Biophysical characterization of the alpha-globin binding protein alpha-hemoglobin stabilizing protein

Alpha-hemoglobin stabilizing protein (AHSP) is a small (12 kDa) and abundant erythroid-specific protein that binds specifically to free alpha-(hemo)globin and prevents its precipitation. When present in excess over beta-globin, its normal binding partner, alpha-globin can have severe cytotoxic effects that contribute to important human diseases such as beta-thalassemia. Because AHSP might act as a chaperone to prevent the harmful aggregation of alpha-globin during normal erythroid cell development and in diseases of globin chain imbalance, it is important to characterize the biochemical properties of the AHSP.alpha-globin complex. Here we provide the first structural information about AHSP and its interaction with alpha-globin. We find that AHSP is a predominantly alpha-helical globular protein with a somewhat asymmetric shape. AHSP and alpha-globin are both monomeric in solution as determined by analytical ultracentrifugation and bind each other to form a complex with 1:1 subunit stoichiometry, as judged by gel filtration and amino acid analysis. We have used isothermal titration calorimetry to show that the interaction is of moderate affinity with an association constant of 1 x 10(7) m(-1) and is thus likely to be biologically significant given the concentration of AHSP (approximately 0.1 mm) and hemoglobin (approximately 4 mm) in the late pro-erythroblast.     

Biophysical characterization of the interaction of high-density lipoprotein (HDL) with endotoxins.

The interaction of bacterial endotoxins [lipopolysaccharide (LPS) and the 'endotoxic principle' lipid A], with high-density lipoprotein (HDL) from serum was investigated with a variety of physical techniques and biological assays. HDL exhibited an increase in the gel to liquid crystalline phase transition temperature Tc and a rigidification of the acyl chains of the endotoxins as measured by Fourier-transform infrared spectroscopy and differential scanning calorimetry. The functional groups of the endotoxins interacting with HDL are the phosphates and the diglucosamine backbone. The finding of phosphates as target groups is in accordance to measurements of the electrophoretic mobility showing that the zeta potential decreases from -50 to -60 mV to -20 mV at binding saturation. The importance of the sugar backbone as further target structure is in accordance with the remaining negative potential and competition experiments with polymyxin B (PMB) and phase transition data of the system PMB/dephosphorylated LPS. Furthermore, endotoxin binding to HDL influences the secondary structure of the latter manifesting in a change from a mixed alpha-helical/beta-sheet structure to a predominantly alpha-helical structure. The aggregate structure of the lipid A moiety of the endotoxins as determined by small-angle X-ray scattering shows a change of a unilamellar/inverted cubic into a multilamellar structure in the presence of HDL. Fluorescence resonance energy transfer data indicate an intercalation of pure HDL, and of [LPS]-[HDL] complexes into phospholipid liposomes. Furthermore, HDL may enhance the lipopolysaccharide-binding protein-induced intercalation of LPS into phospholipid liposomes. Parallel to these observations, the LPS-induced cytokine production of human mononuclear cells and the reactivity in the Limulus test are strongly reduced by the addition of HDL. These data allow to develop a model of the [endotoxin]/[HDL] interaction.     

Biophysical evidence of lipid and carbohydrate binding activities of shrimp high density lipoprotein/beta glucan binding protein

Crustacean High Density Lipoprotein/beta-Glucan Binding Protein (HDL/BGBP) has been studied due to its role in nutrition and immune response via activation of the defense cells (hemocytes) upon binding 1,3-D-beta-glucan carbohydrates. In this study, HDL/BGBP was found to be composed mainly of beta sheets, as determined by circular dichroism. Lipoprotein aggregation resulted when HDL/BGBP interacted with phospolipid vesicles, laminaribiose (1,3-beta-glucan disaccharide) or heparin. HDL/BGBP has similar dissociation constants for laminaribiose (K(d)=22 mM) or heparin (K(d)=46 mM) as determined by 90 degrees light scattering.     

