Glycosaminoglycan-binding properties and kinetic characterization of human heparin cofactor II expressed in Escherichia coli

Irreversible inactivation of α-thrombin (T) by the serpin, heparin cofactor II (HCII), is accelerated by ternary complex formation with the glycosaminoglycans (GAGs) heparin and dermatan sulfate (DS). Low expression of human HCII in Escherichia coli was optimized by silent mutation of 27 rare codons and five secondary Shine-Dalgarno sequences in the cDNA. The inhibitory activities of recombinant HCII, and native and deglycosylated plasma HCII, and their affinities for heparin and DS were compared. Recombinant and deglycosylated HCII bound heparin with dissociation constants (K_D) of 6 ± 1 and 7 ± 1 μM, respectively, ∼6-fold tighter than plasma HCII, with K_D 40 ± 4 μM. Binding of recombinant and deglycosylated HCII to DS, both with K_D 4 ± 1 μM, was ∼4-fold tighter than for plasma HCII, with K_D 15 ± 4 μM. Recombinant HCII, lacking N-glycosylation and tyrosine sulfation, inactivated α-thrombin with a 1:1 stoichiometry, similar to plasma HCII. Second-order rate constants for thrombin inactivation by recombinant and deglycosylated HCII were comparable, at optimal GAG concentrations that were lower than those for plasma HCII, consistent with its weaker GAG binding. This weaker binding may be attributed to interference of the Asn¹⁶⁹N-glycan with the HCII heparin-binding site.     

RGS11 interacts preferentially with R7BP over Gαoa - Characterization of Gβ5-free RGS11

Regulator of G protein signaling 11 (RGS11) is the least characterized member of the R7 family of Gγ-like GGL domain-containing RGS proteins. All R7-RGS proteins of a variety of cell types are found in Gβ5-containing complexes that exhibit a number of unique functional properties. However, presence of Gβ5 reduced the affinity of R7-RGS7 for Gα subunits, also only RGS7 bound to Muscarinic M3-Receptor, but the Gβ5-RGS7 dimer did not, making it difficult to study differential interaction of R7-RGS proteins. Here, we report the successful purification of functionally intact, Gβ5-free recombinant RGS11 (rRGS11), obtained by expressing N- and C-terminally truncated form of RGS11 in Escherichia coli BL 21 (DE3), that differentially interact with R7BP and Gα_oa. rRGS11 was capable of interacting with Gα_oa and R7BP (RGS7 family binding protein) with equilibrium dissociation constants (K_D) of 904 (±208) nM, and 308 (±97) nM, respectively. It also induced several-fold increase in the GTPase activity of Gα_oa. The binding of rRGS11 was differential with a binding preference for R7BP over Gα_oa implying extended roles of R7BP. In addition, we identified a novel interaction between Gα_oa and R7BP with a K_D of 592 (±150) nM. The production of stable and functional rRGS11 would provide chances to discover more functions of RGS11 yet to be identified.     

Comparative analyses of a small molecule/enzyme interaction by multiple users of Biacore technology

To gauge the experimental variability associated with Biacore analysis, 36 different investigators analyzed a small molecule/enzyme interaction under similar conditions. Acetazolamide (222 g/mol) binding to carbonic anhydrase II (CAII; 30,000 Da) was chosen as a model system. Both reagents were stable and their interaction posed a challenge to measure because of the low molecular weight of the analyte and the fast association rate constant. Each investigator created three different density surfaces of CAII and analyzed an identical dilution series of acetazolamide (ranging from 4.1 to 1000 nM). The greatest variability in the results was observed during the enzyme immobilization step since each investigator provided their own surface activating reagents. Variability in the quality of the acetazolamide binding responses was likely a product of how well the investigators’ instruments had been maintained. To determine the reaction kinetics, the responses from the different density surfaces were fit globally to a 1:1 interaction model that included a term for mass transport. The averaged association and dissociation rate constants were 3.1 ± 1.6 × 10⁶ M⁻¹ s⁻¹ and 6.7 ± 2.5 × 10⁻² s⁻¹, respectively, which corresponded to an average equilibrium dissociation constant (K_D) of 2.6 ± 1.4 × 10⁻⁸ M. The results provide a benchmark of variability in interpreting binding constants from the biosensor and highlight keys areas that should be considered when analyzing small molecule interactions.     

