Biosensor measurement of the interaction kinetics between insulin-like growth factors and their binding proteins.

The binding kinetics of human insulin-like growth factor binding protein (IGFBP) 1-6 for recombinant human insulin-like growth factor (IGF) I and II were measured and compared in the present study using surface plasmon resonance biosensor technique. Different concentrations of IGFBPs (5-100 nM) were allowed to interact with the immobilized IGF-I or IGF-II on sensor chip surface. Both des(1-3)IGF-I and insulin are known to bind weakly to the IGFBPs and therefore are used as negative controls for the binding experiments. The resultant sensorgrams were analyzed by using simple 1:1 binding model to derive both the association rate (k(a)) and dissociation rate (k(d)) constants for IGFBP-IGF interactions. The k(a) values of IGFBPs are in the range of 1x10(4) to 9x10(5) M(-1) s(-1) for IGF-I and 7x10(3) to 1.7x10(6) M(-1) s(-1) for IGF-II, respectively. The orders of k(a) for both IGF-I and IGF-II are IGFBP-3>IGFBP-5>IGFBP-6>IGFBP-4>IGFBP-2>++ +IGFBP-1. The k(d) values of IGFBPs are in the range of 1.5x10(-5) to 2x10(-4) s(-1) for IGF-I and 3.6x10(-5) to 3.7x10(-4) s(-1) for IGF-II, respectively. The order of k(d) for IGF-I is IGFBP-6>IGFBP-5>IGFBP-4>IGFBP-3>IGFBP-2>++ +IGFBP-1 and that for IGF-II is IGFBP-5>IGFBP-6>IGFBP-2>IGFBP-4>IGFBP-3>++ +IGFBP-1, respectively. The equilibrium affinity constants (K(A)) were calculated based on the ratio of k(a)/k(d) and were more precise than the published literature values based on competitive radioligand binding assays. The systematic study enables a direct comparison on the IGF-binding properties among the various IGFBPs, and the kinetic data provide additional information to delineate the physiological role of different IGFBPs in vivo.     

Insight into the interaction sites between fatty acid binding proteins and their ligands

Fatty acid binding proteins (FABPs), are evolutionarily conserved small cytoplasmic proteins that occur in many tissue-specific types. One of their primary functions is to facilitate the clearance of the cytoplasmic matrix from free fatty acids and of other detergent-like compounds. Crystallographic studies of FABP proteins have revealed a well defined binding site located deep inside their β-clam structure that is hardly exposed to the bulk solution. However, NMR measurements revealed that, when the protein is equilibrated with its ligands, residues that are clearly located on the outer surface of the protein do interact with the ligand. To clarify this apparent contradiction we applied molecular dynamics simulations to follow the initial steps associated with the FABP–fatty acid interaction using, as a model, the interaction of toad liver basic FABP, or chicken liver bile acid binding protein, with a physiological concentration of palmitate ions. The simulations (~200 ns of accumulated time) show that fatty acid molecules interact, unevenly, with various loci on the protein surface, with the favored regions being the portal and the anti-portal domains. Random encounters with palmitate at these regions led to lasting adsorption to the surface, while encounters at the outer surface of the β-clam were transient. Therefore, we suggest that the protein surface is capable of sequestering free fatty acids from solution, where brief encounters evolve into adsorbed states, which later mature by migration of the ligand into a more specific binding site.     

Electron paramagnetic resonance study of the interactions between steroid hormones and binding proteins]

Interaction of a spin labeled corticosteroid (desoxycorticosterone nitroxyde: DOC -NO) with three purified proteins (albumin, transcortin, progesterone binding protein: PBG) was studied by electron spin resonance (ESR) spectroscopy. DOC-NO was competitive with natural corticosteroids and therefore bound at the same site to specific binding proteins. ESR spectra in the presence of each of the proteins showed an immobilized (bound) form of the spin labeled steroid and allowed the calculation of the corresponding association constant (Ka) at equilibrium. The three binding proteins could be characterized by the ESR parameters of the DOC-NO bound form. The thermodynamic parameters (deltaH, deltaS) of the steroid-protein interactions were calculated from the ESR data obtained within a wide temperature range (3--40 degrees C). The ESR spectra width (2T) was used to evaluate the polarity of the spin label environment within the steroid binding site: a hydrophobic character was observed for transcortin whereas PBG exhibited a more hydrophilic steroid binding sits. The rotational correlation time of the three protein DOC-NO complexes at equilibrium were calculated from ESR data; the results were correlated with the protein molecular size and suggested a non spherical shape for the binding macromolecule in solution. Spin labelling of biologically active steroids thus provides a novel approach for the study of the interaction of these hormones with their binding protein. Providing a suitable spin label, the ESR parameters may allow the characterization of several types of binding sites of different biological significance for the same hormone, in biological fluids as well as in target tissues.     