1,3-beta-D glucan binding protein (BGBP) from the white shrimp,Penaeus vannamei,is also a heparin binding protein

Shrimp BGBP was purified as a 100 kDa glycoprotein by affinity chromatography using immobilised heparin. BGBP bound simple carbohydrates, glycosaminoglycans like heparin sulphate and glycoproteins, but it was unable to agglutinate erythrocytes. Using an ELISA-based microplate assay, it was shown that simple carbohydrates such as n-glucose and D-mannose are competitive inhibitors of heparin sulphate binding to BGBP. Based on these properties BGBP is considered as a new type of heparin binding protein.     

Biophysical characterization of interaction between apolipoprotein A-I and bacterial lipopolysaccharide

We studied the effect of bacterial lipopolysaccharide (LPS)-apolipoprotein A-I (apo A-I) interaction on the structure and function of this protein. The micellization process of dimirystoil phosphatidylcholine liposomes (MLV-DMPC) by apo A-I in the presence of LPS was characterized. Apo A-I may interact with MLV-DMPC at the lipid transition temperature, forming micellar complexes. The kinetics of MLV-DMPC micellization was studied by turbidimetry. In the absence of LPS, a monoexponential decrease in turbidity is observed. Preincubation of apo A-I with LPS impairs the micellization reaction, resulting in biphasic kinetics. The amplitude of the fast phase decreases with increasing concentrations of LPS. In the absence or in the presence of low amounts of LPS (1∶0.1 protein:LPS weight ratio), two major micellization products-containing two and three apo A-I molecules per particle-were observed. However, in the presence of higher amounts of LPS (1∶1 protein:LPS weight ratio), particles mainly contained two apo A-I molecules. In contrast, a decrease in intrinsic fluorescence intensity of the protein was observed in the presence of an increasing LPS concentration. Finally, we studied the effect of LPS on the transition temperature (Tt) of MLV-DMPC without detecting changes in Tt. In conclusion, the changes found in the micellization process are likely to be mainly caused by changes in the apo A-I conformation by LPS interaction in solution.     

Biophysical characterization of the interaction between hepatic glucokinase and its regulatory protein: impact of physiological and pharmacological effectors

Glucokinase (GK) is a key enzyme of glucose metabolism in liver and pancreatic beta-cells, and small molecule activators of GK (GKAs) are under evaluation for the treatment of type 2 diabetes. In liver, GK activity is controlled by the GK regulatory protein (GKRP), which forms an inhibitory complex with the enzyme. Here, we performed isothermal titration calorimetry and surface plasmon resonance experiments to characterize GK-GKRP binding and to study the influence that physiological and pharmacological effectors of GK have on the protein-protein interaction. In the presence of fructose-6-phosphate, GK-GKRP complex formation displayed a strong entropic driving force opposed by a large positive enthalpy; a negative change in heat capacity was observed (Kd = 45 nm, DeltaH = 15.6 kcal/mol, TDeltaS = 25.7 kcal/mol, DeltaCp = -354 cal mol(-1) K(-1)). With k(off) = 1.3 x 10(-2) s(-1), the complex dissociated quickly. The thermodynamic profile suggested a largely hydrophobic interaction. In addition, effects of pH and buffer demonstrated the coupled uptake of one proton and indicated an ionic contribution to binding. Glucose decreased the binding affinity between GK and GKRP. This decrease was potentiated by an ATP analogue. Prototypical GKAs of the amino-heteroaryl-amide type bound to GK in a glucose-dependent manner and impaired the association of GK with GKRP. This mechanism might contribute to the antidiabetic effects of GKAs.     

Biophysical characterization of the complex between human papillomavirus E6 protein and synapse-associated protein 97

The E6 protein of human papillomavirus (HPV) exhibits complex interaction patterns with several host proteins, and their roles in HPV-mediated oncogenesis have proved challenging to study. Here we use several biophysical techniques to explore the binding of E6 to the three PDZ domains of the tumor suppressor protein synapse-associated protein 97 (SAP97). All of the potential binding sites in SAP97 bind E6 with micromolar affinity. The dissociation rate constants govern the different affinities of HPV16 and HPV18 E6 for SAP97. Unexpectedly, binding is not mutually exclusive, and all three PDZ domains can simultaneously bind E6. Intriguingly, this quaternary complex has the same apparent hydrodynamic volume as the unliganded PDZ region, suggesting that a conformational change occurs in the PDZ region upon binding, a conclusion supported by kinetic experiments. Using NMR, we discovered a new mode of interaction between E6 and PDZ: a subset of residues distal to the canonical binding pocket in the PDZ(2) domain exhibited noncanonical interactions with the E6 protein. This is consistent with a larger proportion of the protein surface defining binding specificity, as compared with that reported previously.     

