Get access

Thermodynamics and mechanism of cutinase stabilization by trehalose

Authors

  • Ricardo P. Baptista,

    1. Institute of Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, Lisboa 1049-001, Portugal
    Search for more papers by this author
  • Shona Pedersen,

    1. Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, Aalborg DK-9000, Denmark
    Search for more papers by this author
  • Gonçalo J. M. Cabrita,

    1. Institute of Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, Lisboa 1049-001, Portugal
    Search for more papers by this author
  • Daniel E. Otzen,

    1. Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, Aalborg DK-9000, Denmark
    Search for more papers by this author
  • Joaquim M. S. Cabral,

    1. Institute of Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, Lisboa 1049-001, Portugal
    Search for more papers by this author
  • Eduardo P. Melo

    Corresponding author
    1. Institute of Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, Lisboa 1049-001, Portugal
    2. Institute of Biotechnology and Bioengineering, Centre for Molecular and Structural Biomedicine, Universidade do Algarve, Campus de Gambelas, Faro 8005-139, Portugal
    • Institute of Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, Lisboa 1049-001, Portugal
    Search for more papers by this author

Abstract

Trehalose has been widely used to stabilize cellular structures such as membranes and proteins. The effect of trehalose on the stability of the enzyme cutinase was studied. Thermal unfolding of cutinase reveals that trehalose delays thermal unfolding, thus increasing the temperature at the midpoint of unfolding by 7.2°. Despite this stabilizing effect, trehalose also favors pathways that lead to irreversible denaturation. Stopped-flow kinetics of cutinase folding and unfolding was measured and temperature was introduced as experimental variable to assess the mechanism and thermodynamics of protein stabilization by trehalose. The main stabilizing effect of trehalose was to delay the rate constant of the unfolding of an intermediate. A full thermodynamic analysis of this step has revealed that trehalose induces the phenomenon of entropy–enthalpy compensation, but the enthalpic contribution increases more significantly leading to a net stabilizing effect that slows down unfolding of the intermediate. Regarding the molecular mechanism of stabilization, trehalose increases the compactness of the unfolded state. The conformational space accessible to the unfolded state decreases in the presence of trehalose when the unfolded state acquires residual native interactions that channel the folding of the protein. This residual structure results into less hydrophobic groups being newly exposed upon unfolding, as less water molecules are immobilized upon unfolding. © 2008 Wiley Periodicals, Inc. Biopolymers 89: 538–547, 2008.

This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Ancillary