1,1'-bis(anilino)-4-,4'-bis(naphtalene)-8,8'-disulfonate acts as an inhibitor of lipoprotein lipase and competes for binding with apolipoprotein CII

Lipoprotein lipase (LPL) is dependent on apolipoprotein CII (apoCII), a component of plasma lipoproteins, for function in vivo. The hydrophobic fluorescent probe 1,1'-bis(anilino)-4,4'-bis(naphthalene)-8,8'-disulfonate (bis-ANS) was found to be a potent inhibitor of LPL. ApoCII prevented the inhibition by bis-ANS, and was also able to restore the activity of inhibited LPL in a competitive manner, but only with triacylglycerols with acyl chains longer than three carbons. Studies of fluorescence and surface plasmon resonance indicated that LPL has an exposed hydrophobic site for binding of bis-ANS. The high affinity interaction was characterized by an equilibrium constant Kd of 0.10-0.26 microm and by a relatively high on rate constant kass = 2.0 x 10(4) m(-1) s(-1) and a slow off-rate with a dissociation rate constant kdiss = 1.2 x 10(-4) s(-1). The high affinity binding of bis-ANS did not influence interaction of LPL with heparin or with lipid/water interfaces and did not dissociate the active LPL dimer into monomers. Analysis of fragments of LPL after photoincorporation of bis-ANS indicated that the high affinity binding site was located in the middle part of the N-terminal folding domain. We propose that bis-ANS binds to an exposed hydrophobic area that is located close to the active site. This area may be the binding site for individual substrate molecules and also for apoCII.     

Cross-linking of ribosomal proteins by 4-(6-formyl-3-3-azidophenoxy)butyrimidate, a heterobifunctional, cleavable cross-linker.

For the identification of neighbor relationships between proteins in biological systems 4-(6-formyl-3-azidophenoxy)butyrimidate (FAPB-imidate), a heterobifunctional, cleavable cross-linker was synthesized. The reagent has an imido ester at one end, which is used for the attachment to amino groups of a specific protein whose environment has to be characterized. At the other end, the reagent has both an azido and an aldehyde group. The azido group can be used to cross-link the protein photochemically to a variety of chemical groups of neighboring proteins. The aldehyde group is able to cross-link the protein by reductive alkylatin to amino groups of neighboring proteins. In both cases, the cross-linker can be made radioactive with NaB3H4. the cross-linked complexes can be split at the band originating from the imidate group by treatment with ammonia. Hereby, the radioactive cross-linker remains covalently attached to the unknown neighboring protein, which can be therefore easily identified. In order to explore the usefulness of FAPB-imidate as a cross-linking agent, the compound was attached to ribosomal protein L7. With this modified L7, the existence of the well-known complex between L7 and ribosomal protein L10 could be demonstrated by the photochemical procedure. By the chemical procedure, the presence of dimers of L7 in solution could be shown.     

Molecular dynamics and IR spectroscopy in investigation of phase transitions in molecular crystal 4,4'-bis(11-hydroxy-1-undecyloxy)biphenyl

Molecular dynamics (MD) simulations combined with temperature-dependent IR spectroscopic measurements were used to study phase transitions in molecular crystals of the mesogenic diol 4,4'-bis(11-hydroxy-1-undecyloxy)biphenyl. DSC measurements revealed four phase transitions in this molecular crystal at approximately 327.1 K, 389.8 K, 419.1 K and 431.9 K. Analysis of the dynamic trajectories at temperatures of 300 K, 360 K, 400 K and 480 K revealed changes in conformation of the mesogenic diol molecules and consequently changes in crystal packing and crystal structure in the temperature range 300-480 K and enabled us to understand the mechanism of the phase transitions.     