Identification of a rabbit liver cytosolic binding protein for human growth hormone.

A specific growth hormone (GH) binding protein of Mr approx. 100000 has been demonstrated in the cytosolic fraction (200000g supernatant) of pregnant-rabbit liver by gel filtration techniques. This binding species was detectable by a standard charcoal separation procedure but not by the widely used poly(ethylene glycol) precipitation method. The GH binding protein had similar binding characteristics to those of classical membrane-bound GH receptors. The kinetics of association and dissociation, binding affinity (2.56 X 10(9)1/mol) and hormonal specificity have been established. There appears to be equal or greater amounts of GH binding protein in the cytosol than in the membrane fraction. The presence of the GH binding protein in rabbit liver cytosol was substantiated by its selective purification on a GH-Affigel 15 affinity column. This technique has resulted in a 200-300-fold purification with no substantial change in binding affinity. The ability of a concanavalin A-Sepharose affinity column to also bind the cytosolic binding protein indicates that, like the membrane-bound GH receptor, it is a glycoprotein. This is the first report of a cytosolic binding protein for GH and raises important questions regarding its potential physiological role in the mechanism of action of GH.     

Specific interaction between mouse liver non-histone chromosomal proteins and mouse DNA demonstrated by a sequential DNA-protein binding procedure.

The binding of mouse liver chromosomal proteins to DNA has been investigated using the nitrocellulose filter binding technique. Careful purification of the DNA involving nuclease S1 digestion and prefiltration through a nitrocellulose filter is used to reduce background binding in the absence of protein to less than 1%. Procedures involving direct binding of protein to labeled DNA, competition of binding of labeled DNA by unlabeled DNA, and dissociation of DNA . protein complexes with time do not indicate significant preference for binding to mouse DNA relative to Escherichia coli DNA. This specificity is demonstrated much more clearly by a novel type of procedure, which we call a sequential binding procedure. In this procedure non-specific binding proteins are sequestered by incubation with an excess of unlabeled E. coli DNA prior to addition of labeled DNA. Under these conditions, labeled mouse DNA is bound to filters to a 3- to 4-fold greater extent than labeled E. coli DNA.     

No direct correlation between binding of sex hormones and calcium pump activity by rat liver microsomal preparations

In the previous paper (J. steroid Biochem. 16 (1982) 437–446. [5]), we demonstrated that in vitro liver microsomal preparations of adult male rats possessed binding sites specific for progesterone (Prog) 2 of high affinity (K_D ∼ 25.2 nM) and high capacity (N_max ∼ 6.43 pmol/mg of microsomal protein), using 130 mM NaCl-based incubation buffer. To explore the biological roles of liver microsomal Prog binding, we investigated the effects of such binding on liver microsomal Ca²⁺ pump activity. Firstly, we obtained results similar to those previously obtained concerning the characteristics of microsomal Prog binding using 100 mM KCl-based incubation buffer, usually used for experiments on microsomal Ca²⁺ pump activity. For microsomal ⁴⁵Ca²⁺ uptake we also obtained results similar to those already demonstrated by several investigators. That is to say liver microsomal ⁴⁵Ca²⁺ uptake was markedly increased by the addition of 30 mM oxalate and 5 mM ATP, and was not inhibited by the addition of 5 mM NaN₃ into the incubation buffer. However, the addition of 1.0 μM Prog, as well as 17β-hydroxy-5α-androstan-3-one (5α-DHT) and estradiol-17β (E₂ -17β), which should be a sufficiently saturable concentration for liver microsomal binding sites specific for it, affected little microsomal ⁴⁵Ca²⁺ uptake statistically, though microsomal binding capacity for Prog was 5–10 times higher than that for 5α-DHT and E₂-17β. In addition, Prog (1.0 μM) had little effect on ⁴⁵Ca²⁺ release from prelabeled microsomes. In conclusion, we suggest, therefore, that there is no direct correlation between binding of sex hormones and Ca²⁺ pump activity by rat liver microsomal preparations.     

