Biomedical subjects

M S Lewis

Publications and source records attributed to M S Lewis.

At least 37 records · Page 2Linked to original sources

PubMed1998

Structure-function relations of heparin-mimetic sulfated xylan oligosaccharides: inhibition of human immunodeficiency virus-1 infectivity in vitro.

Heparins/heparan sulfates modulate the function of proteins and cell membranes in numerous biological systems including normal and disease processes in humans. Heparin has been used for many years as an anticoagulant, and anticoagulant heparin-mimetics were developed several decades ago by chemical sulfation of non-mammalian polysaccharides, e.g., an antithrombotic sulfated xylan. This pharmaceutical, which comprises a mixture of sulfated oligoxylans, also mimics most other biological actions of natural heparins in vitro, including inhibition of the human immunodeficiency virus, but the molecular basis for these actions has been unclear. Here, numerous Components of the sulfated oligoxylan mixture were isolated and when bioassayed in the case of anti-HIV-1 infectivity revealed that a structural specificity underlines the capacity of sulfated xylan to inhibit HIV-1, rather than a non-specific mechanism. Components were isolated by chromatographic fractionation through Bio-Gel P10 in 0.5 M ammonium bicarbonate. This fractionation revealed an elution range associated with apparent molecular weights of approximately 22000 to <1500 relative to standard heparin and heparan sulfates and newly prepared sulfated oligosaccharide standards. Components were characterized by metachromatic absorption spectroscopy, ultracentrifugation, GlcA analysis, and potency against HIV-1 infectivity, both in the tetrazolium cytotoxicity assay and in syncytium-forming assays, in CD4-lymphocytes. Structural specificity was indicated by the differential potencies exhibited by the Components: Highest activity (cytotoxicity) was exhibited by Components in the chromatographic region > or = approximately 5500 in mass (50% effective (inhibitory) concentration = 0.5-0.7 microg ml(-1) in the first fractionation series, and 0.1-0.5 microg ml(-1) in a second series). The potency declined sharply below approximately 5400 in mass, but with an exception; a second structure exhibiting relatively high potency eluted among low-mass oligosaccharides which had an average size of approximately a nonomer. Components displayed differential potencies also against the syncytium-forming infectivity of HIV-1. The high potency against syncytium-formation was retained by Components down to a minimum size of about 4500 in mass, smaller than the > or = approximately 5400 required above. One in ten of the beta1,4-linked xyloses in the native xylan are substituted with a monomeric alpha1,2 DGlcA branch. We have speculated that pharmaceutical actions of sulfated xylan might be related to structures involving the alpha-D linked substituents and this was examined using a space-filling model of a sulfated octaxylan and by analyses of Components for GlcA content. Understanding structure/function relations in the heparin-like actions of these agents would be of general significance for the careful examination of their potential clinical usefulness in many human processes modulated by heparins, including AIDS.

Anti-HIV Agents↗

PubMed1997

High affinity binding and allosteric regulation of Escherichia coli glycogen phosphorylase by the histidine phosphocarrier protein, HPr.

The histidine phosphocarrier protein (HPr) is an essential element in sugar transport by the bacterial phosphoenolpyruvate:sugar phosphotransferase system. Ligand fishing, using surface plasmon resonance, was used to show the binding of HPr to a nonphosphotransferase protein in extracts of Escherichia coli; the protein was subsequently identified as glycogen phosphorylase (GP). The high affinity (association constant approximately 10(8) M-1), species-specific interaction was also demonstrated in electrophoretic mobility shift experiments by polyacrylamide gel electrophoresis. Equilibrium ultracentrifugation analysis indicates that HPr allosterically regulates the oligomeric state of glycogen phosphorylase. HPr binding increases GP activity to 250% of the level in control assays. Kinetic analysis of coupled enzyme assays shows that the binding of HPr to GP causes a decrease in the Km for glycogen and an increase in the Vmax for phosphate, indicating a mixed type activation. The stimulatory effect of E. coli HPr on E. coli GP activity is species-specific, and the unphosphorylated form of HPr activates GP more than does the phosphorylated form. Replacement of specific amino acids in HPr results in reduced GP activation; HPr residues Arg-17, Lys-24, Lys-27, Lys-40, Ser-46, Gln-51, and Lys-72 were established to be important. This novel mechanism for the regulation of GP provides the first evidence directly linking E. coli HPr to the regulation of carbohydrate metabolism.