Probing different conformational states of bovine alpha-lactalbumin: fluorescence studies with 4,4'-bis[1-(phenylamino)-8-naphthalenesulfonate]

The binding of the fluorescent probe 4,4'-bis[1-(phenylamino)-8-naphthalenesulfonate] (bis-ANS) to bovine alpha-lactalbumin (alpha-LA) was investigated. A strong dependence of the Kd value with the bound calcium stoichiometry was found, with Kd values ranging from 6.2 +/- 0.4 to 64.6 +/- 5.9 microM for apo-alpha-LA and 1:1 Ca(II)-alpha-LA, respectively. A 350-fold enhancement of the bis-ANS emission was observed in the protein-bis-ANS complex, along with an approximately 33-nm blue shift. Both appeared to be related to the hydrophobicity of the binding site and were independent of the Ca(II) ion content. From the difference in bis-ANS affinity between apo-alpha-LA and Ca(II)-alpha-LA, we demonstrated that Zn(II) and Al(III) were able to "lock" the protein into a new "apo-like" conformation, which was similar to, but not identical with, the apo conformation. The protein could be interconverted between all three conformations in a Mn(II) titration. The first Mn(II) shifted the apoprotein to the Ca(II) conformation; at higher Mn(II) levels, binding to the second site shifted the protein toward the apo-like conformation. The same behavior was observed with calcium in large excess. The evidence supported a model for the mutually nonexclusive binding of metals both to site I ("calcium site") and to site II ("zinc site") simultaneously. The results suggest that alpha-lactalbumin possesses a hydrophobic surface that becomes somewhat less accessible upon 1:1 calcium binding in the absence of metals that also bind to the zinc site.     

Models for superoxide dismutases: characterization of mononuclear Cu(II), Fe(III), and Mn(II) complexes with 4',5'-bis(salicylideneimino)benzo-15-crown-5

Mononuclear Cu(II), Fe(III), and Mn(II) complexes with 4',5'-bis (salicylideneimino)benzo-15-crown-5, (SALH2), were characterized by elemental analysis, IR and UV-Vis spectroscopy and tested spectrometrically as catalysts for superoxide disproportionation by utilizing xanthine-xanthine oxidase (XXO) assays. The results indicate that the examined mononuclear complexes are speculative potent superoxide dismutase mimics.     

Theoretical study on novel nitrogen-rich energetic compounds of bis(amino)-azobis(azoles) with tetrazene unit

Six stereoisomers of 5,5'-bis(amino)-1,1'-azobis(tetrazoles) and 30 other structures, including all possible bis(amino)-azobis(azoles) with an N-N=N-N unit, were designed. The molecular geometries were fully optimized at the DFT-B3LYP level with the 6-31++g (d, p) basis set. From the absence of any imaginary frequency in the infrared vibration frequency spectrum, it is predicted that all these studied structures may exist in stable forms. The results of the total energies of the stereoisomers of 5,5'-bis(amino)-1,1'-azobis(tetrazoles) indicate that the two symmetric trans-form structures are more likely to exist than the other four. The pyrolysis process, chemical stability and molecular electrostatic potential were studied via the investigation of their electronic structure. Heats of formation (HOFs) were calculated using the atomization energy method based on the results of the harmonic vibration frequencies, and a linear relationship was found between the HOF and nitrogen chain or nitrogen content. Densities of the title compounds were predicted with the Monte Carlo method. Finally, according to the results of the calculated HOFs and densities, the explosive parameters of these compounds were calculated using the Kamlet-Jacobs formula. 5,5'-Bis(amino)-1,1'-azobis(tetrazoles) and its isomer 5,5'-bis(amino)-2,2'-azobis(tetrazoles) may have potential for use as energetic compounds.     

Interactions at the nucleic acid binding site of the avian retroviral nucleocapsid protein: studies utilizing the fluorescent probe 4,4'-bis(phenylamino)(1,1'-binaphthalene)-5,5'-disulfonic acid