Biophysical characterization of interactions between the core binding factor alpha and beta subunits and DNA

Core binding factors (CBFs) play key roles in several developmental pathways and in human disease. CBFs consist of a DNA binding CBFalpha subunit and a non-DNA binding CBFbeta subunit that increases the affinity of CBFalpha for DNA. We performed sedimentation equilibrium analyses to unequivocally establish the stoichiometry of the CBFalpha:beta:DNA complex. Dissociation constants for all four equilibria involving the CBFalpha Runt domain, CBFbeta, and DNA were defined. Conformational changes associated with interactions between CBFalpha, CBFbeta, and DNA were monitored by nuclear magnetic resonance and circular dichroism spectroscopy. The data suggest that CBFbeta 'locks in' a high affinity DNA binding conformation of the CBFalpha Runt domain.     

Identification and characterization of syntenin binding protein in the black tiger shrimp Penaeus monodon

Shrimp exhibit a diverse response to viral infection that is manifested in drastic up- and down-regulations of a variety of genes. In our previous work, we identified syntenin of the shrimp Penaeus monodon (Pm) as a dynamic responder to white spot syndrome virus (WSSV) infection, its message being greatly upregulated in the acute phase of the infection. In order to further explore the link between Pm-syntenin and viral infection, we performed a yeast two-hybrid screening of a P. monodon cDNA library, using Pm-syntenin as bait. One of the molecules that specifically interacted with Pm-syntenin was the receptor-binding domain of alpha-2-macroglobulin (alpha2M). A GST pull-down assay showed that GST-alpha2M, but not GST alone, was capable of co-precipitating syntenin. Another GST pull-down assay showed that GST-syntenin, but not GST alone, was capable of co-precipitating alpha2M. In addition, mutant analyses showed that the N-terminal 131 amino acids of syntenin were both necessary and sufficient to bind the C-terminus receptor-binding domain of alpha2M. Furthermore, WSSV-infected Pm showed a significant upregulation of the alpha2M message, suggesting that both syntenin and its protein partner alpha2M are upregulated in the acute phase of a WSSV infection. Taken together with a previous report showing the co-localization of alpha2M and syntenin in the exosome of a dendritic cell line, it is likely that syntenin, through its interaction with alpha2M, plays an important role in the immune defense mechanisms of viral infections of shrimps.     

Demonstration and characterization of a specific interaction between gonococcal transferrin binding protein A and TonB

Iron scavenging by Neisseria gonorrhoeae is accomplished by the expression of receptors that are specific for host iron-binding proteins, such as transferrin and lactoferrin. Efficient transferrin-iron acquisition is dependent on the combined action of two proteins, designated TbpA and TbpB. TbpA is a TonB-dependent outer membrane receptor, whereas TbpB is lipid modified and serves to increase the efficiency of transferrin-iron uptake. Both proteins, together or separately, can be isolated from the gonococcal outer membrane by using affinity chromatography techniques. In the present study, we identified an additional protein in transferrin-affinity preparations, which had an apparent molecular mass of 45 kDa. The ability to copurify this protein by transferrin affinity was dependent upon the presence of TbpA and not TbpB. The amino-terminal sequence of the 45-kDa protein was identical to the amino terminus of gonococcal TonB, indicating that TbpA stably interacted with TonB, without the addition of chemical cross-linkers. Using immunoprecipitation, we could recover TbpA-TonB complexes without the addition of transferrin, suggesting that ligand binding was not a necessary prerequisite for TonB interaction. In contrast, a characterized TonB box mutant of TbpA did not facilitate interaction between these two proteins such that complexes could be isolated. We generated an in-frame deletion of gonococcal TonB, which removed 35 amino acids, including a Neisseria-specific, glycine-rich domain. This mutant protein, like the parental TonB, energized TbpA to enable growth on transferrin. Consistent with the functionality of this deletion derivative, TbpA-TonB complexes could be recovered from this strain. The results of the present study thus begin to define the requirements for a functional interaction between gonococcal TbpA and TonB.     