Allosteric Regulation↗

PubMed1997

Phosphorylation of serine 392 stabilizes the tetramer formation of tumor suppressor protein p53.

Tumor suppressor protein p53 is a tetrameric phosphoprotein that activates transcription from several cell cycle regulating genes in response to DNA damage. Tetramer formation is critical to p53's ability to activate transcription; however, posttranslational modifications and protein stabilization also contribute to p53's ability to activate transcription. To determine if phosphorylation affects tetramer formation, we synthesized phosphopeptides corresponding to residues 303-393 of human p53, which includes the domain responsible for tetramer formation. Phosphate was chemically incorporated at Ser315, Ser378, or Ser392 and also at both Ser315 and Ser392. Equilibrium ultracentrifugal analyses showed that phosphorylation at Ser392 increased the association constant for reversible tetramer formation nearly 10-fold. Phosphorylation of either Ser315 or Ser378 had little effect on tetramer formation, but phosphorylation of Ser315 largely reversed the effect of phosphorylation at Ser392. Analyses by calorimetry demonstrated that phosphorylation may influence subunit affinity (and, in turn, DNA binding) by an enthalpy-driven process, possibly between the C-terminal residues and the region immediately adjacent to Ser315. The Kd for the tetramer-monomer transition of the unphosphorylated p53 C-terminal domain was determined to be approximately 1-10 microM. Thus, in normal, undamaged cells p53 may be largely monomeric. Enhancement of tetramer formation through phosphorylation of Ser392, coupled with a DNA-damage-induced increase in its nuclear concentration, could provide a switch that activates p53 as a transcription factor in response to DNA damage.

Biopolymers↗

PubMed1997

Zn2+ promotes the self-association of human immunodeficiency virus type-1 integrase in vitro.

It has been recently demonstrated that the Mg(2+)-dependent 3'-processing activity of purified human immunodeficiency virus type-1 (HIV-1) integrase is stimulated by the addition of exogenous Zn2+ [Lee, S. P., & Han, M. K. (1996) Biochemistry 35, 3837-3844]. This activation was hypothesized to result from integrase self-association. In this report, we examine the Zn2+ content of purified HIV-1 integrase by atomic absorption spectroscopy and by application of a thiol modification reagent, p-(hydroxymercuri)benzenesulfonate, with a metallochromic indicator, 4-(2-pyridylazo)resorcinol. We find that the Zn2+ content of HIV-1 integrase varies from 0.1 to 0.92 equiv of Zn2+ per monomer depending on the conditions of protein purification. In vitro activity assays, time-resolved fluorescence emission anisotropy, and gel filtration chromatographic analyses all indicate that EDTA yields an apoprotein which is predominantly monomeric and less active with Mg2+. Further, sedimentation equilibrium studies reveal that reconstitution of the apoprotein with Zn2+ results in a monomer-tetramer-octamer transition. These results suggest that Zn2+ promotes a conformation with enhanced oligomerization and thereby stimulates Mg(2+)-dependent 3'-processing. This may also imply that multimers larger than dimers (tetramers and possibly octamers) are required for in vitro activity of integrase in the presence of Zn2+ and Mg2+. It should be noted, however, that the content of Zn2+ did not significantly affect the 3'-processing and strand transfer reactions with Mn2+ in vitro.

Chromatography, Gel↗

PubMed1997

Effect of phosphorylation on tetramerization of the tumor suppressor protein p53.

Human tumor suppressor protein p53 is a 393-amino acid phosphoprotein that enhances transcription in response to DNA damage from several genes that regulate cell cycle progression. The tetrameric state of p53 is critical to wild-type function; the p53 tetramerization element is located in the C-terminal region of the protein. This region is phosphorylated at several evolutionarily conserved serines, suggesting that phosphorylation may be an important regulator of p53 function. In order to determine the effect of phosphorylation on tetramer formation, we synthesized phosphopeptides corresponding to p53(Ser303-Asp393) with phosphate incorporated at Ser315, Ser378, or Ser392, and at both Ser315 and Ser392. Equilibrium ultracentrifugation analysis showed that phosphorylation at Ser392 increased the association constant for tetramer formation nearly ten-fold. By itself, phosphorylation at Ser315 or Ser378 had little effect on tetramer formation, but Ser315 largely reversed the effect of phosphorylation at Ser392. Analysis by calorimetry suggests that phosphorylation may influence subunit affinity by an enthalpy driven process.