Regulation of respiration controlled by mitochondrial creatine kinase in permeabilized cardiac cells in situ: Importance of system level properties

The main focus of this investigation is steady state kinetics of regulation of mitochondrial respiration in permeabilized cardiomyocytes in situ. Complete kinetic analysis of the regulation of respiration by mitochondrial creatine kinase was performed in the presence of pyruvate kinase and phosphoenolpyruvate to simulate interaction of mitochondria with glycolytic enzymes. Such a system analysis revealed striking differences in kinetic behaviour of the MtCK-activated mitochondrial respiration in situ and in vitro. Apparent dissociation constants of MgATP from its binary and ternary complexes with MtCK, K_ia and K_a (1.94 ± 0.86 mM and 2.04 ± 0.14 mM, correspondingly) were increased by several orders of magnitude in situ in comparison with same constants in vitro (0.44 ± 0.08 mM and 0.016 ± 0.01 mM, respectively). Apparent dissociation constants of creatine, K_ib and K_b (2.12 ± 0.21 mM 2.17 ± 0.40 Mm, correspondingly) were significantly decreased in situ in comparison with in vitro mitochondria (28 ± 7 mM and 5 ± 1.2 mM, respectively). Dissociation constant for phosphocreatine was not changed. These data may indicate selective restriction of metabolites' diffusion at the level of mitochondrial outer membrane. It is concluded that mechanisms of the regulation of respiration and energy fluxes in vivo are system level properties which depend on intracellular interactions of mitochondria with cytoskeleton, intracellular MgATPases and cytoplasmic glycolytic system.     

Initial synthesis and characterization of an immobilized heat shock protein 90 column for online determination of binding affinities

Heat shock protein 90α (Hsp90α) was immobilized on aminopropyl silica via the N terminus to create the Hsp90α(NT) column or via the C terminus to create the Hsp90α(CT) column. Binding to the exposed C terminus on the Hsp90α(NT) column was characterized using frontal chromatography and the C-terminus ligands coumermycin A₁ (CA1) and novobiocin (NOVO). The calculated K_d values were 220 ± 110 nM (CA1) and 100 ± 20 nM (NOVO). Nonlinear chromatography was used to determine the association and dissociation rate constants associated with the NOVO-Hsp90α complex: 22.2 ± 8.8 μM⁻¹ s⁻¹ and 2.7 ± 0.6 s⁻¹, respectively. Binding to the exposed N terminus on the Hsp90α(CT) column was characterized using frontal chromatography. The K_d values of the N-terminus ligands geldanamycin (GM, 90 ± 50 nM), 17-allylamino-17-demethoxygeldanamycin (17-AAG, 210 ± 50 nM), and radicicol (RAD, 20 ± 9 nM) were consistent with previously reported values. The effect of the immobilization on ATPase activity was investigated through the determination of IC₅₀ values for inhibition of ATPase activity on the Hsp90α(CT) column. The IC₅₀ for GM was 2.80 ± 0.18 μM, and the relative IC₅₀ values were 17-AAG > GM > RAD, in agreement with previously reported values and indicating that immobilization had not affected ATPase activity or sensitivity to inhibition.     

Fluorescence polarization assay for calmodulin binding to plasma membrane Ca-ATPase: Dependence on enzyme and Ca concentrations

