裂殖壶菌酰基载体蛋白(ACP)基因的克隆与表达

ACP(Acyl carrier protein,酰基载体蛋白)参与高度不饱和脂肪酸的PKS(Polyketide synthase)生物合成途径.从Schizochytrium sp.FJU-512 cDNA文库中获得了ACP基因的cDNA克隆.该序列开放读码框全长429 bp,编码142个氨基酸,等电点为5.04,具有4'-磷酸泛酰巯基乙胺(4'-PP)的结合位点.利用BamH Ⅰ/Hind Ⅲ双酶切,并连接到原核表达载体pET-30a,构建了pET-30a/acp表达载体,转化宿主菌E.coll BL21(DE3),IPTG诱导表达.SDS-PAGE分析表明该蛋白得到高效表达.     

裂殖壶菌酰基载体蛋白(ACP)基因的克隆与表达

ACP (Acyl carrier protein, 酰基载体蛋白) 参与高度不饱和脂肪酸的PKS (Polyketide synthase) 生物合成途径。从Schizochytrium sp.FJU-512 cDNA文库中获得了ACP基因的cDNA克隆。该序列开放读码框全长429 bp, 编码142个氨基酸, 等电点为5.04, 具有4′-磷酸泛酰巯基乙胺(4′-PP)的结合位点。利用BamHⅠ/HindⅢ双酶切, 并连接到原核表达载体pET-30a, 构建了pET-30a/acp表达载体, 转化宿主菌E.coli BL21(DE3), IPTG诱导表达。SDS-PAGE分析表明该蛋白得到高效表达。     

Intein-mediated Cyclization of Bacterial Acyl Carrier Protein Stabilizes Its Folded Conformation but Does Not Abolish Function

Bacterial acyl carrier protein (ACP) is essential for the synthesis of fatty acids and serves as the major acyl donor for the formation of phospholipids and other lipid products. Acyl-ACP encloses attached fatty acyl groups in a hydrophobic pocket within a four-helix bundle, but must at least partially unfold to present the acyl chain to the active sites of its multiple enzyme partners. To further examine the constraints of ACP structure and function, we have constructed a cyclic version of Vibrio harveyi ACP, using split-intein technology to covalently join its closely apposed N and C termini. Cyclization stabilized ACP in a folded helical conformation as indicated by gel electrophoresis, circular dichroism, fluorescence, and mass spectrometry. Molecular dynamics simulations also indicated overall decreased polypeptide chain mobility in cyclic ACP, although no major conformational rearrangements over a 10-ns period were noted. In vivo complementation assays revealed that cyclic ACP can functionally replace the linear wild-type protein and support growth of an Escherichia coli ACP-null mutant strain. Cyclization of a folding-deficient ACP mutant (F50A) both restored its ability to adopt a folded conformation and enhanced complementation of growth. Our results thus suggest that ACP must be able to adopt a folded conformation for biological activity, and that its function does not require complete unfolding of the protein.     

Biophysical Optimization of a Therapeutic Protein by Nonstandard Mutagenesis: STUDIES OF AN IODO-INSULIN DERIVATIVE*

Insulin provides a model for the therapeutic application of protein engineering. A paradigm in molecular pharmacology was defined by design of rapid-acting insulin analogs for the prandial control of glycemia. Such analogs, a cornerstone of current diabetes regimens, exhibit accelerated subcutaneous absorption due to more rapid disassembly of oligomeric species relative to wild-type insulin. This strategy is limited by a molecular trade-off between accelerated disassembly and enhanced susceptibility to degradation. Here, we demonstrate that this trade-off may be circumvented by nonstandard mutagenesis. Our studies employed Lys^B28, Pro^B29-insulin (“lispro”) as a model prandial analog that is less thermodynamically stable and more susceptible to fibrillation than is wild-type insulin. We have discovered that substitution of an invariant tyrosine adjoining the engineered sites in lispro (Tyr^B26) by 3-iodo-Tyr (i) augments its thermodynamic stability (ΔΔG_u 0.5 ±0.2 kcal/mol), (ii) delays onset of fibrillation (lag time on gentle agitation at 37 °C was prolonged by 4-fold), (iii) enhances affinity for the insulin receptor (1.5 ± 0.1-fold), and (iv) preserves biological activity in a rat model of diabetes mellitus. ¹H NMR studies suggest that the bulky iodo-substituent packs within a nonpolar interchain crevice. Remarkably, the 3-iodo-Tyr^B26 modification stabilizes an oligomeric form of insulin pertinent to pharmaceutical formulation (the R₆ zinc hexamer) but preserves rapid disassembly of the oligomeric form pertinent to subcutaneous absorption (T₆ hexamer). By exploiting this allosteric switch, 3-iodo-Tyr^B26-lispro thus illustrates how a nonstandard amino acid substitution can mitigate the unfavorable biophysical properties of an engineered protein while retaining its advantages.     