Amino Acid Sequence↗

PubMed1996

A synthetic conformational epitope from the C4 domain of HIV Gp120 that binds CD4.

The fourth conserved domain of the human immunodeficiency virus type 1 (HIV-1) envelope, the C4 region of glycoprotein 120 (gp120), is believed to be a major part of gp120 that is necessary for binding to CD4. Recently, we found that C4 in gp120 is probably an alpha-helix, because antibodies made against helical constructs of C4 react with native and recombinant gp120 but antibodies against linear C4 constructs do not. For the present study, we performed experiments to determine, first, if CD4 could bind to the helical C4 constructs and, second, if the binding was comparable with CD4 binding to gp120. Immobilized helical constructs derived from the C4s from HIV-1 and HIV-2 bound biotinylated recombinant CD4 with Kd values of 8.59 nM and 14.59 nM, respectively. Recombinant soluble CD4 inhibited the binding of biotinylated CD4 to the C4 construct from HIV-1 with a Kd of 9.88 nM, and recombinant gp120 blocked the binding of CD4 to the immobilized helical construct from C4 of HIV-1 with a Kd of 8.08 nM. The C4 peptide-(419-436) from HIV-1 (KIKQIINMWQEVGKAMYA-NH2) blocked CD4 binding to gp120 but only in a buffer containing 0.03% Brij 35 where the peptide displayed 17 +/- 1% alpha-helix; without the Brij 35, peptide-(419-436) displayed no helical content. The Kd for the peptide-(419-436) blocking CD4 binding to gp120 in Brij 35-containing buffer was found to be 42 microM. These results indicate that C4 constructs from HIV-1 and HIV-2 do bind CD4, but the constructs must display an alpha-helical conformation to do so. In addition, the results reported here will provide answers to key questions about structural requirements for HIV vaccines and therapeutics that hinge on understanding the molecular nature of the gp120-CD4 interaction as the first step in the HIV infection process.

Amino Acid Sequence↗

PubMed1996

Effects of pH and Ca2+ on heterodimer and heterotetramer formation by chromogranin A and chromogranin B.

The two major proteins of the secretory vesicles of neuroendocrine cells, chromogranin A (CGA) and chromogranin B (CGB), have been shown to undergo pH- and Ca2+-dependent conformational changes and aggregation and have been suggested to play essential roles during secretory vesicle biogenesis in the trans-Golgi network. CGA has been shown to exist primarily in a tetrameric state at pH 5.5 and primarily in a dimeric state at pH 7.5, and CGB has been shown to exist in a monomeric state at both pH 5.5 and pH 7.5. Using purified CGA and CGB, it recently has been shown that CGA interacts with CGB at pH 5.5 (Yoo, S. H.(1996) J. Biol. Chem. 271, 1558-1565). In expanding this investigation, we have studied the temperature dependence of the pH-dependent interaction of CGA and CGB by analytical ultracentrifugation and found that two molecules of CGA bound to two molecules of CGB at pH 5.5 with DeltaG0 values of -43.6 kcal/mol in the absence of Ca2+ at 37 degrees C and -40.3 kcal/mol in the presence of 0.1 mM Ca2+. However, one molecule of CGA bound to one molecule of CGB at pH 7.5 with DeltaG0 values of -13.6 kcal/mol in the absence of Ca2+ at 37 degrees C. The magnitude of DeltaG0 values increased with increasing temperatures at both pH values. However, the values for enthalpy and entropy changes decreased with increasing temperatures in both pH levels, suggesting formation of more ordered structures. In the absence of Ca2+ at pH 5. 5, the heterotetramerization reaction at 37 degrees C was entropically driven, whereas in the presence of Ca2+ (0.1 mM) the heterotetramerization was virtually an enthalpic reaction. On the other hand, the heterodimer formation in the absence of Ca2+ at pH 7. 5 showed large negative enthalpy and entropy changes at 37 degrees C, indicating an enthalpic interaction compensated by entropic changes. In view of the interaction of tetrameric CGA with tetrameric inositol 1,4,5-trisphosphate (IP3) receptor and the existence of heterotetrameric IP3 receptor in the cell, the heterotetramer formation by CGA and CGB not only raises the possibility of interaction between the heterotetrameric chromogranin and heterotetrameric IP3 receptor but also appears to reflect their important roles in the cell.