Biophysical characterization of the interaction of bovine seminal plasma protein PDC-109 with phospholipid vesicles

PDC-109 is the major protein of bovine seminal plasma. It binds to the bovine sperm surface at ejaculation and modulates sperm capacitation. PDC-109 displays phosphorylcholine- and heparin-binding activities which are thought to account for its sperm surface coating and glycosaminoglycan-induced sperm capacitating activities, respectively. We have characterized the interaction of isolated PDC-109 with membranes of phospholipid vesicles using a biophysical approach. Our results show that PDC-109 interacts not only with the solvent-exposed phosphorylcholine head group but also with the hydrophobic core of liposomes. Binding of PDC-109 to membranes is a very rapid, biphasic process with half times of less than one second. Maximal binding of PDC-109 to small unilamellar vesicles was achieved with a stoichiometric ratio of 10–11 phosphatidylcholine molecules/PDC-109 molecule. Incorporation of phosphatidylethanolamine or phosphatidylserine into phosphatidylcholine vesicles reduced the binding of PDC-109, suggesting that both the density of phosphorylcholine groups and the surface charge determine the interaction of the seminal plasma protein with the surface of the membrane. Electron spin resonance measurements showed that binding of PDC-109 to phosphatidylcholine vesicles caused a rigidification of the membrane. The relevance of the data for describing the role of PDC-109 in the modulation of sperm capacitation is discussed. Received: 16 June 1997 / Accepted: 10 September 1997     

Biophysical characterization of V3-lipopeptide liposomes influencing HIV-1 infectivity

The V3-loop of the HIV-1 gp120 alters host cell immune function and modulates infectivity. We investigated biophysical parameters of liposome constructs with embedded lipopeptides from the principle neutralizing domain of the V3-loop and their influence on viral infectivity. Dynamic light scattering measurements showed liposome supramolecular structures with hydrodynamic radius of the order of 900 and 1300nm for plain and V3-lipopeptide liposomes. Electron paramagnetic resonance measurements showed almost identical local microenvironment. The difference in liposome hydrodynamic radius was attributed to the fluctuating ionic environment of the V3-lipopeptide liposomes. In vitro HIV-1 infectivity assays showed that plain liposomes reduced virus production in all cell cultures, probably due to the hydrophobic nature of the aggregates. Liposomes carrying V3-lipopeptides with different cationic potentials restored and even enhanced infectivity (p<0.05). These results highlight the need for elucidation of the involvement of lipid bilayers as dynamic components in supramolecular structures and in HIV-1 fusion mechanisms.     

Biophysical characterization of G-protein coupled receptor-peptide ligand binding

G-protein coupled receptors (GPCRs) are ubiquitous membrane proteins allowing intracellular responses to extracellular factors that range from photons of light to small molecules to proteins. Despite extensive exploitation of GPCRs as therapeutic targets, biophysical characterization of GPCR-ligand interactions remains challenging. In this minireview, we focus on techniques that have been successfully used for structural and biophysical characterization of peptide ligands binding to their cognate GPCRs. The techniques reviewed include solution-state nuclear magnetic resonance (NMR) spectroscopy, solid-state NMR, X-ray diffraction, fluorescence spectroscopy and single-molecule fluorescence methods, flow cytometry, surface plasmon resonance, isothermal titration calorimetry, and atomic force microscopy. The goal herein is to provide a cohesive starting point to allow selection of techniques appropriate to the elucidation of a given GPCR-peptide interaction.     