Differences in the Structure and Dynamics of the Apo- and Palmitate-ligated Forms of Aedes aegypti Sterol Carrier Protein 2 (AeSCP-2)

Sterol carrier protein-2 (SCP-2) is a nonspecific lipid-binding protein expressed ubiquitously in most organisms. Knockdown of SCP-2 expression in mosquitoes has been shown to result in high mortality in developing adults and significantly lowered fertility. Thus, it is of interest to determine the structure of mosquito SCP-2 and to identify its mechanism of lipid binding. We report here high quality three-dimensional solution structures of SCP-2 from Aedes aegypti determined by NMR spectroscopy in its ligand-free state (AeSCP-2) and in complex with palmitate. Both structures have a similar mixed α/β fold consisting of a five-stranded β-sheet and four α-helices arranged on one side of the β-sheet. Ligand-free AeSCP-2 exhibited regions of structural heterogeneity, as evidenced by multiple two-dimensional ¹⁵N heteronuclear single-quantum coherence peaks for certain amino acids; this heterogeneity disappeared upon complex formation with palmitate. The binding of palmitate to AeSCP-2 was found to decrease the backbone mobility of the protein but not to alter its secondary structure. Complex formation is accompanied by chemical shift differences and a loss of mobility for residues in the loop between helix αI and strand βA. The structural differences between the αI and βA of the mosquito and the vertebrate SCP-2s may explain the differential specificity (insect versus vertebrate) of chemical inhibitors of the mosquito SCP-2.     

NMR Structure of a Viral Peptide Inserted in Artificial Membranes: A VIEW ON THE EARLY STEPS OF THE BIRNAVIRUS ENTRY PROCESS*

Nonenveloped virus must penetrate the cellular membrane to access the cytoplasm without the benefit of membrane fusion. For birnavirus, one of the peptides present in the virus capsid, pep46 for infectious bursal disease virus, is able to induce pores into membranes as an intermediate step of the birnavirus-penetration pathway. Using osmotic protection experiments, we demonstrate here that pep46 and its pore-forming N-terminal moiety (pep22) form pores of different diameters, 5–8 and 2–4 nm, respectively, showing that both pep46 moieties participate to pore formation. The solution structures of pep46, pep22, and pep24 (the pep46 C-terminal moiety) in different hydrophobic environments and micelles determined by ¹H NMR studies provide structural insights of the pep46 domain interaction. In CDCl₃/CD₃OH mixture and in dodecylphosphocholine micelles, the N-terminal domain of pep46 is structured in a long kinked helix, although the C terminus is structured in one or two helices depending upon the solvents used. We also show that the folding and the proline isomerization status of pep46 depend on the type of hydrophobic environment. NMR spectroscopy with labeled phospholipid micelles, differential scanning calorimetry, and plasmon waveguide resonance studies show the peptides lie parallel to the lipid-water interface, perturbing the fatty acid chain packing. All these data lead to a model in which the two domains of pep46 interact with the membrane to form pores.     

Solution NMR Structure of the Ca2+-bound N-terminal Domain of CaBP7: A REGULATOR OF GOLGI TRAFFICKING*

Calcium-binding protein 7 (CaBP7) is a member of the calmodulin (CaM) superfamily that harbors two high affinity EF-hand motifs and a C-terminal transmembrane domain. CaBP7 has been previously shown to interact with and modulate phosphatidylinositol 4-kinase III-β (PI4KIIIβ) activity in in vitro assays and affects vesicle transport in neurons when overexpressed. Here we show that the N-terminal domain (NTD) of CaBP7 is sufficient to mediate the interaction of CaBP7 with PI4KIIIβ. CaBP7 NTD encompasses the two high affinity Ca²⁺ binding sites, and structural characterization through multiangle light scattering, circular dichroism, and NMR reveals unique properties for this domain. CaBP7 NTD binds specifically to Ca²⁺ but not Mg²⁺ and undergoes significant conformational changes in both secondary and tertiary structure upon Ca²⁺ binding. The Ca²⁺-bound form of CaBP7 NTD is monomeric and exhibits an open conformation similar to that of CaM. Ca²⁺-bound CaBP7 NTD has a solvent-exposed hydrophobic surface that is more expansive than observed in CaM or CaBP1. Within this hydrophobic pocket, there is a significant reduction in the number of methionine residues that are conserved in CaM and CaBP1 and shown to be important for target recognition. In CaBP7 NTD, these residues are replaced with isoleucine and leucine residues with branched side chains that are intrinsically more rigid than the flexible methionine side chain. We propose that these differences in surface hydrophobicity, charge, and methionine content may be important in determining highly specific interactions of CaBP7 with target proteins, such as PI4KIIIβ.