Abstract A series of recombinant peptides, each including the sequence proposed to be the first nucleotide‐binding fold of cystic fibrosis transmembrane conductance regulator (CFTR), has been produced in an attempt to find a model peptide that would autologously fold into a soluble structure with native‐like properties. The peptide NBD 1F , which contains the 267‐amino acid sequence of CFTR from 384 to 650, meets these requirements. The peptide was produced with a high expression bacterial plasmid pRSET, purified from inclusion bodies following solubilization with 6 M guanidine‐HCl and refolded from 8 M urea. Competitive displacement of trinitrophenol‐ATP by nucleotides reveals binding of ATP and related nucleotides with K D s in the low micromolar range; the K D for ATP‐ γ S is 1.0 ± 0.4 μM and for ADP 8.8 ± 3.1 μM. The native‐like character of the model peptide's structure is further supported by the findings that the K D for the ATP analog, 5′‐adenylimidodiphosphate, is fourfold lower than the K D for the methylene analog, 5′‐adenylmethylenediphosphonate, and that ATP binding slows the trypsin proteolysis of NBD 1F . The CD spectra of NBD 1F and the parallel peptide containing the most common cystic fibrosis mutation, deletion of Phe 508, are essentially indistinguishable, both spectra indicating 28% α‐helix and 23% β‐sheet, with insignificant differences in the amounts of β‐turns and random structure. Extensive investigation using multiple conditions with highly purified preparations of the model peptides demonstrates that they do not support ATP hydrolysis. These large recombinant peptides offer practical models for the investigation of the first nucleotide‐binding domain of CFTR.
The circular dichroism of Eco RI restriction endonuclease was measured to 178 nm and analyzed for secondary structure. The results (33% alpha-helix, 25% beta-sheet, 17% turns, and 25% other structures) compare well with our joint prediction from sequence data.
The conformational and spacial configurations of the biologically active undecapeptide, Substance P, were studied using conformational energy calculations. Low energy conformers of residues 1–5 and 6–11 were found by energy minimization and the two fragments were then combined to find low‐energy structures for Substance P. Several configurational classes were found with different backbone conformations. A comparison of the final low‐energy structures with data from biological tests on analogs of Substance P gives some insight into the conformation required for interaction at the biological receptor.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTConformational analysis of thymidylate synthase from amino acid sequence and circular dichroismParthasarathy Manavalan, Denice M. Mittelstaedt, Michael I. Schimerlik, and W. Curtis Johnson, Jr.Cite this: Biochemistry 1986, 25, 21, 6650–6655Publication Date (Print):October 1, 1986Publication History Published online1 May 2002Published inissue 1 October 1986https://pubs.acs.org/doi/10.1021/bi00369a048https://doi.org/10.1021/bi00369a048research-articleACS PublicationsRequest reuse permissionsArticle Views49Altmetric-Citations16LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
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