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At least 811 records · Page 45Linked to original sources
PubMed1986

Interaction of a spin-labeled phenylalanine analogue with normal and sickle hemoglobins: detection of site-specific interactions through spin-label-induced 1H NMR relaxation.

In a preliminary report, we have previously shown that N-[(2,2,5,5-tetramethyl-1-oxypyrrolidin-3-yl)carbonyl]-L-phenyl ala nine tert-butyl ester (SL-Phe) exhibits specific binding to hemoglobin and an antiaggregation activity more than 2 orders of magnitude greater than that of phenylalanine [Lu, H.-Z., Currie, B. L., & Johnson, M. E. (1984) FEBS Lett. 173, 259-263]. Transverse 1H NMR relaxation measurements have been used to investigate the interaction of SL-Phe with hemoglobin molecules by use of the resonances assigned to the C2 protons of the beta 2 His, the beta 143 His, and the beta 146 or beta 97 His residues as intrinsic probes. Distance calculations using the paramagnetically induced relaxation data suggest that the SL-Phe binding site is approximately 12-16 A away from the C2 protons of the beta 2 His and the beta 146 or beta 97 His residues in the (carbonmonoxy)hemoglobin tetramer; for deoxyhemoglobin, the distances are approximately 14-17 A between the SL-Phe binding site and the C2 protons of the beta 2 His, the beta 143 His, and the beta 146 His residues. Calculations using the (carbonmonoxy)hemoglobin crystal atomic coordinates only restrict the probable SL-Phe binding region to the full F and H helices of the beta-chain and a small section of the alpha-chain. For deoxyhemoglobin, the distance calculations provide greater restrictions on the probable binding region, limiting it to small sections of the beta-chain F, G, and H helices near the EF bend and to a few residues on the alpha-chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1988

Secondary enzyme-substrate interactions: kinetic evidence for ionic interactions between substrate side chains and the pepsin active site.

The possibility that pig pepsin has a cation binding specificity in its secondary binding subsites has been examined by the pepsin-catalyzed hydrolysis of a series of synthetic octa- to undecapeptide substrates. These chromophoric substrates are cleaved by pepsin in the phenylalanyl-p-nitrophenylalanyl (Phe-Nph) bond. Lys and Arg residues were placed into seven different positions in the substrates, and their effect on kcat and Km was examined between pH 2.8 and pH 5.8 (I = 0.1 M, 37 degrees C). Kinetic evidence indicates the existence in the enzyme binding subsites S4, S3, S2, S3', S4', and S5' of a group(s) which become(s) negatively charged at higher pH. For most substrates, the magnitude as well as the pH dependence of kcat was unaffected by the presence of Lys or Arg in these peptides. In contrast, changes up to 5 orders of magnitude were observed for Km, depending on the number of basic residues and on their positions in the sequence. Km for a group of substrates at pH greater than 5.5 was lower than 50 nM. Values for kcat/Km for some substrates exceed the level of 10(8) M-1 s-1. Therefore, the free energy derived from ionic interactions in secondary binding sites influences mostly the binding step on the reaction pathway. This result is in contrast to the previous observations that the length and the hydrophobic character of the substrate residues in some positions influence kcat with little effect on Km toward shorter substrates of pepsin [Fruton, J. (1976) Adv. Enzymol. Relat. Areas Mol. Biol. 44, 1-36].

Amino Acid Sequence↗
PubMed1996

Oligomycin sensitivity conferring protein of mitochondrial ATP synthase: deletions in the N-terminal end cause defects in interactions with F1, while deletions in the C-terminal end cause defects in interactions with F0.