Calmodulin (CaM) is a Ca²⁺ signaling protein that binds to a wide variety of target proteins, and it is important to establish methods for rapid characterization of these interactions. Here we report the use of fluorescence polarization (FP) to measure the K_d for the interaction of CaM with the plasma membrane Ca²⁺-ATPase (PMCA), a Ca²⁺ pump regulated by binding of CaM. Previous assays of PMCA-CaM interactions were indirect, based on activity or kinetics measurements. We also investigated the Ca²⁺ dependence of CaM binding to PMCA. FP assays directly detect CaM-target interactions and are rapid, sensitive, and suitable for high-throughput screening assay formats. Values for the dissociation constant K_d in the nanomolar range are readily measured. We measured the changes in anisotropy of CaM labeled with Oregon Green 488 on titration with PMCA, yielding a K_d value of CaM with PMCA (5.8 ± 0.5 nM) consistent with previous indirect measurements. We also report the binding affinity of CaM with oxidatively modified PMCA (K_d = 9.8 ± 2.0 nM), indicating that the previously reported loss in CaM-stimulated activity for oxidatively modified PMCA is not a result of reduced CaM binding. The Ca²⁺ dependence follows a simple Hill plot demonstrating cooperative binding of Ca²⁺ to the binding sites in CaM.     

Single-strand DNA translation initiation step analyzed by Isothermal Titration Calorimetry

Is single-strand DNA translatable? Since the 60s, the question still remains whether or not DNA could be directly translated into protein. Some discrepancies in the results were reported about functional translation of single-strand DNA but all results converged on a similar behavior of RNA and ssDNA in the initiation step. Isothermal Titration Calorimetry method was used to determine thermodynamic constants of interaction between single-strand DNA and S30 extract of Escherichia coli. Our results showed that the binding was not affected by the nature of the template tested and the dissociation constants were in the same range when ssDNA (K_d = 3.62 ± 2.1 × 10⁻⁸ M) or the RNA corresponding sequence (K_d = 2.7 ± 0.82 × 10⁻⁸ M) bearing SD/ATG sequences were used. The binding specificity was confirmed by antibiotic interferences which block the initiation complex formation. These results suggest that the limiting step in translation of ssDNA is the elongation process.     

SPR-based interaction studies with small molecular weight ligands using hAGT fusion proteins

An immobilization procedure for protein on surface plasmon resonance sensor (SPR) chips is described. The target protein, cyclophilin D, is thereby genetically linked to a mutant of the human DNA repair protein O⁶-alkylguanine-DNA-alkyltransferase (hAGT). The procedure includes the immobilization of an alkylguanine derivative on the surface by amine coupling and contact of the surface with a solution of the fusion protein (TCypD-hAGT). TCypD-hAGT could be immobilized using buffer solutions of purified protein or cell extracts. High densities of covalently linked proteins were achieved by either procedure. Binding experiments performed with the ligand cyclosporin A indicate relative binding activities close to 100%. The K_D value (12 nM) and the kinetic rate constants k_on (3 × 10⁵ M⁻¹s⁻¹) and k_off (4 × 10⁻³s⁻¹) are given and compared to values determined for cyclophilin D linked to the surface by amide coupling chemistry. The K_D value is in excellent agreement with the K_D value determined in solution by fluorescence titration.     

Development of a scintillation proximity assay binding method for the human 5-hydroxytryptamine 6 receptor using intact cells

We describe the first validated scintillation proximity assay (SPA) binding method for quantitation of ³H-labeled d-lysergic acid diethylamide (LSD) binding to recombinant human 5-hydroxytryptamine 6 (5-HT₆) receptors expressed in Chinese hamster ovary (CHO)-Dukx and HeLa cells. The assay was developed using intact cells as a receptor source because membrane fractions derived from these cells failed to discern specific binding from a high level of nonspecific binding. The pharmacological binding profile of seven 5-HT₆ agonists and antagonists using intact CHO-Dukx/5-HT₆ cells in the SPA format was similar to data obtained from a filtration binding assay using HeLa/5-HT₆ membranes. K_i values and rank order of potencies obtained in the SPA format were consistent with published filtration data as follows: SB-271046 (K_i = 1.9 nM) > methiothepin (K_i = 6.2 nM) > mianserin (K_i = 74.3 nM) > 5-methoxytryptamine (5-MeOT, K_i = 111 nM) > 5-HT (K_i = 150 nM) > ritanserin (K_i = 207 nM) > 5-carboxamidotryptamine (5-CT, K_i = 704 nM). Additional evaluation with four antipsychotics demonstrated strong agreement with previous literature reports. A high specific binding signal and low assay variability, as determined by Z′ = 0.81 ± 0.017, make the SPA format amenable to automation and higher throughput; hence, this assay can be a viable alternative to the more labor-intensive filtration and centrifugation methods.     

