 7-and 6-fold reduction for Q209A respectively. This shows that Gly203 and Gln209 in IGFBP-5 are important determinants in binding IGF-I, and due to their complete conservation in all IGFBP sequences, we suggest that they are likely to be involved in binding IGF-I in all six binding proteins. In addition, these two non-basic residues lie within the ECM binding region (201-218) of IGFBP-5, demonstrating that the C-terminus contains partially overlapping IGF-I and ECM binding sites. We therefore propose that heparin binding to basic amino acids in IGFBP-5 between 201-218 may physically occlude subsequent interaction between IGF-I and Gly203/Gln209, and that this may explain previous work of others showing reduced affinity of ECM bound IGFBP-5 for IGF-I.
TL;DR: The crystal structure of LFABP contains two bound oleate molecules and provides the explanation for its unusual stoichiometry, which will bind hydrophobic molecules larger than the ligands which will associate with other iLBPs.
Abstract: The crystal and solution structures of all of the intracellular lipid binding proteins (iLBPs) reveal a common β-barrel framework with only small local perturbations. All existing evidence points to the binding cavity and a poorly delimited ‘portal’ region as defining the function of each family member. The importance of local structure within the cavity appears to be its influence on binding affinity and specificity for the lipid. The portal region appears to be involved in the regulation of ligand exchange. Within the iLBP family, liver fatty acid binding protein or LFABP, has the unique property of binding two fatty acids within its internalized binding cavity rather than the commonly observed stoichiometry of one. Furthermore, LFABP will bind hydrophobic molecules larger than the ligands which will associate with other iLBPs. The crystal structure of LFABP contains two bound oleate molecules and provides the explanation for its unusual stoichiometry. One of the bound fatty acids is completely internalized and has its carboxylate interacting with an arginine and two serines. The second oleate represents an entirely new binding mode with the carboxylate on the surface of LFABP. The two oleates also interact with each other. Because of this interaction and its inner location, it appears the first oleate must be present before the second more external molecule is bound.
Journal Article•
TL;DR: Radioiodinated DBP was used as a probe to characterize biochemically the neutrophil DBP binding site and demonstrated that DBP binds to a membrane macromolecule that partitions to the detergent insoluble fraction and does not associate with the cytoskeleton.
Abstract: The vitamin D binding protein (DBP) is a multifunctional plasma protein that can modulate certain immune and inflammatory responses. The diverse cellular functions of DBP appear to require cell surface binding to mediate these processes. Numerous reports have detected DBP bound to the surface of several cell types and would support the concept of a cell surface binding site for DBP. However, direct evidence for such a molecule has been lacking and essentially nothing is known about its basic biochemical properties. In the present study, radioiodinated DBP was used as a probe to characterize biochemically the neutrophil DBP binding site. Radiolabeled DBP binds to and remains associated with the plasma membrane and is not degraded. Quantitation of DBP binding to either intact cells or purified plasma membranes showed nonsaturable (linear) binding with positive cooperativity, possibly suggesting DBP oligomer formation. Solubilization of cell bound 125I-DBP with various nonionic and zwitterionic detergents demonstrated that DBP binds to a membrane macromolecule that partitions to the detergent insoluble fraction. Moreover, this molecule does not associate with the cytoskeleton. Cross-linking of radiolabeled DBP bound to plasma membranes increased the amount of protein that partitioned to the insoluble fraction, and analysis of these complexes by SDS-PAGE revealed that they may be very large since they did not enter the gel. Finally, treatment of plasma membranes with either proteases or chondroitinase ABC completely abrogated membrane binding of DBP, suggesting that the protein binds to a chondroitin sulfate proteoglycan.
TL;DR: Interactions with novel sulfo-oligosaccharides based on the Lex pentasaccharide sequence are investigated and it is observed that the 3′-sulfo-Lex series are more potent than the corresponding 3″-sialyl- Lex series as inhibitors of the paucivalent L-selectin.
TL;DR: As a whole, GST P1–1 represents the first enzyme which displays a temperature-dependent homotropic regulation of substrate (e.g. GSH) binding.
TL;DR: These studies provide new insight into the regulation of TH by the concentration of H4biopterin which may have significant implications for the physiological regulation of catecholamine biosynthesis in neuroendocrine cells.
