An artificial crystalline DDT‐binding polypeptide

Moser, Rudolf; Thomas, Richard M.; Gutte, Bernd

doi:10.1016/0014-5793(83)80555-9

Cited by 63 publications

(24 citation statements)

References 12 publications

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“…The artificial DDTbinding polypeptide (DBP) was synthesized by the solid phase method (Merrifield, 1963;Barany and Merrifield, 1979) and was shown by c.d. measurements in 55% aqueous ethanol to contain 42% /3-sheet (Moser, 1986) which agreed well with the /3-sheet content of the model (Moser et al, 1983). The purified synthetic product was homogeneous by various analytical criteria and could be crystallized; the crystals, however, were not suitable for Xray structural analysis.…”

Section: Introductionmentioning

confidence: 54%

“…We have designed a 24-residue polypeptide (Moser et al, 1983) (Figure 1) capable of binding the insecticide DDT ~ 1000 times more strongly than bovine serum albumin, a well-known carrier of hydrophobic molecules (k D of the peptide-DDT complex in 55% aqueous ethanol = 0.9 x 10~6 M). The tools for the design were model building and secondary structure prediction (Chou and Fasman, 1978;Levitt, 1978).…”

Section: Introductionmentioning

confidence: 99%

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Expression of the synthetic gene of an artificial DDT-binding polypeptide in Escherichia coli

Moser¹,

Frey²,

Münger³

et al. 1987

Protein Eng Des Sel

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This paper reports the expression of an artificial functional polypeptide in bacteria. The gene of a designed 24-residue DDT-binding polypeptide (DBP) was inserted between the BamYQ and PsA cleavage sites of plasmid pUR291. The hybrid plasmid, pUR291-DBP, was cloned in Escherichia coli JM109. After induction by isopropyl-/3-D-thiogalactopyranoside a fusion protein was expressed in which DBP was linked to the COOH-tenninus of (3-galactosidase. DBP, which is stable to trypsin, was obtained by tryptic digestion of the fusion protein and subsequent fractionation of the tryptic peptides by reversed-phase h.p.l.c. Recombinant and chemically synthesized DBP showed identical chromatographic properties, amino add composition, and chymotryptic digestion patterns. Both the /3-galactosidase-DBP fusion and isolated recombinant DBP bound DDT. The fusion protein was 25 times as potent as the designed 24-residue DBP in activating a cytochrome P-450 model system using equimolar catalytic amounts of the two proteins. Key words: protein design/DDT-binding polypeptide/pUR291 hybrid plasmid/fusion protein/recombinant DDT-binding polypeptide could be efficiently expressed in a suitable organism, and (ii) if there was expression, the physicochemical and biological properties of the primary translation product (inevitably a fusion protein containing the DBP sequence) could be studied, and DDT binding and metabolism by a DBP + and a DBP~ strain of E. coli could be compared. Materials and methods Construction of the hybrid plasmid pUC8-DBPThe eight DNA fragments constituting the double-stranded DNA of the DBP gene (Figure 2, nucleotide sequences in boxes) for insertion between the BamHl and Pstl cleavage sites of die polylinker of plasmid pUC8 (Vieira and Messing, 1982) were synthesized by the solid phase phosphoramidite method (Beaucage and Caruthers, 1981) using protected deoxyribonucleotidemorpholino-methoxyphosphines (Dorper and Winnacker, 1983). After synthesis and complete deprotection they were purified by electrophoresis on preparative denaturing 16% polyacrylamide gels. The bands containing the target oligodeoxynucleotides were

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Section: Introductionmentioning

confidence: 54%

Section: Introductionmentioning

confidence: 99%

Expression of the synthetic gene of an artificial DDT-binding polypeptide in Escherichia coli

Moser¹,

Frey²,

Münger³

et al. 1987

Protein Eng Des Sel

Self Cite

View full text Add to dashboard Cite

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“…All of the above design properties are based on statistical preferences ofresidues to form ,/3structure and partition into hydrophobic or hydrophilic environments. The structure formed also Other designed proteins also exhibit characteristics associated with the molten globule state (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)19), such as low cooperativity of unfolding, ANS binding, and poor NMR characteristics. For example, the betabellins have successively improved in both solubility and the appearance of order by NMR, but even for betabellin 12, whose proton resonances were assignable, it turned out that none of the numerous peaks in the two-dimensional nuclear Overhauser effect spectroscopy spectrum represented long-range or tertiary interactions (19).…”

Section: Discussionmentioning

confidence: 99%

“…One approach to expanding this knowledge involves de novo design of protein molecules of specific three-dimensional structure (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). Recent advances in molecular biology and computer-aided design tools have increased the potential for rational design of proteins.…”

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confidence: 99%

Betadoublet: de novo design, synthesis, and characterization of a beta-sandwich protein.

