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Research Themes Protein design

Making invisible proteins visible

SBKB [doi:10.1038/sbkb.2011.44]
Technical Highlight - October 2011
Short description: Protein flexibility allows conversion of an invisible excited state to a ground state with implications for rational protein design.

X-ray crystal structure of L99A mutant T4L, PDB code 3DMV. Reprinted from 1 .

Proteins are intrinsically flexible, and understanding how dynamics contribute to function is challenging. Recent advances in NMR spectroscopy are yielding unprecedented insights into how conformational transitions involving sparsely populated and transient excited states can directly contribute to signaling or catalytic activity.

Because conformations can exchange during an NMR experiment, a transient excited state causes a lifetime broadening of signals from the predominant ground state. NMR relaxation dispersion techniques can quantify this broadening and therefore detect an excited state that is otherwise invisible owing to its low abundance. The data can also be used to recover chemical shift information, directly reporting on structural features of the excited state, as well as kinetic and thermodynamic parameters describing the exchange process. Coupled with the Rosetta method of structure determination, it is now possible to calculate structures of excited states.

Kay and colleagues have used relaxation dispersion to investigate phage T4 lysozyme, a model system for understanding relationships between structure, dynamics, folding and binding. Using a L99A mutant with an engineered cavity that binds benzene, they found that the ground state, which constitutes 97% of the population, exchanges with an excited state that has a lifetime of 1 ms and a population of 3%. As the crystal structure of the L99A mutant is identical to the that of the wild type, the authors resolved this discrepancy by using Rosetta to determine the structure of the excited state. They observed a rearrangement of helices surrounding the cavity and a repositioned residue Phe114 projecting directly into the cavity, abrogating benzene binding.

The structure next allowed the authors to design a double mutant (L99A G113A) by strategically placing a helix-stabilizing residue in the excited structure, increasing its population to 34%. The population shift in the double mutant allowed observation of separate signals for all states (ground, excited and benzene bound), and the authors found that binding occurs only via the ground state by an unknown mechanism. A further substitution identified by Rosetta further perturbed the equilibrium in a L99A G113A R119P triple mutant excited state populated to 96%, completely inverting the population distribution relative to L99A. The observation of functional divergence in an excited state has clear implications for directed-evolution experiments and rational protein design.

Michael A. Durney

References

  1. G. Bouvignies et al. Solution structure of a minor and transiently formed state of a T4 lysozyme mutant.
    Nature 477, 111-114 (2011). doi:10.1038/nature10349

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