Spider acetylcholine binding proteins: An alternative model to study the interaction between insect nAChRs and neonicotinoids

Acetylcholine binding proteins (AChBPs) are homologs of extracellular domains of nicotinic acetylcholine receptors (nAChRs) and serve as models for studies on nAChRs. Particularly, studies on invertebrate nAChRs that are limited due to difficulties in their heterologous expression have benefitted from the discovery of AChBPs. Thus far, AChBPs have been characterized only in aquatic mollusks, which have shown low sensitivity to neonicotinoids, the insecticides targeting insect nAChRs. However, AChBPs were also found in spiders based on the sequence and tissue expression analysis. Here, we report five AChBP subunits in Pardosa pseudoannulata, a predator enemy against rice insect pests. Spider AChBP subunits shared higher sequence similarities with nAChR subunits of both insects and mammals compared with mollusk AChBP subunits. The AChBP1 subunit of P. pseudoannulata (Pp-AChBP) was then expressed in Sf9 cells. The Ls-AChBP from Lymnaea stagnalis was also expressed for comparison. In both AChBPs, one ligand site per subunit was present at each interface between two adjacent subunits. Neonicotinoids had higher affinities (7.9–18.4 times based on K_d or K_i values) for Pp-AChBP than for Ls-AChBP, although epibatidine and α-bungarotoxin showed higher affinities for Ls-AChBP. These results indicate that spider AChBP could be used as an alternative model to study the interaction between insect nAChRs and neonicotinoids.     

Arrestin binding to calmodulin: a direct interaction between two ubiquitous signaling proteins

Arrestins serve as multi-functional regulators of G-protein coupled receptors, interacting with hundreds of different receptor subtypes and a variety of other signaling proteins. Here we identify calmodulin as a novel arrestin interaction partner using three independent methods in vitro and in cells. Arrestin preferentially binds calcium-loaded calmodulin with a Kd value of approximately 7 microM, which is within range of endogenous calmodulin concentrations. The calmodulin binding site is localized on the concave side of the C-domain and a loop in the center of the arrestin molecule, significantly overlapping with receptor and microtubule-binding sites. Using purified proteins, we found that arrestins sequester calmodulin, preventing its binding to microtubules. Nanomolar affinity of arrestins for their cognate receptors makes calmodulin an ineffective competitor for arrestin binding at relatively high receptor concentrations. The arrestin-calmodulin interaction likely regulates the localization of both proteins and their availability for other interaction partners.     

Free-energy linkage between folding and calcium binding in EF-hand proteins

Troponin is the singular Ca²⁺-sensitive protein in the contraction of vertebrate striated muscles. Troponin C (TnC), the Ca²⁺-binding subunit of the troponin complex, has two distinct domains, C and N, which have different properties despite their extensive structural homology. In this work, we analyzed the thermodynamic stability of the isolated N-domain of TnC using a fluorescent mutant with Phe 29 replaced by Trp (F29W/N-domain, residues 1-90). The complete unfolding of the N-domain of TnC in the absence or presence of Ca²⁺ was achieved by combining high hydrostatic pressure and urea, a maneuver that allowed us to calculate the thermodynamic parameters (ΔV and ΔG_atm). In this study, we propose that part of the affinity for Ca²⁺ is contributed by the free-energy change of folding of the N- and C-domains that takes place when Ca²⁺ binds. The importance of the free-energy change for the structural and regulatory functions of the TnC isolated domains was evaluated. Our results shed light on how the coupling between folding and ion binding contributes to the fine adjustment of the affinity for Ca²⁺ in EF-hand proteins, which is crucial to function.     

Molecular dynamics study of the interaction between fatty acid binding proteins with palmitate mini-micelles

The fatty acid binding proteins (FAPBs) function as intracellular carriers of fatty acid (FA) and related compounds. During the digestion of lipids, the local concentration of FA exceeds their critical micellar concentration; the excess ratio of FA/FABP can be as high as ~1,000/1, consequently building micelles. Considering that the micelle formation is a rapid process, the FABP must be able to remove the mini-micelle. In this study, we describe the results of molecular dynamics simulations of liver basic FABP (Lb-FABP), carried out in the presence of ~20 mM palmitate ions, all in the presence of explicit water and at ionic strength of ~100 mM, approximating physiological conditions. The Lb-FABP appears to react, along with a free FA, with mini-micelle creating a stable complex (on the time scale of the simulations), which is attached to the anti-portal domain of the protein. The complex may be formed by the stepwise addition of free FA or through the interaction of a pre-formed mini-micelle with the free protein. The driving force of the mini-micelle-FABP complex is a combination of electrostatic attraction between the negative carboxylates of the mini-micelle with the positive charge of the N terminal amine residues and Lennard-Jones FA–protein interactions. The preferred tendency of the mini-micelle to react with the anti-portal domain retains the α-helixes of the portal region free for its electrostatic interaction with the membrane, ensuring a rapid unloading of the cargo on the membrane.     

