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Lim Amareth Saderholm Matthew J. Kroll Mathias Yan Yibing Erickson Bruce W. Guy Philippe A. Anderegg Robert J. Makhov Alexander M. Griffith Jack D. 《International journal of peptide research and therapeutics》1999,6(1):3-14
Summary The inverse protein-folding problem has been explored by designing de novo the betabellin target structure (a 64-residue β-sandwich
protein), synthesizing a 32-residue peptide chain (HSLTAKIpkLTFSIAphTYTCAVpkYTAKVSH, wherep=DPro,k=DLys, andh=DHis) that might fold into this structure, and studying how its disulfide-bridged form (betabellin 15D) folds in 10 mM ammonium
acetate with and without Cu2+. Circular dichroic spectropolarimetry indicated that at pH 5.8, 6.4, or 6.7 betabellin 15D exhibited β-sheet structure in
the presence of Cu2+ but not in its absence. Electrospray mass spectrometry demonstrated that at pH 6.3 each molecule of betabellin 15D bound
one or two Cu(II) ions. Electron microscopy showed that at pH 6.7 betabellin 15D formed short broad fibrils in the presence
of Cu2+ but not in its absence. The observed width of the fibrils (7±2 nm) was consistent with the width (6.8nm) of a structural
model of a fibril that contained two adjacent rows of betabellin 15D β-sandwiches joined lengthwise by multiple intersheet
hydrogen bonds and widthwise by multiple Cu(II)-imidazole bonds. Electron paramagnetic resonance spectrometry revealed that
some pairs of Cu(II) ions in a Cu(II)/betabellin 15D complex were magnetically coupled, which is consistent with the structural
model of the Cu(II)/betabellin 15D fibril. 相似文献
2.
Engineering of betabellin-15D: a 64 residue beta sheet protein that forms long narrow multimeric fibrils.
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A. Lim M. J. Saderholm A. M. Makhov M. Kroll Y. Yan L. Perera J. D. Griffith B. W. Erickson 《Protein science : a publication of the Protein Society》1998,7(7):1545-1554
The betabellin target structure is a beta-sandwich protein consisting of two 32 residue beta-sheets packed against one another by interaction of their hydrophobic faces. The 32 residue chain of betabellin-15S (HSLTAKIpkLTFSIAphTYTCAV pkYTAKVSH, where p=DPro, k=DLys, and h=DHis) did not fold in water at pH 6.5. Air oxidation of betabellin-15S provided betabellin-15D, the 64 residue disulfide bridged two-chain molecule, which also remained unfolded in water at pH 6.5. By circular dichroic spectropolarimetry, the extent of beta structure observed for betabellin-15D increased with the pH and ionic strength of the solution and the betabellin-15D concentration. By electron microscopy, in 5.0 mM MOPS and 0.25 M NaCl at pH 6.9, betabellin-15D formed long narrow multimeric fibrils. A molecular model was constructed to show that the dimensions of these betabellin-15D fibrils are consistent with a single row of beta-sandwich molecules joined by multiple intersheet H-bonds. 相似文献
3.
Amareth Lim Matthew J. Saderholm Mathias Kroll Yibing Yan Bruce W. Erickson Philippe A. Guy Robert J. Anderegg Alexander M. Makhov Jack D. Griffith 《Letters in Peptide Science》1999,6(1):3-14
The inverse protein-folding problem has been explored by designing de novo the betabellin target structure (a 64-residue -sandwich protein), synthesizing a 32-residue peptide chain (HSLTAKIpkLTFSIAphTYTCAVpkYTAKVSH, where p = DPro, k = DLys, and h = DHis) that might fold into this structure, and studying how its disulfide-bridged form (betabellin 15D) folds in 10 mM ammonium acetate with and without Cu2+. Circular dichroic spectropolarimetry indicated that at pH 5.8, 6.4, or 6.7 betabellin 15D exhibited -sheet structure in the presence of Cu2+ but not in its absence. Electrospray mass spectrometry demonstrated that at pH 6.3 each molecule of betabellin 15D bound one or two Cu(II) ions. Electron microscopy showed that at pH 6.7 betabellin 15D formed short broad fibrils in the presence of Cu2+ but not in its absence. The observed width of the fibrils (7 ± 2 nm) was consistent with the width (6.8 nm) of a structural model of a fibril that contained two adjacent rows of betabellin 15D -sandwiches joined lengthwise by multiple intersheet hydrogen bonds and widthwise by multiple Cu(II)-imidazole bonds. Electron paramagnetic resonance spectrometry revealed that some pairs of Cu(II) ions in a Cu(II)/betabellin 15D complex were magnetically coupled, which is consistent with the structural model of the Cu(II)/betabellin 15D fibril. 相似文献
4.