Quinn

Tweedy²,

Williams³

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

158

109

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How an amino acid sequence encodes the information necessary for a protein to adopt a unique tertiary structure remains unresolved. We are addressing this problem by designing "from scratch" protein molecules that will adopt predetermined three-dimensional structures. Based on this strategy, two identical four-stranded 3-sheets were designed to dimerize and form a 1sandwich protein, called betadoublet. A synthetic gene encoding half the 13-sandwich protein was expressed in Escherichia colt, and the protein was purified to homogeneity. Biophysical characterization of betadoublet in aqueous solution demonstrated that the disulfide formed between the two sheets and that the dimer was a compact unaggregated globular protein, consing predominantly of 1-sheet and stable to thermal denaturation. It has some backbone amide protons whose exchange is slow enough to be measured by NMR but binds more of the dye 1-anilinonaphthalene-8-sulfonate than a well-folded protein.Our understanding of the relationship between a protein's tertiary structure and its amino acid sequence directly effects our comprehension of protein folding and the connection between protein structure and function and our ability to design macromolecules with new functions. One approach to expanding this knowledge involves de novo design of protein molecules of specific three-dimensional structure (1-10). Recent advances in molecular biology and computer-aided design tools have increased the potential for rational design of proteins. We have undertaken the design, synthesis, and structural characterization of a (3-sandwich protein, betadoublet.Betadoublet was designed to form a pair of identical four-stranded (3-sheets that, facing one another, produce a sandwich. This motif is found in many naturally occurring proteins, such as immunoglobulins (11), concanavalin A (12), exotoxin A (13), and superoxide dismutase (14). A disulfide bond connecting the sheets was added to mimic the disulfide in the immunoglobulin fold and as a design diagnostic tool. The challenges of designing a (-sandwich molecule include accounting for the nonlocal interactions inherent in (3-structure (vs. a-structure) and production of a soluble small ,(protein using only naturally occurring amino acids in a nonrepetitive native-like distribution. Our goal was to produce a stable three-dimensional (-sandwich molecule based on a specific design model.Our approach has been one of a continuing cycle of design and evaluation beginning with the betabellins, a series of proteins based on this (3-sandwich motif that have been prepared by solid-phase peptide synthesis (15, 16). Successive versions of betabellin have improved in total amount of (3-structure and solubility. These molecules have also provided the opportunity to test the resolution of tight-turn geometry using D-amino acids (17)(18)(19). Most recently, betabellin 12 has been shown to have fully assignable NMR spectra in dimethyl sulfoxide, with many local but no longrange nuclear Overhauser effects observable (19). The beta...

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“…A more ambitious undertaking, the design and synthesis of an anti-parallel 'sandwich' of , strands, has been discussed by Richardson & Richardson (1987). An analogous but more empiric approach to the construction of a functional polypeptide was taken by Gutte's group (Moser et al, 1983) in their chemical synthesis and subsequent characterization of a 24-residue polypeptide intended to consist of four short antiparallel strands in a sheet which functions as a surface to bind the hydrophobic ligand DDT with high affinity. All three projects had as their goal the construction from first principles of frameworks or scaffolds onto which function might be grafted.…”

Section: Structures Of Factitious Proteins: Inferences From Crystallomentioning

confidence: 99%

Protein engineering. The design, synthesis and characterization of factitious proteins

Shaw

1987

Biochemical Journal

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An artificial crystalline DDT‐binding polypeptide

Cited by 63 publications

References 12 publications

Expression of the synthetic gene of an artificial DDT-binding polypeptide in Escherichia coli

Expression of the synthetic gene of an artificial DDT-binding polypeptide in Escherichia coli

Betadoublet: de novo design, synthesis, and characterization of a beta-sandwich protein.

Protein engineering. The design, synthesis and characterization of factitious proteins

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