Biochemical characterization of a low molecular weight aspartic protease inhibitor from thermo-tolerant Bacillus licheniformis: Kinetic interactions with Pepsin

The present article reports a low molecular weight aspartic protease inhibitor, API, from a newly isolated thermo-tolerant Bacillus licheniformis. The inhibitor was purified to homogeneity as shown by rp-HPLC and SDS-PAGE. API is found to be stable over a broad pH range of 2–11 and at temperature 90 °C for 2 1/2 h. It has a Mr (relative molecular mass) of 1363 Da as shown by MALDI-TOF spectra and 1358 Da as analyzed by SDS-PAGE .The amino acid analysis of the peptide shows the presence of 12 amino acid residues having Mr of 1425 Da. The secondary structure of API as analyzed by the CD spectra showed 7% α-helix, 49% β-sheet and 44% aperiodic structure. The Kinetic studies of Pepsin–API interactions reveal that API is a slow-tight binding competitive inhibitor with the IC₅₀ and K_i values 4.0 nM and (3.83 nM–5.31 nM) respectively. The overall inhibition constant K_i? value is 0.107 ± 0.015 nM. The progress curves are time-dependent and consistent with slow-tight binding inhibition: E + I ? (k₄, k₅) EI ? (k₆, k₇) EI?. Rate constant k₆ = 2.73 ± 0.32 s^− 1 reveals a fast isomerization of enzyme–inhibitor complex and very slow dissociation as proved by k₇ = 0.068 ± 0.009 s^− 1. The Rate constants from the intrinsic tryptophanyl fluorescence data is in agreement with those obtained from the kinetic analysis; therefore, the induced conformational changes were correlated to the isomerization of EI to EI?.     

Ibuprofen modulates allosterically NO dissociation from ferrous nitrosylated human serum heme-albumin by binding to three sites

Human serum albumin (HSA) is a monomeric allosteric protein. Here, the effect of ibuprofen on denitrosylation kinetics (k_off) and spectroscopic properties of HSA-heme-Fe(II)-NO is reported. The k_off value increases from (1.4 ± 0.2) × 10⁻⁴ s⁻¹, in the absence of the drug, to (9.5 ± 1.2) × 10⁻³ s⁻¹, in the presence of 1.0 × 10⁻² M ibuprofen, at pH 7.0 and 10.0 °C. From the dependence of k_off on the drug concentration, values of the dissociation equilibrium constants for ibuprofen binding to HSA-heme-Fe(II)-NO (K₁ = (3.1 ± 0.4) × 10⁻⁷ M, K₂ = (1.7 ± 0.2) × 10⁻⁴ M, and K₃ = (2.2 ± 0.2) × 10⁻³ M) were determined. The K₃ value corresponds to the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(II)-NO determined by monitoring drug-dependent absorbance spectroscopic changes (H = (2.6 ± 0.3) × 10⁻³ M). Present data indicate that ibuprofen binds to the FA3-FA4 cleft (Sudlow’s site II), to the FA6 site, and possibly to the FA2 pocket, inducing the hexa-coordination of HSA-heme-Fe(II)-NO and triggering the heme-ligand dissociation kinetics.     

Quantitative measurement of binding kinetics in sandwich assay using a fluorescence detection fiber-optic biosensor

