Letter | Published:

Solution structure of a minor and transiently formed state of a T4 lysozyme mutant

Nature volume 477, pages 111114 (01 September 2011) | Download Citation

Abstract

Proteins are inherently plastic molecules, whose function often critically depends on excursions between different molecular conformations (conformers)1,2,3. However, a rigorous understanding of the relation between a protein’s structure, dynamics and function remains elusive. This is because many of the conformers on its energy landscape are only transiently formed and marginally populated (less than a few per cent of the total number of molecules), so that they cannot be individually characterized by most biophysical tools. Here we study a lysozyme mutant from phage T4 that binds hydrophobic molecules4 and populates an excited state transiently (about 1 ms) to about 3% at 25 °C (ref. 5). We show that such binding occurs only via the ground state, and present the atomic-level model of the ‘invisible’, excited state obtained using a combined strategy of relaxation-dispersion NMR (ref. 6) and CS-Rosetta7 model building that rationalizes this observation. The model was tested using structure-based design calculations identifying point mutants predicted to stabilize the excited state relative to the ground state. In this way a pair of mutations were introduced, inverting the relative populations of the ground and excited states and altering function. Our results suggest a mechanism for the evolution of a protein’s function by changing the delicate balance between the states on its energy landscape. More generally, they show that our approach can generate and validate models of excited protein states.

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Data deposits

The structural ensembles of the L99A excited state and L99A,G113A,R119P T4L have been deposited with PDB (accession codes 2LCB, 2LC9).

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Acknowledgements

We thank J. Forman-Kay for providing laboratory space, and J. Forman-Kay and T. Alber for discussions. G.B. acknowledges The European Molecular Biology Organization and The Canadian Institutes of Health Research (CIHR) for postdoctoral fellowships. L.E.K. holds a Canada Research Chair in Biochemistry. This work was supported by the CIHR and the Natural Sciences and Engineering Research Council of Canada, with computations performed on the GPC supercomputer at the SciNet HPC Consortium.

Author information

Author notes

    • Guillaume Bouvignies
    •  & Pramodh Vallurupalli

    These authors contributed equally to this work.

Affiliations

  1. Department of Molecular Genetics, The University of Toronto, Toronto, Ontario M5S 1A8, Canada

    • Guillaume Bouvignies
    • , Pramodh Vallurupalli
    • , D. Flemming Hansen
    •  & Lewis E. Kay
  2. Department of Biochemistry, The University of Toronto, Toronto, Ontario M5S 1A8, Canada

    • Guillaume Bouvignies
    • , Pramodh Vallurupalli
    • , D. Flemming Hansen
    •  & Lewis E. Kay
  3. Department of Chemistry, The University of Toronto, Toronto, Ontario M5S 1A8, Canada

    • Guillaume Bouvignies
    • , Pramodh Vallurupalli
    • , D. Flemming Hansen
    •  & Lewis E. Kay
  4. Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA

    • Bruno E. Correia
    • , Oliver Lange
    • , Robert M. Vernon
    •  & David Baker
  5. Program in Computational Biology, Instituto Gulbenkian de Ciência, P-2780-156 Oeiras, Portugal

    • Bruno E. Correia
  6. Hospital for Sick Children, Program in Molecular Structure and Function, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada

    • Alaji Bah
    • , Robert M. Vernon
    •  & Lewis E. Kay
  7. Department of Chemistry and Biochemistry, University of California Santa Barbara, Santa Barbara, California 93106, USA

    • Frederick W. Dahlquist

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Contributions

G.B., P.V. and D.F.H. made samples. G.B., P.V., D.F.H. and L.E.K. designed and performed all NMR experiments. G.B., P.V., B.E.C., O.L. and R.M.V. performed structure calculations. G.B., P.V. and A.B. carried out initial crystallization trials. F.W.D, D.B., G.B., P.V. and L.E.K. designed experiments and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Lewis E. Kay.

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    Supplementary Information

    The file contains Supplementary Figures 1-10 with legends, Supplementary Tables 1-9 and additional references.

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DOI

https://doi.org/10.1038/nature10349

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