The structural and functional properties of the nucleocapsid (NC) protein of the avian myeloblastosis virus were examined by steady-state fluorescence and fluorescence anisotropy measurements of the complex between the NC and the extrinsic fluorophore 4,4'-bis(phenylamino)(1,1'-binaphthalene)-5,5'-disulfonic acid (bis-ANS). The intrinsic fluorescence of bis-ANS is enhanced many fold upon forming a complex with the NC. Between 2 and 10 molecules of bis-ANS bind strongly to the NC, with an overall Kd of less than 10(-6) M. The emission of bis-ANS in the complex can also be induced by excitation at 298 nm, indicating that energy is transferred from Trp 80, the sole tryptophan in the NC protein, to bis-ANS. The energy transferred between the Trp 80 and bis-ANS was analyzed to yield a calculated distance of separation between these fluorophores of 28 +/- 3 A; thus, Trp 80 is well removed from the nearest bound bis-ANS. The fluorescence emission of bis-ANS in the NC.bis-ANS complex is efficiently quenched by added salts and by poly(A), suggesting that salt (presumably anions), nucleic acid, and bis-ANS bind to the same, positively charged region on the NC protein. A site size of six nucleotides was determined for nucleic acid binding to the NC protein, with an estimated Kd of less than 10(-6) M. Salt (anion) binding is strong, but nonspecific, with a Kapp of 4 mM, raising the possibility that anion binding to the NC protein might regulate the interaction of the NC with viral RNA inside the host cell.     

Covalent immobilization of proteins on carbon nanotubes using the cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide--a critical assessment

Functionalization of carbon nanotubes (CNTs) with proteins is often a key step in their biological applications, particularly in biosensing. One popular method has used the cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to covalently conjugate proteins onto carboxylated CNTs. In this article, we critically assess the evidence presented in these conjugation studies in the literature. As CNTs have a natural affinity for diverse proteins through hydrophobic and electrostatic interactions, it is therefore important to differentiate protein covalent attachment from adsorption in the immobilization mechanism. Unfortunately, many studies of conjugating proteins onto CNTs using EDC lacked essential controls to eliminate the possibility of protein adsorption. In studies where the attachment was claimed to be covalent, discrepancies existed and the observed immobilization appeared to be due to adsorption. So far, bond analysis has been lacking to ascertain the nature of the attachment using EDC. We recommend that this approach of covalent immobilization of proteins on CNTs be re-evaluated and treated with caution.     

Synthesis and biological activity of tricyclic analogues of 9-[[cis-1',2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl]guanine

The base moiety of the potent antiherpetic agent 9-[[cis-1',2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl]guanine 3 was transformed into that of the tricyclic 3,9-dihydro-9-oxo-6-R-5H-imidazo[1,2-a]purine system. The tricyclic analogues 5a-d were evaluated for their activity against herpes viruses as well as for cytostatic activity against HSV-1 thymidine kinase (TK) gene-transduced human osteosarcoma tumor cells. Marked activity was found against VZV. The 6-phenyl-substituted fluorescent analogues 5c and d were comparable to that of parent 3 in activity against the VZV strain YS and were 3-fold less active against the VZV strain OKA. The compounds 5a-d also showed marked activity against HSV-1 (KOS) and HSV-2 (G)-against the former generally approximately comparable to that of acyclovir 1a and one order of magnitude lower than 3; against the latter comparable to that of 1a and approximately 6- to 30-fold lower than that of 3. The most pronounced cytostatic activity (5-fold lower than that of 3) was exhibited by compounds 5c and d. Tricyclic analogues with pseudosugar moieties are intrinsically bio-active.     

Interaction of non-histone proteins HMG1 and HMG2 with core histones in nucleosomes and core particles revealed by chemical cross-linking

Chemical cross-linking was used to study the interaction of the non-histone chromosomal proteins HMG1 and HMG2 with core histones in H1,H5-depleted nucleosomes or core particles. Cross-linking with a 'zero-length' cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and with a longer (cleavable) cross-linker dimethyl-3,3'-dithiobispropionimidate revealed an interaction of HMG1 and HMG2 with (or proximity to) core histones in both types of particles. These results indicated that the presence of the 40-50-base-pairs-long segment of the 'linker' DNA in nucleosomes was not necessary for the establishment of mutual contacts of HMG1 and HMG2 proteins with core histones. Possible implications of the interaction of HMG1 and HMG2 proteins with histones for the structure and functioning of chromatin are discussed.     