Fiber-optic biosensors have been studied intensively because they are very useful and important tools for monitoring biomolecular interactions. Here we describe a fluorescence detection fiber-optic biosensor (FD-FOB) using a sandwich assay to detect antibody-antigen interaction. In addition, the quantitative measurement of binding kinetics, including the association and dissociation rate constants for immunoglobulin G (IgG)/anti-mouse IgG, is achieved, indicating 0.38 × 10⁶ M⁻¹ s⁻¹ for k_a and 3.15 × 10⁻³ s⁻¹ for k_d. These constants are calculated from the fluorescence signals detected on fiber surface only where the excited evanescent wave can be generated. Thus, a confined fluorescence-detecting region is achieved to specifically determine the binding kinetics at the vicinity of the interface between sensing materials and uncladded fiber surface. With this FD-FOB, the mathematical deduction and experimental verification of the binding kinetics in a sandwich immunoassay provide a theoretical basis for measuring rate constants and equilibrium dissociation constants. A further measurement to study the interaction between human heart-type fatty acid-binding protein and its antibody gave the calculated kinetic constants k_a, k_d, and K_D as 8.48 × 10⁵ M⁻¹ s⁻¹, 1.7 × 10⁻³ s⁻¹, and 2.0 nM, respectively. Our study is the first attempt to establish a theoretical basis for the florescence-sensitive immunoassay using a sandwich format. Moreover, we demonstrate that the FD-FOB as a high-throughput biosensor can provide an alternative to the chip-based biosensors to study real-time biomolecular interaction.     

Kinetics of the Ca, H, and Mg Interaction with the Ion-Binding Sites of the SR Ca-ATPase

Electrochromic styryl dyes were used to investigate mutually antagonistic effects of Ca²⁺ and H⁺ on binding of the other ion in the E₁ and P-E₂ states of the SR Ca-ATPase. On the cytoplasmic side of the protein in the absence of Mg²⁺ a strictly competitive binding sequence, H₂E₁?HE₁?E₁?CaE₁?Ca₂E₁, was found with two Ca²⁺ ions bound cooperatively. The apparent equilibrium dissociation constants were in the order of K_1/2(2 Ca) = 34 nM, K_1/2(H) = 1 nM and K_1/2(H₂) = 1.32 μM. Up to 2 Mg²⁺ ions were also able to enter the binding sites electrogenically and to compete with the transported substrate ions (K_1/2(Mg) = 165 μM, K_1/2(Mg₂) = 7.4 mM). In the P-E₂ state, with binding sites facing the lumen of the sarcoplasmatic reticulum, the measured concentration dependence of Ca²⁺ and H⁺ binding could be described satisfactorily only with a branched reaction scheme in which a mixed state, P-E₂CaH, exists. From numerical simulations, equilibrium dissociation constants could be determined for Ca²⁺ (0.4 mM and 25 mM) and H⁺ (2 μM and 10 μM). These simulations reproduced all observed antagonistic concentration dependences. The comparison of the dielectric ion binding in the E₁ and P-E₂ conformations indicates that the transition between both conformations is accompanied by a shift of their (dielectric) position.     

Effect of ethanol on spectral binding, inhibition, and activity of CYP3A4 with an antiretroviral drug nelfinavir

Cytochrome P450 3A4 (CYP3A4) is the most abundant CYP enzyme in the liver and metabolizes approximately 50% of the drugs, including antiretrovirals. Although CYP3A4 induction by ethanol and impact of CYP3A4 on drug metabolism and toxicity is known, CYP3A4-ethanol physical interaction and its impact on drug binding, inhibition, or metabolism is not known. Therefore, we studied the effect of ethanol on binding and inhibition of CYP3A4 with a representative protease inhibitor, nelfinavir, followed by the effect of alcohol on nelfinavir metabolism. Our initial results showed that methanol, ethanol, isopropanol, isobutanol, and isoamyl alcohol bind in the active site of CYP3A4 and exhibit type I spectra. Among these alcohol compounds, ethanol showed the lowest K_D (5.9 ± 0.34 mM), suggesting its strong binding affinity with CYP3A4. Ethanol (20 mM) decreased the K_D of nelfinavir by >5-fold (0.041 ± 0.007 vs. 0.227 ± 0.038 μM). Similarly, 20 mM ethanol decreased the IC₅₀ of nelfinavir by >3-fold (2.6 ± 0.5 vs. 8.3 ± 3.1 μM). These results suggest that ethanol facilitates binding of nelfinavir with CYP3A4. Furthermore, we performed nelfinavir metabolism using LCMS. Although ethanol did not alter k_cat, it decreased the K_m of nelfinavir, suggesting a decrease in catalytic efficiency (k_cat/K_m). This is an important finding because alcoholism is prevalent in HIV-1-infected persons and alcohol is shown to decrease the response to antiretroviral therapy.     