The structure/function relationships of oligomycin sensitivity conferring protein (OSCP) of bovine mitochondrial ATP synthase were studied by nested deletion mutagenesis, followed by analyses of the resultant OSCPs for their ability to restore partial reactions of ATP synthesis in OSCP-depleted F1-F0 complexes. Our results indicate that, from the N-terminus of OSCP, up to 13 amino acid residues could be deleted without any effect on OSCP coupling activity. However, deletion of 16 or more residues led to a slow decline in the ability of resultant mutant forms to restore ATP synthesis. Compared to the wild-type form of OSCP, deletion mutant ND-28 (deletion of residues 1-28) is 50% as active in its ability to reconstitute ATP-Pi exchange activity. Detailed analyses of mutant ND-28 revealed that it was able to bind to the membrane segment (F0) of ATP synthase and restore oligomycin-sensitive ATPase activity in OSCP-depleted F1-F0 complexes. However, it did not bind to soluble segment F1, nor did it confer cold stability to either soluble F1 or reconstituted F1-F0 complex. On the other hand, studies on nested deletions on the C-terminal end indicate that three residues could be deleted without compromising the energy-coupling activity of OSCP. However, truncations of five or more residues caused an impairment in the ability of resultant mutant forms to restore ATP-Pi exchange activity in OSCP-depleted complexes. Mutant CD-10 (deletion of amino acids 181-190) was completely ineffective as a coupling factor. Detailed analyses of this mutant revealed that the subunit was able to bind to soluble F1 segment and confer cold stability to the enzyme but was neither able to associate with the membrane segment (F0) nor able to reconstitute high oligomycin sensitivity in depleted F1-F0 complexes. We take these data to suggest that the N-terminal end of OSCP corresponding to residues G16-N28 is essential for binding of the coupling factor to soluble F1 but not for coupling the energy of proton translocation to the synthesis of ATP; on the other hand, the carboxyl-terminal end of OSCP containing amino acids K181-M186 is important for F0-OSCP interactions as well as for the coupling of the energy of delta microH+ during the synthesis of ATP. These results suggest a model for OSCP in which the N-terminus is associated with the F1 segment and the C-terminus is associated with the F0 segment, while the central part of the polypeptide forms three or more helices constituting the stalk in the intact F1F0 enzyme.

Adenosine Triphosphatases↗
PubMed1997

Interactions of cyclins with cyclin-dependent kinases: a common interactive mechanism.

The formation of cdk-cyclin complexes has been investigated at the molecular level and quantified using spectroscopic approaches. In the absence of phosphorylation, cdk2, cdc2, and cdk7 form highly stable complexes with their "natural" cyclin partners with dissociation constants in the nanomolar range. In contrast, nonphosphorylated cdc2-cyclin H, cdk2-cyclin H, and cdk7-cyclin A complexes present a 25-fold lower stability. On the basis of both the structure of the cdk2-cyclin A complex and on our kinetic results, we suggest that interaction of any cyclin with any cdk involves the same hydrophobic contacts and induces a marked conformational change in the catalytic cleft of the cdks. Although cdks bind ATP strongly, they remain in a catalytically inactive conformation. In contrast, binding of the cyclin induces structural rearrangements which result in the selective reorientation of ATP, a concomitant 3-fold increase in its affinity, and a 5-fold decrease of its release from the active site of cdks.

Adenosine Triphosphate↗
PubMed1999

Replication protein A interactions with DNA. 2. Characterization of double-stranded DNA-binding/helix-destabilization activities and the role of the zinc-finger domain in DNA interactions.

Human replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein that is composed of subunits of 70, 32, and 14 kDa. RPA is required for multiple processes in cellular DNA metabolism. RPA has been reported to (1) bind with high affinity to single-stranded DNA (ssDNA), (2) bind specifically to certain double-stranded DNA (dsDNA) sequences, and (3) have DNA helix-destabilizing ("unwinding") activity. We have characterized both dsDNA binding and helix destabilization. The affinity of RPA for dsDNA was lower than that of ssDNA and precisely correlated with the melting temperature of the DNA fragment. The rates of helix destabilization and dsDNA binding were similar, and both were slow relative to the rate of binding ssDNA. We have previously mapped the regions required for ssDNA binding [Walther et al. (1999) Biochemistry 38, 3963-3973]. Here, we show that both helix-destabilization and dsDNA-binding activities map to the central DNA-binding domain of the 70-kDa subunit and that other domains of RPA are needed for optimal activity. We conclude that all types of RPA binding are manifestations of RPA ssDNA-binding activity and that dsDNA binding occurs when RPA destabilizes a region of dsDNA and binds to the resulting ssDNA. The 70-kDa subunit of all RPA homologues contains a highly conserved putative (C-X2-C-X13-C-X2-C) zinc finger. This motif directly interacts with DNA and contributes to dsDNA-binding/unwinding activity. Evidence is presented that a metal ion is required for the function of the zinc-finger motif.