Binding of 5,5'-bis[8-(phenylamino)-1-naphthalenesulfonate] by the regulatory subunits of adenosine cyclic 3',5'-phosphate dependent protein kinase

Binding to the regulatory subunits of types I and II adenosine cyclic 3',5'-phosphate (cAMP) dependent protein kinase (RI and RII, respectively) produces large distinctive increases in fluorescence and optical activity of 5,5'-bis[8-(phenylamino)-1-naphthalenesulfonate] [bis(ANS)]. Both specific and nonspecific interactions are involved. Association of the regulatory subunits with either the catalytic subunit or cAMP results in dissociation of a major portion of the bound bis(ANS) as detected by changes in fluorescence and circular dichroism. The results are consistent with the accepted cAMP binding properties of RI and RII, showing cooperativity in case of RI and two heterologous binding sites for RII. cGMP has the same overall effect on bis(ANS) binding as cAMP. However, very high concentrations are required for complete dissociation of bis(ANS) from RII, consistent with the observation that cGMP is inefficient in bringing about the dissociation of the type II holoenzyme. Magnesium binding to sites having dissociation constants of ca. 12 mM increases the interaction of bis(ANS) with both of the isolated regulatory subunits. Experiments involving the 37 000-dalton fragment of RII indicate that the limited proteolytic cleavage was heterogeneous, with only 24-39% of the resulting population interacting strongly with the catalytic subunit.     

Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions

A substantial range of protein-protein interactions can be readily monitored in real time using bioluminescence resonance energy transfer (BRET). The procedure involves heterologous coexpression of fusion proteins, which link proteins of interest to a bioluminescent donor enzyme or acceptor fluorophore. Energy transfer between these proteins is then detected. This protocol encompasses BRET1, BRET2 and the recently described eBRET, including selection of the donor, acceptor and substrate combination, fusion construct generation and validation, cell culture, fluorescence and luminescence detection, BRET detection and data analysis. The protocol is particularly suited to studying protein-protein interactions in live cells (adherent or in suspension), but cell extracts and purified proteins can also be used. Furthermore, although the procedure is illustrated with references to mammalian cell culture conditions, this protocol can be readily used for bacterial or plant studies. Once fusion proteins are generated and validated, the procedure typically takes 48-72 h depending on cell culture requirements.     

6,6'-Bis(2-hydroxyphenyl)-2,2'-bipyridine manganese(III) complexes: a novel series of superoxide dismutase and catalase mimetics

A series of novel manganese(III) complexes is described based on a 6,6'-bis(2-hydroxyphenyl)-2,2'-bipyridine template. These complexes show superoxide dismutase and catalase activity. The effect of the aromatic substitution pattern on the SAR is described.     

Guanosine tetraphyosphate and its analogues. Chemical synthesis of guanosine 3',5'-dipyrophosphate, deoxyguanosine 3',5'-dipyrophosphate, guanosine 2',5'-bis(methylenediphosphonate), and guanosine 3',5'-bis(methylenediphosphonate).

A new procedure for the synthesis of the pyrophosphate bond has been employed in the preparation of nucleoside dipyrophosphates from nucleoside 3',5'-diphosphates. The method makes use of a powerful phosphorylating agent generated in a mixture of cyanoethyl phosphate, dicyclohexylcarbodiimide, and mesitylenesulfonyl chloride in order to avoid possible intramolecular reactions between the two phosphate groups on the sugar ring. That such reactions can readily occur was shown by the facile cyclization of deoxyguanosine 3',5'-diphosphate to P1,P2-deoxyguanosine 3',5'-cyclic pyrophosphate in the presence of dicyclohexylcarbodiimide alone. The phosphorylation reagent was initially tested in the conversion of deoxyguanosine 3',5'-diphosphate to the corresponding 3',5'-dipyrophosphate and was then used to phosphorylate 2'-O-(alpha-methoxyethyl)guanosine 3',5'-diphosphate, which had been prepared from 2'-O-(alpha-methoxyethyl)guanosine. In the latter case, the addition of the two beta phosphate groups was accomplished in 40% yield. Removal of the methoxyethyl group from the phosphorylated product gave guanosine 3',5'-dipyrophosphate, which was shown to be identical with guanosine tetraphosphate prepared enzymatically from a mixture of GDP and ATP. A modification of published procedures was also necessary to effect the synthesis of guanosine bis(methylenediphosphonate). Guanosine was treated with methylenediphosphonic acid and dicyclohexylcarbodiimide in the absence of added base. The product consisted of a mixture of guanosine 2',5' - and 3',5'-bis(methylenediphosphonate), which was resolved by anion-exchange chromatography. The 2',5' and 3',5' isomers are interconvertible at low pH, with the ultimate formation of an equilibrium mixture having a composition ratio of 2:3. The predominant constituent of this mixture has been unequivocally identified as the 3',5' isomer by synthesis from 2'-O-tetrahydropyranylguanosine.     