Nakhle Bassam M. Vestal Tracie L. Saderholm Matthew J. Erickson Bruce W. 《International journal of peptide research and therapeutics》1999,6(1):33-43
Summary This paper describes the engineering of braced tripod proteins for use as molecular frameworks. Specifically, a 30-residue
tripod-shaped protein with three proline-II helical legs braced by an iron(II)tris(bipyridine) complex was modularly designed,
chemically synthesized, and biophysically characterized. Three copies of a 10-residue leg peptide were covalently linked through
sulfide bonds to an N-terminal apex (1,3,5-tris(methylene)benzene) and by amide bonds to the brace (FeII (Mbc)3: Mbc is 4′-methyl-2,2′-bipyridine-4-carbonyl). The leg peptide (H-Cys-Pro5-Pra(Mbc)-Pro3-NH2: Pra iscis-4-amino-l-proline) was assembled by the solid-phase method using Boc-Pra(Mbc)-OH, which was synthesized in 75% overall yield by coupling
Mbc-OH to the 4-amino group of Boc-Pra-OCH3 and saponifying the methyl ester group. The iron(II)-braced tripod was assembled by S-alkylation of three copies of the leg
peptide with 1,3,5-tris(bromomethyl)benzene followed by ligation of Fe2+ to the resulting unbraced tripod. The CD spectrum of the iron(II)-braced tripod showed a positive MLCT band at 570 nm and
a negative π-π* band at 312 nm, so its FeII(Mbc)3 brace was predominantly in the Δ configuration. In a mostly acetonitrile solution at 25°C, the leg peptide and the unbraced
tripod isomerized from the proline-II helical form into the proline-I helical form but the iron(II)-braced tripod remained
in the proline-II helical form. 相似文献
5.
Bassam M. Nakhle Tracie L. Vestal Matthew J. Saderholm Bruce W. Erickson 《Letters in Peptide Science》1999,6(1):33-43
This paper describes the engineering of braced tripod proteins for use as molecular frameworks. Specifically, a 30-residue tripod-shaped protein with three proline-II helical legs braced by an iron(II)tris(bipyridine) complex was modularly designed, chemically synthesized, and biophysically characterized. Three copies of a 10-residue leg peptide were covalently linked through sulfide bonds to an N-terminal apex (1,3,5-tris(methylene)benzene) and by amide bonds to the brace (FeII(Mbc)3: Mbc is 4-methyl-2,2-bipyridine-4-carbonyl). The leg peptide (H-Cys-Pro5-Pra(Mbc)-Pro3-NH2: Pra is cis-4-amino-l-proline) was assembled by the solid-phase method using Boc-Pra(Mbc)-OH, which was synthesized in 75% overall yield by coupling Mbc-OH to the 4-amino group of Boc-Pra-OCH3 and saponifying the methyl ester group.The iron(II)-braced tripod was assembled by S-alkylation of three copies of the leg peptide with 1,3,5-tris(bromomethyl)benzene followed by ligation of Fe2+ to the resulting unbraced tripod. The CD spectrum of the iron(II)-braced tripod showed a positive MLCT band at 570 nm and a negative –* band at 312 nm, so its FeII(Mbc)3 brace was predominantly in the configuration. In a mostly acetonitrile solution at 25 °C, the leg peptide and the unbraced tripod isomerized from the proline-II helical form into the proline-I helical form but the iron(II)-braced tripod remained in the proline-II helical form. 相似文献
6.