DNA↗
PubMed1999

pKa values and the pH dependent stability of the N-terminal domain of L9 as probes of electrostatic interactions in the denatured state. Differentiation between local and nonlocal interactions.

pKa values were measured for the 6 carboxylates in the N-terminal domain of L9 (NTL9) by following NMR chemical shifts as a function of pH. The contribution of each carboxylate to the pH dependent stability of NTL9 was estimated by comparing the pKa values for the native and denatured state of the protein. A set of peptides with sequences derived from NTL9 were used to model the denatured state. In the protein fragments, the pKa values measured for the aspartates varied between 3.8 and 4.1 and the pKa values measured for the glutamates varied between 4.1 and 4.6. These results indicate that the local sequence can significantly influence pKa values in the denatured state and highlight the difficulties in using standard pKa values derived from small compounds. Calculations based on the measured pKa values suggest that the free energy of unfolding of NTL9 should decrease by 4.4 kcal mol-1 when the pH is lowered from 6 to 2. In contrast, urea and thermal denaturation experiments indicate that the stability of the protein decreases by only 2.6 kcal mol-1 when the carboxylates are protonated. This discrepancy indicates that the protein fragments are not a complete representation of the denatured state and that nonlocal sequence effects perturb the pKa's in the denatured state. Increasing the salt concentration from 100 to 750 mM NaCl removes the discrepancy between the stabilities derived from denaturation experiments and the stability changes calculated from the pKa values. At high concentrations of salt there is also less variation of the pKa values measured in the protein fragments. Our results argue that in the denatured state of NTL9 there are electrostatic interactions between groups both local and nonlocal in primary sequence.

Amino Acid Sequence↗
PubMed2007

Weak interaction induces an ON/OFF switch, whereas strong interaction causes gradual change: folding transition of a long duplex DNA chain by poly-L-lysine.

A large-scale conformational change in genomic DNA is an essential feature of gene activation in living cells. Considerable effort has been applied to explain the mechanism in terms of key-lock interaction between sequence-specific regulatory proteins and DNA, in addition to the modification of DNA and histones such as methylation and acetylation. However, it is still unclear whether these mechanisms can explain the ON/OFF switching of a large number of genes that accompanies differentiation, carcinogenesis, etc. In this study, using single-molecule observation of DNA molecules by fluorescence microscopy with the addition of poly-L-lysine with different numbers of monomer units (n = 3, 5, 9, and 92), we found that an ON/OFF discrete transition in the higher-order structure of long duplex DNA is induced by short poly-L-lysine, whereas a continuous gradual change is induced by long poly-L-lysine. On the other hand, polycations with a lower positive charge have less potential to induce DNA compaction. Such a drastic difference in the conformational transition of a giant DNA between short and large oligomers is discussed in relation to the mechanisms of gene regulation in a living cell.

Biophysics↗
PubMed2002

The laser-induced blue state of bacteriorhodopsin: mechanistic and color regulatory roles of protein-protein interactions, protein-lipid interactions, and metal ions.

