Enhanced Protein Thermostability by Ala → Aib Replacement†

In order to evaluate the potential stabilizing effect of Aib at the protein level, we have studied the conformational and stability properties of Aib-containing analogs of the carboxy-terminal subdomain 255−316 of thermolysin

Vincenzo De Filippis


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  • The Aib analogs of fragment 255−316 were prepared by a semisynthetic approach in which the natural fragment 255−316 was coupled to synthetic analogs of peptide 303−316 using V8-protease in 50% aqueous glycerol [De Filippis, V., and Fontana, A. Int
  • Far- and near-UV circular dichroism measurements demonstrated that both secondary and tertiary structures of the natural fragment 255−316 are fully retained upon Ala → Aib substitution(s)
  • The enhanced stability of the Ala304Aib analog can be quantitatively explained on the basis of a reduced backbone entropy of unfolding due to the restriction of the conformational space allowed to Aib in respect to Ala, while the larger stabilization observed for the Ala309Aib analog can be accounted for by both entropic and hydrophobic effects
  • The slightly destabilizing effect of the Ala → Aib exchange in position 312 appears to derive from unfavorable strain energy effects, since φ and ψ values for Ala312 are out of the allowed angles for Aib
  • The results of this study indicate that the rational incorporation of Aib into a polypeptide chain can be a general procedure to significantly stabilize proteins

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