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Dissecting the energetics of protein α-helix C-cap termination through chemical protein synthesis

Nature Chemical Biology volume 2, pages 139143 (2006) | Download Citation

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Abstract

The α-helix is a fundamental protein structural motif and is frequently terminated by a glycine residue1,2,3,4,5. Explanations for the predominance of glycine at the C-cap terminal portions of α-helices have invoked uniquely favorable energetics of this residue in a left-handed conformation4 or enhanced solvation of the peptide backbone because of the absence of a side chain6. Attempts to quantify the contributions of these two effects have been made previously, but the issue remains unresolved. Here we have used chemical protein synthesis to dissect the energetic basis of α-helix termination by comparing a series of ubiquitin variants containing an L-amino acid or the corresponding D-amino acid at the C-cap Gly35 position. D-Amino acids can adopt a left-handed conformation without energetic penalty, so the contributions of conformational strain and backbone solvation can thus be separated. Analysis of the thermodynamic data revealed that the preference for glycine at the C′ position of a helix is predominantly a conformational effect.

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Acknowledgements

This research was supported by the US Department of Energy Genomes to Life Genomics Program grant DE-FG02-04ER63786 (to S.B.K.), the US National Science Foundation Materials Research Science and Engineering Centers Program at the University of Chicago (grant DMR-0213745), and by US National Institutes of Health grant GM54537 (to G.I.M.). Use of the Advanced Photon Source was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under Contract No. W-31-109-Eng-38. Portions of this work were performed at the Industrial Macromolecular Crystallography Association (IMCA-CAT) and at the DuPont-Northwestern-Dow Collaborative Access Team (DND-CAT) located in 17ID and 5ID, respectively, of the Advanced Photon Source. Use of the IMCA-CAT was supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with the Center for Advanced Radiation Sources at the University of Chicago. DND-CAT is supported by the E.I. DuPont de Nemours & Co., The Dow Chemical Company, the US National Science Foundation through grant DMR-9304725 and the State of Illinois through the Department of Commerce and the Board of Higher Education grant IBHE HECA NWU 96.

Author information

Affiliations

  1. Institute for Biophysical Dynamics, Center for Integrative Science, 929 East 57th Street, The University of Chicago, Chicago, Illinois 60637, USA.

    • Duhee Bang
    • , Valentina Tereshko
    • , Anthony A Kossiakoff
    •  & Stephen B Kent
  2. Department of Chemistry, Center for Integrative Science, 929 East 57th Street, The University of Chicago, Chicago, Illinois 60637, USA.

    • Duhee Bang
    •  & Stephen B Kent
  3. Department of Biochemistry & Molecular Biology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, Pennsylvania 17033, USA.

    • Alexey V Gribenko
    •  & George I Makhatadze
  4. Department of Biochemistry and Molecular Biology, Center for Integrative Science, 929 East 57th Street, The University of Chicago, Chicago, Illinois 60637, USA.

    • Valentina Tereshko
    • , Anthony A Kossiakoff
    •  & Stephen B Kent

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Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Stephen B Kent or George I Makhatadze.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Electrospray mass spectrometry analysis for each chemically synthesized ubiquitin molecule is shown.

  2. 2.

    Supplementary Fig. 2

    The Ramachandram plot of the incorporated D-Gln35, D-Val35 and L-Gln35 residues.

  3. 3.

    Supplementary Fig. 3

    The SigmaA-weighted omit (left) and final (right) electron density maps 2F(obs)–F(calc) plotted at 1σ level around the residues D-Gln35 (panel A, molecule B), D-Val35 (panel B, molecule A) and L-Gln35 (panel C, molecule A) in the cubic crystal forms reported in this work.

  4. 4.

    Supplementary Fig. 4

    The conformation of residue 35 and its neighbors in the cubic crystal form.

  5. 5.

    Supplementary Fig. 5

    The conformation of residue 35 and its neighbors in the orthorhombic crystal form.

  6. 6.

    Supplementary Table 1

    Data collection and refinement statistics for ubiquitin molecules in the cubic crystal form.

  7. 7.

    Supplementary Table 2

    Thermodynamic parameters of unfolding of the position 35 variants of ubiquitin.

  8. 8.

    Supplementary Methods

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DOI

https://doi.org/10.1038/nchembio766

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