Adrenal Medulla↗

PubMed1996

Biophysical characterization of a recombinant soluble interleukin 2 receptor (Tac). Evidence for a monomeric structure.

The interleukin 2 receptor (IL2R) plays a prominent role in the biology of T cells, B cells, and NK cells during activation. Of the three chains described, the alpha-chain of the receptor (Tac; IL2R alpha; CD25) is the most subject to regulation and is shed from the surface of activated cells to generate a soluble form in serum and tissues. Conflicting results have been reported on the native structure of soluble Tac, suggesting variously a monomer, a dimer, or higher noncovalent forms, spawning different models for its mechanism of action. We similarly show a large M(r)(app) by HPLC sieving chromatography, suggesting a tetrameric form. However, stoichiometry-ordered size (SOS) analysis of antibody-antigen complexes indicated only a single epitope per Tac molecule, compatible with a monomeric form. This larger M(r)(app) also conflicted with prior in vivo data showing rapid filtration of soluble Tac through the renal glomerulus that was not expected of a larger complex. Using different solvents, denaturants, and columns in the chromatography suggested that the elevated M(r)(app) values were an artifact of solute-column interactions, termed "ionic exclusion", rather than reflecting larger native structures. Analytical ultracentrifugation using a new type of analysis specific to glycoproteins demonstrated monomeric masses under all salt conditions with no tendency to form dimers or higher aggregates. Finally, circular dichroism spectroscopy showed no salt-dependent changes to suggest conformational alterations that might correlate with mobility changes on high pressure liquid chromatography. We conclude therefore that Tac is monomeric under physiologic conditions. Assessments of higher molecular weight for the purified soluble protein by other methods may be explained by the highly acidic nature of the molecule, which hampers matrix penetration with chromatographic media and by the high carbohydrate content and low partial specific volumes that accelerate the molecule in sedimentation media relative to pure protein standards.

Chromatography, Gel↗

PubMed1996

Dissociation kinetics of RepA dimers: implications for mechanisms of activation of DNA binding by chaperones.

BACKGROUND: The replication initiator of plasmid P1, RepA, binds DNA as monomer. The binding is stimulated by the chaperones DnaJ, DnaK and GrpE of Escherichia coli. Two models of chaperone action have been proposed. (i) Chaperones dissociate RepA dimers, which are inactive in DNA binding, into active monomers. (ii) The dissociation occurs spontaneously but the monomeric products require the chaperones for refolding into the active form. The latter model was based on the observation that RepA diluted 1000-fold below the K(D) for dimer dissociation, still required the chaperones for DNA binding. RESULTS: We have confirmed that under the condition of DNA binding experiments, the RepA dimers dissociate reversibly into monomers with a K(D) value of 1.1 +/- 0.1 microM. In the vicinity of this concentration, the sedimentation coefficient of RepA was concentration dependent, allowing estimation of s(20,w) coefficients for the RepA monomer (2.95 S) and dimer (4.01 S). Dynamic light scattering experiments indicated an increase of the monomer fraction within 5 min of RepA dilution. Circular dichroism (CD) measurements were consistent with these results. CONCLUSION: RepA monomerization is efficient without the mediation of chaperones. They are required to activate RepA most likely because they are needed to re-fold RepA monomers.

Circular Dichroism↗

PubMed1996

Chemical synthesis of phosphorylated peptides of the carboxy-terminal domain of human p53 by a segment condensation method.