The perturbations of the native state of goat alpha-lactalbumin induced by 1,1'-bis(4-anilino-5-naphthalenesulfonate) are Ca2+-dependent.

In this work we have studied the interaction of the hydrophobic fluorescent probe 1,1'-bis(4-anilino-5-naphthalenesulfonate) (bis-ANS), with the native state of apo- and Ca2+-bound goat alpha-lactalbumin (GLA). In 10 mM Tris-HCl, pH 7.5, at 4 degrees C in 2 mM EGTA as well as at 37 degrees C in 2 mM Ca2+, the native protein is close to its thermal transition. Therefore, it can be expected that in both conditions the protein is equally susceptible to interaction with bis-ANS. Nevertheless, we have observed a number of interesting differences in the interaction of the dye with the apo and Ca2+ form. Native apo-GLA binds two bis-ANS molecules and in the complex with bis-ANS, the far-UV circular dichroism (CD) spectrum of apo-GLA becomes similar to that of the protein in the molten globule state. In contrast, native Ca2+-GLA binds five bis-ANS molecules and the far-UV CD spectrum of native Ca2+-GLA is conserved for the complex. In both cases, the high activation energies observed in kinetic experiments indicate that upon binding, large parts of the protein structure have to be reorganized. The reduced perturbation of the protein structure in the presence of Ca2+ can be attributed to local stabilization effects.     

Introduction of the carbohydrate-activated promoter P(malK) for recombinant protein production

A production protocol for the use of the malK promoter was established. The protocol includes two phases: an initial fed-batch phase on glucose to reach a high cell density and a fed-batch phase on maltose for production of the desired recombinant protein. It is suggested that this cultivation scheme could be used for all promoters that are catabolite repressed by glucose and where growth and production need to be separated. The specific feature of this system is shown by its ability to control the rate of synthesis of the product protein, ss-galactosidase. In the production phase with a constant feed or an exponential feeding of 0.1 h(-1) it took 4 h longer to reach the maximum specific production rate than with the higher dilution rates of 0.25 h(-1) and 0.4 h(-1), respectively. In the above experiments a dilution rate of 0.3 h(-1) in the growth phase was used. The volumetric production of this system could furthermore be extended to 40 h. All protocol procedures so far tested resulted in the same maximum production rate, but reached in different lengths of time. It is argued that this system is particularly well suited for the production of proteins that have a complex structure and/or need to be produced in a soluble form or to be exported to the periplasm.     

Synthesis and characterization of a photocleavable cross-linker and its application on tunable surface modification and protein photodelivery

A heterobifunctional photocleavable cross-linker based on an o-nitrobenzyl ester moiety was synthesized. The cross-linker has N-hydroxysuccinimidyl and disulfide groups attached at each end and thus can anchor a protein to a gold-coated substrate surface. Steady-state spectroscopic studies suggest that the cross-linker undergoes a clean C-O fragmentation upon irradiation with a quantum yield of 0.1. Consequently, immobilized proteins (such as avidin or antibodies) on a substrate surface can be released efficiently (>95%) under UV irradiation (lambda > 300 nm) without degrading the protein functionality. We also demonstrated protein delivery via bioconjugation of protein molecules to a gold-coated atomic-force microscope (AFM) tip. When the proteins are photoreleased from the AFM tip, they are delivered to the substrate surface as protein clusters of uniform size. This has been confirmed using both AFM and fluorescence microscopy. The application of bioconjugation in this study opens a new avenue for tunable surface modification and controllable protein delivery in studies of biological systems on the nanometer scale.     

