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Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding.

B W Matthews1, H Nicholson, W J Becktel

  • 1Institute of Molecular Biology, University of Oregon, Eugene 97403.

Proceedings of the National Academy of Sciences of the United States of America
|October 1, 1987
PubMed
Summary
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Scientists enhanced protein stability by altering amino acid sequences. Specific substitutions in T4 lysozyme increased thermal stability without affecting enzymatic function, offering a new strategy for protein engineering.

Area of Science:

  • Biochemistry
  • Protein Engineering
  • Structural Biology

Background:

  • Protein stability is crucial for biological function and therapeutic applications.
  • Decreasing the configurational entropy of unfolding is a proposed strategy to enhance protein stability.
  • Amino acid substitutions can modulate protein stability.

Purpose of the Study:

  • To investigate the effect of specific amino acid substitutions on the stability of bacteriophage T4 lysozyme.
  • To determine if these substitutions impact protein enzymatic activity.
  • To analyze the structural consequences of these mutations using crystallographic methods.

Main Methods:

  • Site-directed mutagenesis was used to introduce proline (Pro) and alanine (Ala) substitutions into T4 lysozyme.

Related Experiment Videos

  • Thermal denaturation assays (reversible and irreversible) were performed to assess protein stability.
  • Enzymatic activity assays were conducted to evaluate the functional impact of mutations.
  • High-resolution X-ray crystallography was employed to determine the three-dimensional structures of mutant proteins.

    • Main Results:

    • Both Xaa----Pro and Gly----Xaa substitutions significantly stabilized T4 lysozyme against thermal denaturation.
    • The introduced mutations did not alter the enzymatic activity of the lysozyme.
    • Crystallographic analysis revealed that the proline substitution (Ala-82----Pro) resulted in a structure nearly identical to wild-type.
    • The glycine substitution (Gly-77----Ala) also maintained overall structural similarity, with minor localized conformational adjustments.

    Conclusions:

    • Selected amino acid substitutions can effectively enhance protein stability by reducing configurational entropy.
    • These stabilizing mutations do not necessarily compromise protein function.
    • Combining multiple such substitutions may offer a general approach for significantly improving protein stability, with potential applications in biotechnology and medicine.