In this paper we characterize the mechanistic roles of the crystalline purple membrane (PM) lattice, the earliest bacteriorhodopsin (BR) photocycle intermediates, and divalent cations in the conversion of PM to laser-induced blue membrane (LIBM; lambda(max)= 605 nm) upon irradiation with intense 532 nm pulses by contrasting the photoconversion of PM with that of monomeric BR solubilized in reduced Triton X-100 detergent. Monomeric BR forms a previously unreported colorless monomer photoproduct which lacks a chromophore band in the visible region but manifests a new band centered near 360 nm similar to the 360 nm band in LIBM. The 360 nm band in both LIBM and colorless monomer originates from a Schiff base-reduced retinyl chromophore which remains covalently linked to bacterioopsin. Both the PM-->LIBM and monomer-->colorless monomer photoconversions are mediated by similar biphotonic mechanisms, indicating that the photochemistry is localized within single BR monomers and is not influenced by BR-BR interactions. The excessively large two-photon absorptivities (> or =10(6) cm(4) s molecule(-1) photon(-1)) of these photoconversions, the temporal and spectral characteristics of pulses which generate LIBM in high yield, and an action spectrum for the PM-->LIBM photoconversion all indicate that the PM-->LIBM and Mon-->CMon photoconversions are both mediated by a sequential biphotonic mechanism in which is the intermediate which absorbs the second photon. The purple-->blue color change results from subsequent conformational perturbations of the PM lattice which induce the removal of Ca(2+) and Mg(2+) ions from the PM surface.

Bacteriorhodopsins↗
PubMed2001

Assessing the balance between protein-protein interactions and enzyme-substrate interactions in the channeling of intermediates between polyketide synthase modules.

6-Deoxyerythronolide B synthase (DEBS) is the modular polyketide synthase (PKS) that catalyzes the biosynthesis of 6-deoxyerythronolide B (6-dEB), the aglycon precursor of the antibiotic erythromycin. The biosynthesis of 6-dEB exemplifies the extraordinary substrate- and stereo-selectivity of this family of multifunctional enzymes. Paradoxically, DEBS has been shown to be an attractive scaffold for combinatorial biosynthesis, indicating that its constituent modules are also very tolerant of unnatural substrates. By interrogating individual modules of DEBS with a panel of diketides activated as N-acetylcysteamine (NAC) thioesters, it was recently shown that individual modules have a marked ability to discriminate among certain diastereomeric diketides. However, since free NAC thioesters were used as substrates in these studies, the modules were primed by a diffusive process, which precluded involvement of the covalent, substrate-channeling mechanism by which enzyme-bound intermediates are directly transferred from one module to the next in a multimodular PKS. Recent evidence pointing to a pivotal role for protein-protein interactions in the substrate-channeling mechanism has prompted us to develop novel assays to reassess the steady-state kinetic parameters of individual DEBS modules when primed in a more "natural" channeling mode by the same panel of diketide substrates used earlier. Here we describe these assays and use them to quantify the kinetic benefit of linker-mediated substrate channeling in a modular PKS. This benefit can be substantial, especially for intrinsically poor substrates. Examples are presented where the k(cat) of a module for a given diketide substrate increases >100-fold when the substrate is presented to the module in a channeling mode as opposed to a diffusive mode. However, the substrate specificity profiles for individual modules are conserved regardless of the mode of presentation. By highlighting how substrate channeling can allow PKS modules to effectively accept and process intrinsically poor substrates, these studies provide a rational basis for examining the enormous untapped potential for combinatorial biosynthesis via module rearrangement.

Acyl Carrier Protein↗
PubMed2006

Computational study of matrix-peptide interactions in MALDI mass spectrometry: Interactions of 2,5- and 3,5-dihydroxybenzoic acid with the tripeptide valine-proline-leucine.

The mechanism of matrix-to-analyte proton transfer in matrix-assisted laser desorption and ionization mass spectrometry (MALDI-MS) has been investigated computationally by modeling the matrix-analyte interaction of potential MALDI matrixes such as 2,5-dihydroxybenzoic acid (2,5-DHB) and 3,5-DHB with the tripeptide valine-proline-leucine (VPL). A combination of molecular dynamics/simulated annealing calculations followed by density functional theory geometry optimization using a reasonably large basis set has been done on a large number of clusters in an attempt to study the ionization energy of each matrix in the cluster environment and the intracluster proton transfer from the matrix to the tripeptide. The calculations show a substantial reduction in the IP for both matrixes in their cluster environments. In the 2,5-system, proton transfer can sometimes occur in the neutral clusters (preformed ions), whereas proton transfer in the cationic clusters, which is actually a double proton transfer, is spontaneous and exoergic. Even though it is more acidic from a thermodynamic perspective, the radical cation of 3,5-DHB is a less efficient proton donor to VPL. The thermodynamics of proton transfer in the cationic clusters is discussed in detail.