A segment condensation method was developed for the chemical synthesis of large (> 90 amino acid) phosphopeptides and was used to produce phosphorylated and non-phosphorylated derivatives of the C-terminal tetramerization and regulatory domains of human p53 (residues 303-393). Efficient condensation synthesis of the 91 residue p53 domain was achieved in two steps. The non-phosphorylated N-terminal segment p53(303-334) (1) and its derivative phosphorylated at serine 315 (1P315), and the non-phosphorylated middle segment p53(335-360) (2), were synthesized as partially protected peptide thioesters in the solid phase using Boc chemistry. The C-terminal segment p53(361-393) (3) and its derivative phosphorylated at serine 392 (3P392) were synthesized as partially protected peptides in the solid phase using Fmoc chemistry. Phosphoamino acid was incorporated into the N-terminal segment (1P315) at the residue corresponding to p53 serine 315 as Boc-Ser(PO3(Bzl)2)-OH during synthesis. Serine 392 in the C-terminal segment was selectively phosphorylated after synthesis by phosphitylation followed by oxidation. A derivative phosphorylated at serine 378 was synthesized in a one-step condensation of the unphosphorylated N-terminal segment (1) and the phosphorylated long C-terminal segment p53(335-393) (2-3P378). Yields of the ligated peptides after removal of the protecting groups and HPLC purification averaged 60% for the first condensation and 35% for the second condensation. All five p53 peptides exhibited monomer-tetramer association as determined by analytical ultracentrifugation. Circular dichroism spectroscopy revealed that phosphorylation at Ser315 increased the alpha-helical content, which was abolished when Ser392 also was phosphorylated, suggesting an interaction between N-terminal and C-terminal residues of the C-terminal domain of p53.

Amino Acid Sequence↗

PubMed1995

Stoichiometry and thermodynamics of the interaction between the Fc fragment of human IgG1 and its low-affinity receptor Fc gamma RIII.

IgG-Fc receptors, cell surface glycoproteins binding the Fc region of antibodies, play a crucial role in the immune system. To better understand the nature of the recognition process, we have examined the interaction between huIgG1-Fc and a soluble fragment of huFc gamma RIII (sCD16). Analytical ultracentrifugation experiments clearly demonstrate that IgG1-Fc and sCD16 interact weakly to form a 1:1 complex with an association constant of 1.7 x 10(5) M-1 in PBS at 22.0 degrees C. The thermodynamic parameters, obtained from the temperature dependence of the equilibrium binding constants, exhibit an enthalpy-entropy compensation with a favorable enthalpy at physiological temperatures. The value of -360 cal mol-1 K-1 for delta Cp zero possibly identifies the process as one in which local folding/rearrangement is coupled to complex formation. The 1:1 stoichiometry and thermodynamic parameters provide a basis for understanding the nature of the Fc gamma R-IgG interactions.

Amino Acid Sequence↗

PubMed1995

Four p53 DNA-binding domain peptides bind natural p53-response elements and bend the DNA.

Recent structural studies of the minimal core DNA-binding domain of p53 (p53DBD) complexed to a single consensus pentamer sequence and of the isolated p53 tetramerization domain have provided valuable insights into their functions, but many questions about their interacting roles and synergism remain unanswered. To better understand these relationships, we have examined the binding of the p53DBD to two biologically important full-response elements (the WAF1 and ribosomal gene cluster sites) by using DNA circularization and analytical ultracentrifugation. We show that the p53DBD binds DNA strongly and cooperatively with p53DBD to DNA binding stoichiometries of 4:1. For the WAF1 element, the mean apparent Kd is (8.3 +/- 1.4) x 10(-8) M, and no intermediate species of lower stoichiometries can be detected. We show further that complex formation induces an axial bend of at least 60 degrees in both response elements. These results, taken collectively, demonstrate that p53DBD possesses the ability to direct the formation of a tight nucleoprotein complex having the same 4:1 DNA-binding stoichiometry as wild-type p53 which is accompanied by a substantial conformational change in the response-element DNA. This suggests that the p53DBD may play a role in the tetramerization function of p53. A possible role in this regard is proposed.

Base Sequence↗

PubMed1995

Thermodynamic study of the pH-dependent interaction of chromogranin A with an intraluminal loop peptide of the inositol 1,4,5-trisphosphate receptor.