Binding of phospholipids to β-Lactoglobulin and their transfer to lipid bilayers

The bovine milk lipocalin, β-Lactoglobulin (β-LG), has been associated with the binding and transport of small hydrophobic and amphiphilic compounds, whereby it is proposed to increase their bioavailability. We have studied the binding of the fluorescent phospholipid-derivative, NBD-didecanoylphosphatidylethanolamine (NBD-diC₁₀PE) to β-LG by following the increase in amphiphile fluorescence upon binding to the protein using established methods. The equilibrium association constant, K_B, was (1.2 ± 0.2) × 10⁶ M^− 1 at 25 °C, pH 7.4 and I = 0.15 M. Dependence of K_B on pH and on the monomer-dimer equilibrium of β-LG gave insight on the nature of the binding site which is proposed to be the hydrophobic calyx formed by the β-barrel in the protein. The monomer-dimer equilibrium of β-LG was re-assessed using fluorescence anisotropy of Tryptophan. The equilibrium constant for dimerization, K_D, was (7.0 ± 1.5) × 10⁵ M^− 1 at 25 °C, pH 7.4, and 0.15 M ionic strength. The exchange of NBD-diC₁₀PE between β-LG and POPC lipid bilayers was followed by the change in NBD fluorescence. β-LG was shown to be a catalyst of phospholipid exchange between lipid bilayers, the mechanism possibly involving adsorption of the protein at the bilayer surface.     

Kinetic study of dissociation of Eu(III) complex with H8dotp (H8dotp = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylphosphonic acid))

The dissociation kinetics of the europium(III) complex with H₈dotp ligand was studied by means of molecular absorption spectroscopy in UV region at ionic strength 3.0 mol dm⁻³ (Na,H)ClO₄ and in temperature region 25-60 °C. Time-resolved laser-induced fluorescence spectroscopy (TRLIFS) was employed in order to determine the number of water molecules in the first coordination sphere of the europium(III) reaction intermediates and the final products. This technique was also utilized to deduce the composition of reaction intermediates in course of dissociation reaction simultaneously with calculation of rate constants and it demonstrates the elucidation of intimate reaction mechanism. The thermodynamic parameters for the formation of kinetic intermediate (ΔH⁰ = 11 ± 3 kJ mol⁻¹, ΔS⁰ = 41 ± 11 J K⁻¹ mol⁻¹) and the activation parameters (E_a = 69 ± 8 kJ mol⁻¹, ΔH^≠ = 67 ± 8 kJ mol⁻¹, ΔS^≠ = −83 ± 24 J K⁻¹ mol⁻¹) for the rate-determining step describing the complex dissociation were determined. The mechanism of proton-assisted reaction was proposed on the basis of the experimental data.     

Sensory rhodopsin II/transducer complex formation in detergent and in lipid bilayers studied with FRET

The photophobic receptor from Natronomonas pharaonis (NpSRII) forms a photo-signalling complex with its cognate transducer (NpHtrII). In order to elucidate the complex formation in more detail, we have studied the intermolecular binding of both constituents (NpSRII and NpHtrII₁₅₇; truncated at residue 157) in detergent buffers, and in lipid bilayers using FRET. The data for hetero-dimer formation of NpSRII/NpHtrII in detergent agrees well with K_D values (∼ 200 nM) described in the literature. In lipid bilayers, the binding affinity between proteins in the NpSRII/NpHtrII complex is at least one order of magnitude stronger. In detergent the strength of binding is similar for both homo-dimers (NpSRII/NpSRII and NpHtrII/NpHtrII) but significantly weaker (K_D  ∼ 16 μM) when compared to the hetero-dimer. The intermolecular binding is again considerably stronger in lipid bilayers; however, it is not as strong as that observed for the hetero-dimer. At a molar transducer/lipid ratio of 1:2000, which is still well above physiological concentrations, only 40% homo-dimers are formed. Apparently, in cell membranes the formation of the assumed functionally active oligomeric 2:2 complex depends on the full-length transducer including the helical cytoplasmic part, which is thought to tighten the transducer-dimer association.     