Computational Biology↗
PubMed2006

Arene-cation interactions of positive quadrupole moment aromatics and arene-anion interactions of negative quadrupole moment aromatics.

Intermolecular interactions involving aromatic pi-electron density are widely believed to be governed by the aromatic molecular quadrupole moment, Theta(zz). Arene-cation binding is believed to occur primarily with negative Theta(zz) aromatics, and arene-anion binding is believed to occur largely with positive Theta(zz) aromatics. We have performed quantum mechanical computations that show the cation binding of positive Theta(zz) aromatics and the anion binding of negative Theta(zz) aromatics is quite common in the gas phase. The pi-electron density of hexafluorobenzene, the prototypical positive Theta(zz ) aromatic (experimental Theta(zz) = 9.5 +/- 0.5 DA), has a Li+ binding enthalpy of -4.37 kcal/mol at the MP2(full)/6-311G**level of theory. The RHF/6-311G** calculated Theta(zz) value of 1,4-dicyanobenzene is +11.81 DA, yet it has an MP2(full)/6-311G** Li+ binding enthalpy of -12.65 kcal/mol and a Na+ binding enthalpy of -3.72 kcal/mol. The pi-electron density of benzene, the prototypical negative Theta(zz) aromatic (experimental Theta(zz) = -8.7 +/- 0.5 DA), has a F- binding enthalpy of -5.51 kcal/mol. The RHF/6-311G** calculated Theta(zz) of C6H2I4 is -10.45 DA, yet it has an MP2(full)/6-311++G** calculated F- binding enthalpy of -20.13 kcal/mol. Our results show that as the aromatic Theta(zz) value increases the cation binding enthalpy decreases; a plot of cation binding enthalpies versus aromatic Theta(zz) gives a line of best of fit with R2 = 0.778. No such correlation exists between the aromatic Theta(zz) value and the anion binding enthalpy; the line of best fit has R2 = 0.297. These results are discussed in terms of electrostatic and polarizability contributions to the overall binding enthalpies.

Benzene↗
PubMed2007

Dynamics of interactions involving deformable drops: hydrodynamic dimpling under attractive and repulsive electrical double layer interactions.

A model developed previously to analyze force measurements between two deformable droplets in the atomic force microscope [Langmuir 2005, 21, 2912-2922] is used to model the drainage of an aqueous film between a mica plate and a deformable mercury drop for both repulsive and attractive electrical double-layer interactions between the mica and the mercury. The predictions of the model are compared with previously published data [Faraday Discuss. 2003, 123, 193-206] on the evolution of the aqueous film whose thickness has been measured with subnanometer precision. Excellent agreement is found between theoretical results and experimental data. This supports the assumptions made in the model which include no-slip boundary conditions at both interfaces. Furthermore, the successful fit attests to the utility of the model as a tool to explore details of the drainage mechanisms of nanometer-thick films in which fluid flow, surface deformations, and colloidal forces are all involved. One interesting result is that the model can predict the time at which the aqueous film collapses when attractive mica-mercury forces are present without the need to invoke capillary waves or other local instabilities of the mercury/electrolyte interface.

Aluminum Silicates↗
PubMed2002

Particle interactions involved in aerosol dispersion of ternary interactive mixtures.