The secretory vesicles of adrenal chromaffin cells have previously been identified as a major inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store, and their Ca2+ store role has been attributed to the presence of chromogranin A, a high capacity, low affinity Ca2+ binding protein. Chromogranin A has since been shown to exist primarily in a dimeric state at pH 7.5 and primarily in a tetrameric state at the intravesicular pH of 5.5 and has also been shown to interact with the membrane proteins of secretory vesicles at pH 5.5, including a 260-kDa protein reactive to IP3 receptor antibody [Yoo, S. H. (1994) J. Biol. Chem. 269, 12001-12006]. In a recent study, chromogranin A was shown to interact with one of the intraluminal loop regions of the IP3 receptor at pH 5.5 but not at pH 7.5 [Yoo, S. H., & Lewis, M. S. (1994) FEBS Lett. 341, 28-32]. To gain further insight, we have studied the temperature dependence of the pH-dependent interaction of chromogranin A with the intraluminal peptide of the the IP3 receptor by analytical ultracentrifugation, using multiwavelength scan analysis, and found that four molecules of the intraluminal domain peptide of the IP3 receptor bound to each chromogranin A tetramer with delta Go values ranging from -23.6 to -27.6 kcal mol-1 in the absence and presence of 35 mN Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Late pulmonary sequela following burns: persistence of hyperprocalcitonemia using a 1-57 amino acid N-terminal flanking peptide assay.

Seven patients were evaluated at a mean duration of 8.4 yr after sustaining inhalational injury associated with burns. At the time of re-examination, the patients were asymptomatic and had normal chest X-rays, and arterial blood gases. Three of the seven patients had abnormally elevated serum calcitonin levels. The spirometry (FEV1) measurements showed an inverse trend to that of the serum calcitonin levels. The elevated calcitonin levels had an abnormal predominance of the procalcitonin component as assessed by several region specific antisera. The serum calcitonin also showed a significant correlation with the hormone level which had been obtained at the time of prior discharge from the hospital (r = 0.91). Although there appears to be no or minimal chronic pulmonary sequela to inhalational injury in burns by pulmonary testing, we speculate that the hyperprocalcitonemia in some of the patients may reflect a long-term hyperplastic response of the bronchio-epithelial pulmonary neuroendocrine cells. The potential significance of this and other lung-associated endocrine markers is discussed.

Biomarkers↗

PubMed1994

Characterization of the tryptophan fluorescence and hydrodynamic properties of rat DNA polymerase beta.

We have examined the biophysical properties of DNA polymerase beta (beta-pol) in solution. Time-resolved and steady-state fluorescence were used to investigate the microenvironment of the lone tryptophanyl residue (Trp324), and a combination of sedimentation equilibrium, sedimentation velocity and fluorescence anisotropy decay measurements were used to study the hydrodynamic properties of the enzyme. Trp324 appears to be exposed to water as judged by the tryptophan emission and steady-state and lifetime quenching experiments. The fluorescence is easily quenched by a neutral quencher acrylamide (kq = 1.59 x 10(9)M-1S-1), and by a negatively charged ionic quencher, I- (kq = 1.60 x 10(9) M-1S-1), but not by a positively charged ionic quencher, Cs+ (kq = 0.2 x 10(9) M-1S-1). The fluorescence lifetime of beta-pol is best described by the sum of two exponentials with a longer lifetime component of 8.4 ns and a shorter lifetime component of 1.3 ns. Decay associated spectra (DAS) show emission maxima at 340 nm and at 345 nm for the shorter lifetime and longer lifetime components, respectively, with corresponding centers of gravity at 347 nm and 348 nm. Sedimentation equilibrium experiments show that the enzyme exists as a monomer at the KCl concentrations (> 0.05 M) studied in the absence of divalent metals. Zn2+ causes higher order aggregation, but no such aggregates are seen with Mg2+ and Mn2+. In the presence of 1 mM manganese, the average lifetime decreased approximately 10%, from 8.14 ns to 7.38 ns, with a concomitant increase of average rotational correlational time (phi) from 24 ns to 28 ns. The accessibility of the positively charged quencher (Cs+) to tryptophan also decreases approximately 50%, indicating alteration of the tryptophan microenvironment. By contrast, Mg2+ causes minor changes in fluorescence properties. The hydrodynamic shape of the intact enzyme and its single-stranded (8 kDa) and double-stranded (31 kDa) DNA binding domains were further investigated by sedimentation velocity measurements. The value of S0(20),W for the intact enzyme is 2.97 S, and the calculated axial ratio is 5.0. In contrast to the 8 kDa domain, which has a less asymmetric shape with an axial ratio of 2.3, the 31 kDa domain shows an elongated structure with an axial ratio of 5.5. These data suggest that the axial ratio of the intact enzyme may be the result of marked bending of the molecule at the flexible hinge region between the two domains.