Abstract: Kinetic studies of tetrameric recombinant human tyrosine hydroxylase isoform 1 (hTH1) have revealed properties so far not reported for this enzyme. Firstly, with the natural cofactor (6R)-lerythro-5,6,7,8-tetrahydrobiopterin (H4biopterin) a time-dependent change (burst) in enzyme activity was observed, with a half-time of about 20 s for the kinetic transient. Secondly, nonhyperbolic saturation behaviour was found for H4biopterin with a pronounced negative cooperativity (0.39 < h < 0.58; [S]0.5 = 24 ± 4 μm). On phosphorylation of Ser40 by protein kinase A, the affinity for H4biopterin increased ([S]0.5 = 11 ± 2 μm) and the negative cooperativity was amplified (h = 0.27 ± 0.03). The dimeric C-terminal deletion mutant (Δ473–528) of hTH1 also showed negative cooperativity of H4biopterin binding (h = 0.4). Cooperativity was not observed with the cofactor analogues 6-methyl-5,6,7,8-tetrahydropterin (h = 0.9 ± 0.1; Km = 62.7 ± 5.7 μm) and 3-methyl-5,6,7,8-tetrahydropterin (H43-methyl-pterin)(h = 1.0 ± 0.1; Km = 687 ± 50 μm). In the presence of 1 mm H43-methyl-pterin, used as a competitive cofactor analogue to BH4, hyperbolic saturation curves were also found for H4biopterin (h = 1.0), thus confirming the genuine nature of the kinetic negative cooperativity. This cooperativity was confirmed by real-time biospecific interaction analysis by surface plasmon resonance detection. The equilibrium binding of H4biopterin to the immobilized iron-free apoenzyme results in a saturable positive resonance unit (ΔRU) response with negative cooperativity (h = 0.52–0.56). Infrared spectroscopic studies revealed a reduced thermal stability both of the apo-and the holo-hTH1 on binding of H4biopterin and lerythro-dihydrobiopterin (H2biopterin). Moreover, the ligand-bound forms of the enzyme also showed a decreased resistance to limited tryptic proteolysis. These findings indicate that the binding of H4biopterin at the active site induces a destabilizing conformational change in the enzyme which could be related to the observed negative cooperativity. Thus, our studies provide new insight into the regulation of TH by the concentration of H4biopterin which may have significant implications for the physiological regulation of catecholamine biosynthesis in neuroendocrine cells.
TL;DR: The overexpression and purification of the N-terminal end of the Menkes disease protein expressed in Escherichia coli, containing one, two and six metal binding domains (MBD), respectively, reveal a unique regulation of Menkes protein upon a change in copper(I) concentration, suggesting that the cooperativity does not occur as an 'all-or-none' cooperativity.
TL;DR: Findings that C(7)/3-phth is a potent allosteric modulator at mAChRs, particularly the M2 subtype, are extended and the effects of cooperativity on apparent drug-receptor subtype selectivity are highlighted.
TL;DR: These data provide the most extensive characterization of the loop A ligand binding domain available to date and define two new residue locations, Ile93 and Asn102, as contributing to the four-loop model of ligandbinding.
Abstract: Four discontinuous extracellular sequence domains have been proposed to form the ligand binding sites of the ligand-gated ion channel receptor superfamily. In this study, we investigated the role of 12 contiguous residues of the inhibitory glycine receptor that define the proposed "loop A" ligand binding domain. Using the techniques of site-directed mutagenesis and patch-clamp electrophysiology, four of the 12 residues were shown to have impaired ligand binding. Three mutants, 193A, A101H, and N102A, resulted in significant (17-44-fold) increases in the agonist EC50 values as compared with the wild-type glycine receptor, whereas Hill coefficients, ImaX values, and antagonist affinity remained largely unaffected. Consideration of receptor efficacy values indicates that these residues are involved in ligand binding rather than channel activation. A fourth mutant, W94A, failed to give rise to any glycine-activated currents, although cell-surface expression was observed, suggesting that this residue may also be involved in agonist binding. These data provide the most extensive characterization of the loop A ligand binding domain available to date and define two new residue locations, Ile93 and Asn102, as contributing to the four-loop model of ligand binding.
TL;DR: The cooperative nature of interaction of cationic surfactants with short oligonucleotides leading to eventual stabilization of DNA duplexes is demonstrated and may have implications for rational design of DNA binding drugs and DNA delivery systems.
TL;DR: The recognition of the hidden CB/RS/11 epitope, as well as the cooperative binding behaviour of the two antibodies, provides evidence that IL‐10 can adopt a conformational state other than that observed in the crystal structure.
Abstract: We present the mapping of two anti-human interleukin-10 (hIL-10) antibodies (CB/RS/2 and CB/RS/11) which have been described as binding their antigen cooperatively. The epitopes were identified using hIL-10-derived overlapping peptide scans prepared by spot synthesis. To identify residues essential for binding within the two epitopes, each position was replaced by all other L-amino acids. The epitope-derived peptides were further characterized with respect to antibody affinity and their inhibition of the antibody-hIL-10 interaction. One antibody (CB/RS/11) binds to residues which are completely buried in the X-ray structure of IL-10. Accessibility of this hidden epitope is enhanced upon binding of the antibody CB/RS/2, which recognizes a discontinuous epitope located nearby. The recognition of the hidden CB/RS/11 epitope, as well as the cooperative binding behaviour of the two antibodies, provides evidence that IL-10 can adopt a conformational state other than that observed in the crystal structure.
TL;DR: The results indicate that high concentrations of purified AR are required for solution dimerization and that cooperative DNA binding stabilizes two dimer forms that differ in redox state.
Abstract: Full-length histidine-tagged, dihydrotestosterone-bound human androgen receptor (AR) was purified to homogeneity by affinity and gel-filtration chromatography for antibody production and analysis of AR dimerization and DNA binding properties. A monoclonal antibody was raised that recognized human and rat AR epitope 360ArgAspTyrTyrAsnPheProLeuAla368 in the NH2-terminal domain and slowed migration of AR−DNA complexes in mobility shift assays. AR binding to androgen response element DNA had a Kd of 2.0 nM and a Hill coefficient of 2.1, indicating high-affinity, cooperative binding. AR solution dimerization was detected only at ≥0.2 μM AR, and DNA binding increased dimerization up to 30-fold. Slow- and fast-migrating AR−DNA complexes were detected under different reducing conditions that differed 5-fold in their dissociation rates from DNA. Treatment with the sulfhydryl oxidizing reagent diamide formed the faster migrating, slower dissociating complex, indicating it represents disulfide-linked AR dimers bound...