The binding of Ni(II) ions to hexahistidine as a model system of the interaction between nickel and His-tagged proteins

The aim of this work is to study the binding of nickel ions to hexahistidine (His(6)) combining potentiometric titrations and spectroscopic (UV-Vis and circular dichroism) determinations in order to establish the species distribution as a function of the pH, their stoichiometry, stability and geometry. For comparative purposes, the same procedure was applied to the Ni-histidine (His) system. His behaves as a tridentate ligand, coordinating the carboxyl group, the imidazole and the amino nitrogen atoms to Ni(II) ions in an octahedral coordination and a bis(histidine) complex is formed at pH higher than 5. For the Ni-His(6) system, the complex formation starts at pH 4 and five different species (Ni(His(6))H, Ni(His(6)), Ni(n)(His(6))(n), Ni(n)(His(6))(n)H(-n/2), Ni(n)(His(6))(n)H(-n)) are formed as a function of the pH. Ni(His(6))H involves the coordination of the imidazole nitrogen and a deprotonated amide nitrogen (N(Im), N(-)) resulting in an octahedral geometry. In Ni(His(6)), an imidazole nitrogen is deprotonated and coordinated (2N(Im), N(-)) to the metal ion with a square planar geometry. The aggregated forms result from the extra Ni-N(Im) coordination, resulting in a 4N square planar geometry that is stabilized by inter/intramolecular hydrogen bonds. This coordination mode is not altered during the deprotonation steps from Ni(n)(His(6))(n).     

Role of insulin-like growth factors and their binding proteins in growth control and carcinogenesis

Interest in the role of the insulin-like growth factor (IGF) axis in growth control and carcinogenesis has recently been increased by the finding of elevated serum insulin-like growth factor I (IGF-I) levels in association with three of the most prevalent cancers in the United States: prostate cancer, colorectal cancer, and lung cancer. IGFs serve as endocrine, autocrine, and paracrine stimulators of mitogenesis, survival, and cellular transformation. These actions are mediated through the type 1 IGF-receptor (IGF-1R), a tyrosine kinase that resembles the insulin receptor. The availability of free IGF for interaction with the IGF-1R is modulated by the insulin-like growth factor-binding proteins (IGFBPs). IGFBPs, especially IGFBP-3, also have IGF-independent effects on cell growth. IGF-independent growth inhibition by IGFBP-3 is believed to occur through IGFBP-3-specific cell surface association proteins or receptors and involves nuclear translocation. IGFBP-3-mediated apoptosis is controlled by numerous cell cycle regulators in both normal and disease processes. IGFBP activity is also regulated by IGFBP proteases, which affect the relative affinities of IGFBPs, IGFs and IGF-1R. Perturbations in each level of the IGF axis have been implicated in cancer formation and progression in various cell types.     

Magnesium fluoride-dependent binding of small G proteins to their GTPase-activating proteins.

GTPase-activating proteins (GAPs) enhance the intrinsic GTPase activity of small G proteins, such as Ras and Rho, by contributing a catalytic arginine to the active site. An intramolecular arginine plays a similar role in heterotrimeric G proteins. Aluminum fluoride activates the GDP form of heterotrimeric G proteins, and enhances binding of the GDP form of small G proteins to their GAPs. The resultant complexes have been interpreted as analogues of the transition state of the hydrolytic reaction. Here, equilibrium binding has been measured using scintillation proximity assays to provide quantitative information on the fluoride-mediated interaction of Ras and Rho proteins with their respective GAPs, neurofibromin (NF1) and RhoGAP. High-affinity fluoride-mediated complex formation between Rho.GDP and RhoGAP occurred in the absence of aluminum; however, under these conditions, magnesium was required. Additionally, the novel observation was made of magnesium-dependent, fluoride-mediated binding of Ras.GDP to NF1 in the absence of aluminum. Aluminum was required for complex formation when the concentration of magnesium was low. Thus, either aluminum fluoride or magnesium fluoride can mediate the high-affinity binding of Rho. GDP or Ras.GDP to GAPs. It has been reported that magnesium fluoride can activate heterotrimeric G proteins. Thus, magnesium-dependent fluoride effects might be a general phenomenon with G proteins. Moreover, these data suggest that some protein.nucleotide complexes previously reported to contain aluminum fluoride may in fact contain magnesium fluoride.