Myosin binding protein C interaction with actin: characterization and mapping of the binding site

Myosin binding protein C (MyBPC) is a multidomain protein associated with the thick filaments of striated muscle. Although both structural and regulatory roles have been proposed for MyBPC, its interactions with other sarcomeric proteins remain obscure. The current study was designed to examine the actin-binding properties of MyBPC and to define MyBPC domain regions involved in actin interaction. Here, we have expressed full-length mouse cardiac MyBPC (cMyBPC) in a baculovirus system and shown that purified cMyBPC binds actin filaments with an affinity of 4.3 ± 1.1 μM and a 1:1 molar ratio with regard to an actin protomer. The actin binding by cMyBPC is independent of protein phosphorylation status and is not significantly affected by the presence of tropomyosin and troponin on the actin filament. In addition, cMyBPC-actin interaction is not modulated by calmodulin. To determine the region of cMyBPC that is responsible for its interaction with actin, we have expressed and characterized five recombinant proteins encoding fragments of the cMyBPC sequence. Recombinant N-terminal fragments such as C0-C1, C0-C4, and C0-C5 cosediment with actin in a linear, nonsaturable manner. At the same time, MyBPC fragments lacking either the C0-C1 or C0-C4 region bind F-actin with essentially the same properties as full-length protein. Together, our results indicate that cMyBPC interacts with actin via a single, moderate affinity site localized to the C-terminal region of the protein. In contrast, certain basic regions of the N-terminal domains of MyBPC may act as small polycations and therefore bind actin via nonspecific electrostatic interactions.     

The construction and characterization of a bifunctional EGFP/sAPRIL fusion protein

A fusion protein of enhanced green fluorescent protein (EGFP) and soluble domain of human a proliferation-inducing ligand (sAPRIL) was efficiently expressed in Escherichia coli BL 21 (DE3). The soluble EGFP/sAPRIL, around 43 kDa, was purified in milligram amounts using metal chellate affinity chromatography and detected with anti-His₆ and anti-hsAPRIL monoclonal antibody. The chimeric protein exhibited similar fluorescence spectra with free EGFP. In vitro, purified EGFP/sAPRIL specifically bound receptor B cell maturation antigen (BCMA) detected by enzyme linked immunosorbent assay (ELISA) and receptors [including heparan sulfate proteoglycan (HSPGs)]-positive cell lines analyzed by fluorescence-activated cell sorting (FACS). Confocal laser microscopy images visibly showed the HSPGs’-dependent binding of EGFP/sAPRIL to NIH-3T3 cell. In addition, the chimera retained the bioactivity to stimulate/co-stimulate proliferation of NIH-3T3 and Jurkat cell/human B cell in vitro. Therefore, the fusion protein shows a readily obtainable source of biologically active sAPRIL which has considerable potential for single-step fluorescence detection assay in the study of APRIL and its receptors.     

Biophysical characterization of the DNA binding domain of gpNu1, a viral DNA packaging protein

Terminase enzymes are common to double-stranded DNA viruses. These enzymes "package" the viral genome into a pre-formed capsid. Terminase from bacteriophage lambda is composed of gpA (72.4 kDa) and gpNu1 (20.4 kDa) subunits. We have described the expression and biochemical characterization of gpNu1DeltaK100, a construct comprising the N-terminal 100 amino acids of gpNu1 (Yang, Q., de Beer, T., Woods, L., Meyer, J., Manning, M., Overduin, M., and Catalano, C. E. (1999) Biochemistry 38, 465-477). Here we present a biophysical characterization of this construct. Thermally induced loss of secondary and tertiary structures is fully reversible. Surprisingly, although loss of tertiary structure is cooperative, loss of secondary structure is non-cooperative. NMR and limited proteolysis data suggest that approximately 30 amino acids of gpNu1DeltaK100 are solvent-exposed and highly flexible. We therefore constructed gpNu1DeltaE68, a protein consisting of the N-terminal 68 residues of gpNu1. gpNu1DeltaE68 is a dimer with no evidence of dissociation or further aggregation. Thermally induced unfolding of gpNu1DeltaE68 is reversible, with concomitant loss of both secondary and tertiary structure. The melting temperature increases with increasing protein concentration, suggesting that dimerization and folding are, at least in part, coupled. The data suggest that gpNu1DeltaE68 represents the minimal DNA binding domain of gpNu1. We further suggest that the C-terminal approximately 30 residues in gpNu1DeltaK100 adopt a pseudo-stable alpha-helix that extends from the folded core of the protein. A model describing the role of this helix in the assembly of the packaging apparatus is discussed.     