Animals↗

PubMed1994

Actobindin binds with high affinity to a covalently cross-linked actin dimer.

Actobindin, a 9.8-kDa protein purified from Acanthamoeba castellanii, contains two actin-binding sites that can simultaneously bind two actin monomers. However, actobindin inhibits actin polymerization to a greater extent than can be explained by its affinity for actin monomers (site-specific KD = 3.3 microM). This paradox would be resolved if actobindin could interfere with the nucleation phase of polymerization by using both binding sites to bind simultaneously to an actin oligomer because the interaction with oligomer would be thermodynamically favored over that with actin monomer. We now show that a covalently cross-linked actin dimer prepared from cross-linked F-actin binds to actobindin with high affinity (apparent KD = 11 nM) in accordance with theoretical predictions for simultaneous binding of two actin subunits per single actobindin and consistent with the hypothesis that actobindin might bind to native actin oligomers and prevent them from nucleating polymerization. Furthermore, the interaction with cross-linked dimer exhibits specificity in that an isomeric cross-linked actin dimer with more rapid electrophoretic mobility binds weakly to actobindin. However, only this isomeric dimer is produced when cross-linking reagents are added to actin undergoing polymerization in the presence of actobindin. Therefore, if actobindin inhibits polymerization by interacting with a native dimer whose conformation is similar to that of the cross-linked dimer with slower electrophoretic mobility, then actobindin must either block the cross-linking sites or convert the dimer to a different conformation.

Acanthamoeba↗

PubMed1994

Specific sequences from the carboxyl terminus of human p53 gene product form anti-parallel tetramers in solution.

Human p53 is a tumor-suppressor gene product associated with control of the cell cycle and with growth suppression, and it is known to form homotetramers in solution. To investigate the relationship of structure to tetramerization, nine peptides corresponding to carboxyl-terminal sequences in human p53 were chemically synthesized, and their equilibrium associative properties were determined by analytical ultracentrifugation. Secondary structure, as determined by circular dichroism measurements, was correlated with oligomerization properties of each peptide. The sedimentation profiles of peptides 319-393 and 319-360 fit a two-state model of peptide monomers in equilibrium with peptide tetramers. Successive deletion of amino- and carboxyl-terminal residues from 319-360 reduced tetramer formation. Further, substitution of alanine for Leu-323, Tyr-327, and Leu-330 abolished tetramerization. Circular dichroism studies showed that peptide 319-351 had the highest alpha-helix content, while the other peptides that did not form tetramers had low helical structure. These studies define a minimal region and identify certain critical residues involved in tetramerization. Cross-linking studies between monomer units in the tetramer suggest that the helices adopt an anti-parallel arrangement. We propose that conformational shifts in the helical structure of the p53 tetramerization domain result in a repositioning of subunits relative to one another. This repositioning provides an explanation relating conformational changes at the carboxyl terminus with changes in sequence-specific DNA binding by the highly conserved central domain.

Circular Dichroism↗

PubMed1994

pH-dependent interaction of an intraluminal loop of inositol 1,4,5-trisphosphate receptor with chromogranin A.

The inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store role of the secretory vesicles of adrenal medullary chromaffin cells is attributed to the presence of high capacity, low affinity Ca2+ binding protein chromogranin A. Chromogranin A has recently been shown to interact with the protein component(s) on the intraluminal side of the secretory vesicle membrane at the intravesicular pH of 5.5 but to dissociate from them at the near physiological pH of 7.5. Further, one of the chromogranin A-interacting membrane proteins was tentatively identified as the IP3 receptor. Therefore, the pH-dependent potential interaction of the intraluminal loop domains of the IP3 receptor with chromogranin A was studied by analytical ultracentrifugation utilizing synthetic intraluminal loop peptides of the IP3 receptor labeled with 5-hydroxy-tryptophan at the N-terminus as a chromophore. One of the intraluminal loop domains was found to interact with chromogranin A at pH 5.5 but not at pH 7.5, suggesting the importance of the intraluminal loop domain in transmitting Ca2+ mobilization signals to chromogranin A.

Amino Acid Sequence↗