Protein farnesyltransferase catalyzes the posttranslational farnesylation of several proteins involved in signal transduction, including Ras, and is a target enzyme for antitumor therapies. Efficient product formation catalyzed by protein farnesyltransferase requires an enzyme-bound zinc cation and high concentrations of magnesium ions. In this work, we have measured the pH dependence of the chemical step of product formation, determined under single-turnover conditions, and have demonstrated that the prenylation rate constant is enhanced by two deprotonations. Substitution of the active site zinc by cadmium demonstrated that one of the ionizations reflects deprotonation of the metal-coordinated thiol of the peptide "CaaX" motif, pK(a1) = 6.0. These data provide additional evidence for the direct involvement of a metal-coordinated sulfur nucleophile in catalysis. The second ionization was assigned to a hydroxyl on the pyrophosphate moiety of farnesyl pyrophosphate, pK(a2) = 7.4. Deprotonation of this group is important for binding of magnesium. This second ionization is not observed for catalysis in the absence of magnesium or when the substrate is farnesyl monophosphate. These data indicate that the maximal rate constant for prenylation requires formation of a zinc-coordinated thiolate nucleophile and enhancement of the electrophilic character at C1 of the farnesyl chain by magnesium ion coordination of the pyrophosphate leaving group. 相似文献
7.
Both forms of the hepatitis delta antigen (HDAg) encoded by hepatitis delta virus are active only as oligomers. Previous studies showed that quadrin, a synthetic 50-residue peptide containing residues 12-60 from the N-terminus of HDAg, interferes with HDAg oligomerization, forms an alpha-helical coiled coil in solution, and forms a novel square octamer in the crystal consisting of four antiparallel coiled-coil dimers joined at the corners by hydrophobic binding of oligomerization sites located at each end of the dimers. We designed and synthesized deltoid (CH3CO-[Cys23]HDAg-(12-27)-seryl-tRNA synthetae-(59-65)-[Cys42]HDAg-(34-60)-Tyr-NH2), a chimeric protein that structurally resembles one end of the quadrin dimer and contains a single oligomerization site. The 51-residue chain of deltoid contains a seven-residue alpha-hairpin loop in place of the remainder of the quadrin dimer plus Cys12 and Cys31 for forming an intrachain disulfide bridge. Reduced, unbridged deltoid (Tm=61 degrees C, DeltaG(H2O)=-1.7 kcal mol(-1)) was less stable to denaturation by heat or guanidine HCl than oxidized, intrachain disulfide-bridged deltoid (Tm>80 degrees C, DeltaG(H2O)=-2.6 kcal mol(-1)). Each form is an alpha-helical dimer that reversibly dissociates into two monomers (Kd=80 microM). 相似文献
8.
Hepatitis delta antigen (HDAg) must form oligomers to be biologically active. Quadrin (HDAg-(12–60)-Tyr) is a 50-residue protein segment from the oligomerization domain of HDAg. The crystal structure of quadrin shows an octamer consisting of four identical copies of a dimer containing an antiparallel -helical coiled coil. Each end of the dimer contains an oligomerization site that interacts isologously with the oligomerization site of another dimer to form a right-angled corner. The resulting quadrin octamer is a 400-residue square protein surrounding a large aqueous hole. We have designed, chemically synthesized, and characterized deltoid and reduced deltoid, two 51-residue chimeric proteins that structurally and functionally mimic one of the two oligomerization sites of the quadrin dimer. Dimerization of deltoid or reduced deltoid should emulate the dimerization of two quadrin dimers to form one right-angled corner of the square. Deltoid and reduced deltoid were designed by molecular modeling, mechanics, and dynamics and synthesized by the solid-phase method. The amino acid sequence of deltoid (GREDILEQWVSCRKKL + PKAPPEE + LRKLKKKCKKLEEDNPWLGNIKGIIGKY) is a chimera of three protein segments: HDAg-(12–28), Thermus thermophilus serine tRNA synthase-(59–65), and HDAg-(34–60)-Tyr. Cysteine (C) was introduced at two positions to explore the effects of the presence (deltoid) or absence (reduced deltoid) of an interhelical disulfide bond. Circular dichroic spectropolarimetry revealed that both synthetic proteins form an -helical structure that is stable over a wide range of pH and KCl concentrations. Size-exclusion chromatography indicated that deltoid and reduced deltoid each form a dimer. Interconversion of these monomers and dimers should be useful model systems for studying the structural features of the right-angled corners of the quadrin octamer that contribute to HDAg oligomerization. If, like quadrin, deltoid or reduced deltoid interferes with HDAg oligomerization, it might serve as a lead compound for the design of potent HDV inhibitors. 相似文献
9.