Molecular characterization of the bifunctional VHDL-CP from the hemolymph of white shrimp Penaeus vannamei

A very high-density lipoprotein (VHDL) purified from the hemolymph of the white shrimp Penaeus vannamei is shown to be identical to the clotting protein (CP) previously reported from the same organism based on size, subunits and N-terminal amino acid sequence. The approximately 440-kDa protein, a homodimer of approximately 200-kDa subunits, was present in KBr gradient fractions ranging in density from 1.155 to 1.212 g/ml. Samples of VHDL after purification by strong cation exchange chromatography were subjected to electrophoresis on native polyacrylamide gels. Lipids associated with the VHDL were detected by Sudan Black and Oil Red O staining and comprise 9-15% of the purified protein. Circular dichroism of VHDL-CP indicates that the alpha-helix content of the VHDL-CP is 32%, while beta-sheets correspond to 33%, closely resembling the secondary structure of CP from the shrimp Penaeus monodon and, remarkably, the secondary structure of very high-density lipophorin E (VHDLpE) from the tobacco hornworm, Manduca sexta.     

Thermodynamic characterization of the binding interaction between the histone demethylase LSD1/KDM1 and CoREST

Flavin-dependent histone demethylases catalyze the posttranslational oxidative demethylation of mono- and dimethylated lysine residues, producing formaldehyde and hydrogen peroxide in addition to the corresponding demethylated protein. In vivo, histone demethylase LSD1 (KDM1; BCH110) is a component of the multiprotein complex that includes histone deacetylases (HDAC 1 and 2) and the scaffolding protein CoREST. Although little is known about the affinities of or the structural basis for the interaction between CoREST and HDACs, the structure of CoREST(286-482) bound to an α-helical coiled-coil tower domain within LSD1 has recently been reported. Given the significance of CoREST in directing demethylation to specific nucleosomal substrates, insight into the molecular basis of the interaction between CoREST and LSD1 may suggest a new means of inhibiting LSD1 activity by misdirecting the enzyme away from nucleosomal substrates. Toward this end, isothermal titration calorimetry studies were conducted to determine the affinity and thermodynamic parameters characterizing the binding interaction between LSD1 and CoREST(286-482). The proteins tightly interact in a 1:1 stoichiometry with a dissociation constant (K(d)) of 15.9 ± 2.07 nM, and their binding interaction is characterized by a favorable enthalpic contribution near room temperature with a smaller entropic penalty at pH 7.4. Additionally, one proton is transferred from the buffer to the heterodimeric complex at pH 7.4. From the temperature dependence of the enthalpy change of interaction, a constant-pressure heat capacity change (ΔC(p)) of the interaction was determined to be -0.80 ± 0.01 kcal mol(-1) K(-1). Notably, structure-driven truncation of CoREST revealed that the central binding determinant lies within the segment of residues 293-380, also known as the CoREST "linker" region, which is a central isolated helix that interacts with the LSD1 coiled-coil tower domain to create a triple-helical bundle. Thermodynamic parameters obtained from the binding between LSD1 and the linker region of CoREST are similar to those obtained from the interaction between LSD1 and CoREST(286-482). These results provide a framework for understanding the molecular basis of protein-protein interactions that govern nucleosomal demethylation.     

Purification and characterization of the clotting protein from the white shrimp Penaeus vannamei

The protein responsible for clot formation was isolated from plasma of the white shrimp Penaeus vannamei by affinity chromatography in a heparin–agarose column. The protein, named clotting protein (CP), was found to be a lipoglycoprotein, composed of two 210-kDa subunits covalently bound by disulfide bridges. CP formed large polymers when incubated with hemocyte lysate. Dansylcadaverine can be incorporated into CP by a hemocyte lysate or guinea pig transglutaminase mediated reaction. The amino acid composition and the amino terminal sequence were determined and compared with the clotting protein of the crayfish and the spiny lobster.