Use of the heterobifunctional cross-linker m-maleimidobenzoyl N-hydroxysuccinimide ester to affinity label cholecystokinin binding proteins on rat pancreatic plasma membranes

The binding of 125I-cholecystokinin-33 (125I-CCK-33) to its receptors on rat pancreatic membranes was decreased by modification of membrane protein sulfhydryl groups. Sulfhydryl modifying reagents also caused an accelerated release of bound 125I-CCK-33 from its receptor. Because of the presence of an essential sulfhydryl group(s) in CCK receptor binding we studied the application of the heterobifunctional (SH,NH2) cross-linker, m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS), to affinity label 125I-CCK-33 binding proteins on rat pancreatic plasma membranes. Analysis of the cross-linked products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that this heterobifunctional cross-linker affinity labeled a major Mr = 80,000-95,000 protein previously identified as part of the CCK receptor on the basis of affinity labeling using homobifunctional and heterobifunctional photoreactive cross-linkers. Additional proteins of Mr greater than 200,000, and Mr = 130,000-140,000 were affinity labeled using MBS. The efficiency of the cross-linking reaction between 125I-CCK-33 and its membrane binding proteins with MBS was significantly greater than that obtained with NH2-directed homobifunctional reagents such as disuccinimidyl suberate. The efficiency of cross-linking could be dramatically improved by reduction of membrane proteins with low-molecular weight thiols prior to binding and cross-linking. The differential labeling patterns of the CCK binding proteins obtained with chemical cross-linkers of similar length but different chemical reactivity underscores the need for caution in predicting native receptor structure from affinity labeling data alone. Using the same pancreatic plasma membrane preparation and 125I-insulin, the Mr = 125,000 alpha-subunit of the insulin receptor was affinity labeled using MBS as cross-linker, demonstrating its utility in identifying other peptide hormone receptors.     

Quantitative analysis of the effects of photoswitchable distance constraints on the structure of a globular protein

Photoswitchable distance constraints in the form of photoisomerizable chemical cross-links offer a general approach to the design of reversibly photocontrolled proteins. To apply these effectively, however, one must have guidelines for the choice of cross-linker structure and cross-linker attachment sites. Here we investigate the effects of varying cross-linker structure on the photocontrol of folding of the Fyn SH3 domain, a well-studied model protein. We develop a theoretical framework based on an explicit-chain model of protein folding, modified to include detailed model linkers, that allows prediction of the effect of a given linker on the free energy of folding of a protein. Using this framework, we were able to quantitatively explain the experimental result that a longer, but somewhat flexible, cross-linker is less destabilizing to the folded state than a shorter more rigid cross-linker. The models also suggest how misfolded states may be generated by cross-linking, providing a rationale for altered dynamics seen in nuclear magnetic resonance analyses of these proteins. The theoretical framework is readily portable to any protein of known folded state structure and thus can be used to guide the design of photoswitchable proteins generally.     

In vitro synthesis and characterization of guanosine 3',5'-bis(diphosphate)

The intracellular alarmone guanosine 3′,5′-bis(diphosphate) (ppGpp) has been thoroughly investigated over the past 40 years and has become one of the best-known effectors in bacterial physiology. ppGpp is also of great importance for biotechnological applications. Systems biology research, involving experimental and mathematical approaches, has contributed a great deal to uncovering the alarmone’s complex regulatory effects. HPLC analysis and UV detection are used to quantify intracellular ppGpp. The samples analyzed also contain other phosphorylated guanine nucleotides and, therefore, are spiked with a standard ppGpp solution. A rapidly growing number of laboratories are turning to synthesizing the nucleotide in vitro involving time-consuming protocols and yielding only low amounts of ppGpp. The current article provides a protocol for the preparation of large quantities of a ribosome extract that contains high ppGpp synthesis activity. The demonstrated upscaling from shaking flask to bioreactor cultivation involves the continuous and refrigerated harvest of the biomass. ¹³C NMR analysis enabled the structural characterization of the synthesis product and was complemented by mass spectrometry and methods that are commonly used to identify ppGpp.