PURPOSE: To investigate the mechanism of action of ternary components within dry powder aerosols. METHODS: Ternary interactive mixtures were prepared containing salbutamol sulphate (SS), coarse lactose carriers and either micronized lactose (ML) or micronized glucose (MG). In vitro drug and excipient aerosol deposition was performed using a twin-stage impinger (TSI) at 60 L/min with a Rotahaler device. Adhesional properties of the lactose carrier were examined using an atomic force microscope (AFM) colloidal probe technique. RESULT: The fine particle fraction (FPF) from ternary mixtures were dependent upon carrier type (p < 0.001), ternary concentration (p < 0.001) and ternary component type (p < 0.05). Ternary mixtures produced higher FPF than binary mixtures, except those containing Superfine (SF), which was attributed to the high proportion of intrinsic fine carrier particles. The higher FPF obtained from ternary mixtures was independent of the mixing order (p = 0.08). Increased adhesion force was observed on the carrier surface following the addition of ternary components (p < 0.001). CONCLUSION: The results confirm that ternary components increase aerosol deposition of powder mixtures. Some results were not entirely consistent with the saturation of active site theory and a hypothesis involving competitive and multilayer adhesion was proposed and requires further testing.

Aerosols↗
PubMedDate not supplied

[Interaction of alpha-chymotrypsin with dimethylsulfoxide: a change of substrate could change the mechanism of interaction].

The kinetic behavior of alpha-chymotrypsin was studied in water-DMSO mixtures at concentrations of the organic solvent that do not cause irreversible denaturation of the enzyme. Various substrates (N-substituted derivatives of L-tyrosine) were found to display substantially different kinetic patterns of interaction with alpha-chymotrypsin, which can be described by totally different kinetic schemes. The differences were ascribed to competition between the N-acyl group of the substrate and the DMSO molecule at the S2-site of substrate binding to the active site of the enzyme.

Chymotrypsin↗
PubMed2006

Direct observation of adsorbed H2-framework interactions in the Prussian Blue analogue MnII3[CoIII(CN)6]2: the relative importance of accessible coordination sites and van der Waals interactions.

Selective recovery of the guest-framework interactions for H(2) adsorbed in a nanoporous Prussian Blue analogue, through differential X-ray and neutron pair distribution function analysis at ca. 77 K, suggests that the H(2) molecule is disordered about a single position at the centre of the pore, ((1/4),(1/4),(1/4)), without binding at accessible Mn(II) sites.

Journal Article↗
PubMed1970

Protein-hydrocarbon interactions. Interactions of various proteins with decane in the presence of alcohols.

1. Solutions of proteins were subjected to gentle agitation in the presence of small quantities of decane containing different alcohols. 2. Some of the protein was lost from solution and adsorbed on the surface of the emulsion formed; at the same time some decane was bound to the protein remaining in solution. 3. Comparison of these results with those obtained with pure decane suggests that a mixed film of protein+alcohol is formed on the surface of the emulsion. 4. If the concentration of alcohol in decane is increased the amount of protein adsorbed on the emulsion is decreased. This phenomenon was used to compare the effect of different alcohols in disrupting the hydrophobic interactions between proteins and hydrocarbons.

Adsorption↗
PubMed2002

Modulation of protein-protein interactions by synthetic receptors: design of molecules that disrupt serine protease-proteinaceous inhibitor interaction.

In the present article we describe the design and evaluation of a synthetic receptor that binds to the exterior surface of chymotrypsin and disrupts its interaction with proteinaceous inhibitors, such as soybean trypsin inhibitor, basic pancreatic trypsin inhibitor, ovomucoid turkey inhibitor, and Bowman-Birk inhibitor. Using enzyme kinetics, nondenaturing gel electrophoresis, and gel filtration chromatography we show that the receptor is particularly effective at blocking the chymotrypsin-soybean trypsin inhibitor complex and that the mechanism involves formation of an initial ternary complex followed by a time-dependent displacement of the proteinaceous inhibitor.

Chromatography, Gel↗
PubMed1973

Cell interactions between histoincompatible T and B lymphocytes. The H-2 gene complex determines successful physiologic lymphocyte interactions.

We used congenic-resistant mouse strains to answer questions concerning the respective roles of genes coding for major histocompatibility and background genotypes in T (thymus-derived)-B (bone marrow-derived) lymphocyte cooperative responses to hapten-protein conjugates. These studies demonstrate conclusively that the gene or genes present in the H-2 complex control the capacity of antigen-specific T and B cells to effectively interact. These findings led us to postulate that there exists on the B-lymphocyte surface an "acceptor" molecule for the active T-cell product or for the T cell itself.

Animals↗