Human mevalonate diphosphate decarboxylase: characterization, investigation of the mevalonate diphosphate binding site, and crystal structure

Expression in Escherichia coli of his-tagged human mevalonate diphosphate decarboxylase (hMDD) has expedited enzyme isolation, characterization, functional investigation of the mevalonate diphosphate binding site, and crystal structure determination (2.4 Å resolution). hMDD exhibits V_max = 6.1 ± 0.5 U/mg; K_m for ATP is 0.69 ± 0.07 mM and K_m for (R,S) mevalonate diphosphate is 28.9 ± 3.3 μM. Conserved polar residues predicted to be in the hMDD active site were mutated to test functional importance. R161Q exhibits a ∼1000-fold diminution in specific activity, while binding the fluorescent substrate analog, TNP-ATP, comparably to wild-type enzyme. Diphosphoglycolyl proline (K_i = 2.3 ± 0.3 uM) and 6-fluoromevalonate 5-diphosphate (K_i = 62 ± 5 nM) are competitive inhibitors with respect to mevalonate diphosphate. N17A exhibits a V_max = 0.25 ± 0.02 U/mg and a 15-fold inflation in K_m for mevalonate diphosphate. N17A’s K_i values for diphosphoglycolyl proline and fluoromevalonate diphosphate are inflated (>70-fold and 40-fold, respectively) in comparison with wild-type enzyme. hMDD structure indicates the proximity (2.8 Å) between R161 and N17, which are located in an interior pocket of the active site cleft. The data suggest the functional importance of R161 and N17 in the binding and orientation of mevalonate diphosphate.     

Distinct interactions between actin and essential myosin light chain isoforms

Binding of the utmost N-terminus of essential myosin light chains (ELC) to actin slows down myosin motor function. In this study, we investigated the binding constants of two different human cardiac ELC isoforms with actin. We employed circular dichroism (CD) and surface plasmon resonance (SPR) spectroscopy to determine structural properties and protein–protein interaction of recombinant human atrial and ventricular ELC (hALC-1 and hVLC-1, respectively) with α-actin as well as α-actin with alanin-mutated ELC binding site (α-actin^ala3) as control. CD spectroscopy showed similar secondary structure of both hALC-1 and hVLC-1 with high degree of α-helicity. SPR spectroscopy revealed that the affinity of hALC-1 to α-actin (K_D = 575 nM) was significantly (p < 0.01) lower compared with the affinity of hVLC-1 to α-actin (K_D = 186 nM). The reduced affinity of hALC-1 to α-actin was mainly due to a significantly (p < 0.01) lower association rate (k_on: 1018 M⁻¹ s⁻¹) compared with k_on of the hVLC-1/α-actin complex interaction (2908 M⁻¹ s⁻¹). Hence, differential expression of ELC isoforms could modulate muscle contractile activity via distinct α-actin interactions.     

Using S-adenosyl-l-homocysteine capture compounds to characterize S-adenosyl-l-methionine and S-adenosyl-l-homocysteine binding proteins

S-Adenosyl-l-methionine (SAM) is recognized as an important cofactor in a variety of biochemical reactions. As more proteins and pathways that require SAM are discovered, it is important to establish a method to quickly identify and characterize SAM binding proteins. The affinity of S-adenosyl-l-homocysteine (SAH) for SAM binding proteins was used to design two SAH-derived capture compounds (CCs). We demonstrate interactions of the proteins COMT and SAHH with SAH–CC with biotin used in conjunction with streptavidin–horseradish peroxidase. After demonstrating SAH-dependent photo-crosslinking of the CC to these proteins, we used a CC labeled with a fluorescein tag to measure binding affinity via fluorescence anisotropy. We then used this approach to show and characterize binding of SAM to the PR domain of PRDM2, a lysine methyltransferase with putative tumor suppressor activity. We calculated the K_d values for COMT, SAHH, and PRDM2 (24.1 ± 2.2 μM, 6.0 ± 2.9 μM, and 10.06 ± 2.87 μM, respectively) and found them to be close to previously established K_d values of other SAM binding proteins. Here, we present new methods to discover and characterize SAM and SAH binding proteins using fluorescent CCs.