A Lim A M Makhov M J Saderholm J D Griffith B W Erickson 《Biochemical and biophysical research communications》1999,264(2):498-504
The betabellin structure is a de novo designed beta-sandwich protein consisting of two 32-residue beta sheets packed against one another by hydrophobic interactions. Betabellin 16S (B16S), a 32-residue peptide chain (HSLTAKIakLTFSIAahTYTCAVakYTAKVSH, where a is DAla, h is DHis, and k is DLys), did not have beta structure in water at pH 6.5. Air oxidation of B16S furnished betabellin 16D (B16D), a 64-residue disulfide-bridged two-chain protein, which also did not fold in water at pH 6.5. However, the extent of beta structure observed for B16D increased with pH and ionic strength of the solution and the B16D concentration as observed by circular dichroism spectropolarimetry. Transmission electron microscopy showed that B16D formed narrow fibrils that associated into broad ribbons in 5.0 mM Mops and 0.25 M NaCl at pH 6.9. 相似文献
10.
Saderholm Matthew J. Erickson Bruce W. 《International journal of peptide research and therapeutics》1999,6(1):23-32
Summary Hepatitis delta antigen (HDAg) must form oligomers to be biologically active. Quandrin (HDAg-(12–60)-Tyr) is a 50-residue
protein segment from the oligomerization domain of HDAg. The crystal structure of quadrin shows an octamer consisting of four
identical copies of a dimer containing an antiparallel α-helical coiled coil. Each end of the dimer contains an oligomerization
site that interacts isologously with the oligomerization site of another dimer to form a right-angled corner. The resulting
quadrin octamer is a 400-residue square protein surrounding a large aqueous hole. We have designed, chemically synthesized,
and characterized deltoid and reduced deltoid, two 51-residue chimeric proteins that structurally and functionally mimic one
of the two oligomerization sites of the quadrin dimer. Dimerization of deltoid or reduced deltoid should emulate the dimerization
of two quadrin dimers to form one right-angled corner of the square. Deltoid and reduced deltoid were designed by molecular
modeling, mechanics, and dynamics and synthesized by the solid-phase method. The amino acid sequence of deltoid (GREDILEQWVSCRKKL+PKAPPEE+LRKLKKKCKKLEEDNPWLGNIKGIIGKY) is a chimera of three protein segments: HDAg-(12–28),Thermus thermophilus serine tRNA synthase-(59–65), and HDAg-(34–60)-Tyr. Cysteine (C) was introduced at two positions to explore the effects of the presence (deltoid) or absence (reduced deltoid) of an interhelical
disulfide bond. Circular dichroic spectropolarimetry revealed that both synthetic proteins from an α-helical structure that
is stable over a wide range of pH and KCl concentrations. Size-exclusion chromatography indicated that deltoid and reduced
deltoid each form a dimer. Interconversion of these monomers and dimers should be useful model systems for studying the structural
features of the right-angled corners of the quandrin octamer that contribute to HDAg oligomerization. If, like quadrin, deltoid
or reduced deltoid interferes with HDAg oligomerization, it might serve as a lead compound for the design of potent HDV inhibitors. 相似文献
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