ENHANCED PROTEIN THERMOSTABILITY BY ALA-]AIB REPLACEMENT

The introduction into peptide chains of alpha-aminoisobutyric acid (Ai b) has proven to stabilize the helical structure in short peptides by restricting the available range of polypeptide backbone conformations. In order to evaluate the potential stabilizing effect of Aib at the p rotein level, we have studied the conformational and stability propert ies of Aib-containing analogs of the carboxy-terminal subdomain 255-31 6 of thermolysin. Previous NMR studies have shown that this disulfide- free 62-residue fragment forms a dimer in solution and that the global 3D structure of each monomer (3 alpha-helices encompassing residues 2 60-274, 281-295, and 301-311) is largely coincident with that of the c orresponding region in the X-ray structure of intact thermolysin. The Aib analogs of fragment 255-316 were prepared by a semisynthetic appro ach in which the natural fragment 255-316 was coupled to synthetic ana logs of peptide 303-316 using V8-protease in 50% (v/v) aqueous glycero l [De Filippis, V., and Fontana, A. (1990) Int. J. Pept. Protein Res, 35, 219-227]. The Ala residue in position 304, 309, or 312, of fragmen t 255-316 was replaced by Aib, leading to the singly substituted fragm ents Ala304Aib, Ala309Aib, and Ala312Aib. Moreover, fragment Ala304Aib /Ala309Aib with a double Ala --> Aib exchange in positions 304 and 309 was produced. Far- and near-UV circular dichroism measurements demons trated that both secondary and tertiary structures of the natural frag ment 255-316 are fully retained upon Ala --> Aib substitution(s). Ther mal unfolding measurements, carried out by recording the ellipticity a t 222 nm upon heating, showed that the melting temperatures (T-m) of a nalogs Ala304Aib and Ala309Aib were 2.2 and 5.4 degrees C higher than that of the Ala-containing natural species (T-m = 63.5 degrees C), res pectively, whereas the T-m of the Ala312Aib analog was lowered by -0.6 degrees C. The enhanced stability of the Ala304Aib analog can be quan titatively explained on the basis of a reduced backbone entropy of unf olding due to the restriction of the conformational space allowed to A ib in respect to Ala, while the larger stabilization observed for the Ala309Aib analog can be accounted for by both entropic and hydrophobic effects. In fact, whereas Ala304 is a surface residue, Ala309 is shie lded from the solvent, and thus the enhanced stability of fragment Ala 309Aib is also due to the burial of an additional -CH3 group with resp ect to the natural fragment. The slightly destabilizing effect of the Ala --> Aib exchange in position 312 appears to derive from unfavorabl e strain energy effects, since phi and psi, values for Ala312 are out of the allowed angles for Aib. Of interest, the simultaneous incorpora tion of Aib at positions 304 and 309 leads to a significant and additi ve increase of +8 degrees C in T-m. The results of this study indicate that the rational incorporation of Aib into a polypeptide chain can b e a general procedure to significantly stabilize proteins.