Design of a monomeric 23-residue polypeptide with defined tertiary structure

Science

Volumn 271, Issue 5247, 1996, Pages 342-345

  Struthers, Mary D ^a   Cheng, Richard P ^a   Imperiali, Barbara ^a  

^a California Institute of Technology ^*  (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BBA1 PROTEIN, SYNTHETIC; DNA BINDING PROTEIN; PEPTIDE; PROTEIN; SYNTHETIC DNA; TRANSCRIPTION FACTOR; ZINC FINGER PROTEIN;

AMINO ACID SEQUENCE; ARTICLE; CHEMICAL STRUCTURE; CHEMISTRY; CIRCULAR DICHROISM; MOLECULAR GENETICS; NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY; PROTEIN ENGINEERING; PROTEIN FOLDING; PROTEIN SECONDARY STRUCTURE; PROTEIN TERTIARY STRUCTURE;

AMINO ACID SEQUENCE; CIRCULAR DICHROISM; DNA-BINDING PROTEINS; GENES, SYNTHETIC; MAGNETIC RESONANCE SPECTROSCOPY; MODELS, MOLECULAR; MOLECULAR SEQUENCE DATA; PEPTIDES; PROTEIN ENGINEERING; PROTEIN FOLDING; PROTEIN STRUCTURE, SECONDARY; PROTEIN STRUCTURE, TERTIARY; PROTEINS; TRANSCRIPTION FACTORS; ZINC FINGERS;

EID: 0030593029     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5247.342     Document Type: Article

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32. αN coupling constants were obtained from cross peak measurements in a DQFCOSY experiment at 25°C to avoid the line broadening observed at 7°C. The structure of BBA1 was stable with respect to changes in temperature. Denaturation studies indicate that the loss of secondary structure (as assessed by CD) between 7°C and 20°C is at most 13% and more likely less than 5% The peptide does not fully unfold at temperatures as high as 80°C, and complete denaturation requires urea concentrations in excess of 8 M. This behavior parallels that of native zinc fingers [M. A. Weiss and H T. Keutmann, Biochemistry 29, 9808 (1990)].
33. αN coupling constants were obtained from cross peak measurements in a DQFCOSY experiment at 25°C to avoid the line broadening observed at 7°C. The structure of BBA1 was stable with respect to changes in temperature. Denaturation studies indicate that the loss of secondary structure (as assessed by CD) between 7°C and 20°C is at most 13% and more likely less than 5% The peptide does not fully unfold at temperatures as high as 80°C, and complete denaturation requires urea concentrations in excess of 8 M. This behavior parallels that of native zinc fingers [M. A. Weiss and H T. Keutmann, Biochemistry 29, 9808 (1990)].
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36. 13β)
37. 4.
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41. The presence of discrete tertiary structure is often probed by studying the interaction of this hydrophobic, fluorescent dye with proteins Although ANS will bind to the apolar regions of partially folded or "molten globule" states of proteins, native folded proteins do not bind ANS. Many designed proteins to date have demonstrated significant ANS binding, which is attributed to poor tertiary packing interaction and the adoption of "molten globule" rather than native-like states (7, 11). BBA1 did not enhance the fluores-cence of ANS, indicating that this designed peptide does not bind ANS under conditions (5) previously shown to establish the presence of these poorly folded states. Experiments were performed in 20 mM acetate buffer, pH 4.5, and 30 μM BBA1 with ANS concentrations ranging from 20 to 600 μM The results indicate that the apolar side chains of BBA1 are sufficiently buried in the folded structure to preclude interaction with the dye.
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44. This work was supported by NSF grant CHE-910445, a Parsons Foundation predoctoral fellowship (M.D.S), an NIH traineeship (R P C), and a Camille and Henry Dreyfus Teacher Scholar Award (B.I.). We gratefully acknowledge the Dorothy Chandler, Camilla Chandler Frost Laboratory in Biology at the California Institute of Technology for use of the Vanan Unity Plus 600 NMR Spectrometer