title: Enhanced protein thermostability by Ala-->Aib replacement.
abstract: The introduction into peptide chains of alpha-aminoisobutyric acid (Aib) 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 protein level, we have studied the conformational and stability properties of Aib-containing analogs of the carboxy-terminal subdomain 255-316 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 260-274, 281-295, and 301-311) is largely coincident with that of the corresponding region in the X-ray structure of intact thermolysin. 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% (v/v) aqueous glycerol [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 fragment 255-316 was replaced by Aib, leading to the singly substituted fragments 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 demonstrated that both secondary and tertiary structures of the natural fragment 255-316 are fully retained upon Ala-->Aib substitution(s). Thermal unfolding measurements, carried out by recording the ellipticity at 222 nm upon heating, showed that the melting temperatures (Tm) of analogs Ala304Aib and Ala309Aib were 2.2 and 5.4 degrees C higher than that of the Ala-containing natural species (Tm = 63.5 degrees C), respectively, whereas the Tm of the Ala312Aib analog was lowered by -0.6 degree C. 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. In fact, whereas Ala304 is a surface residue, Ala309 is shielded from the solvent, and thus the enhanced stability of fragment Ala309Aib is also due to the burial of an additional -CH3 group with respect to the natural fragment. The slightly destabilizing effect of the Ala-->Aib exchange in position 312 appears to derive from unfavorable strain energy effects, since phi and psi values for Ala312 are out of the allowed angles for Aib. Of interest, the simultaneous incorporation of Aib at positions 304 and 309 leads to a significant and additive increase of +8 degrees C in Tm. 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.
authors.affiliation: CRIBI Biotechnology Centre, University of Padua, Italy.
authors.forename: V
authors.initials: V
authors.lastname: De Filippis
authors.forename: F
authors.initials: F
authors.lastname: De Antoni
authors.forename: M
authors.initials: M
authors.lastname: Frigo
authors.forename: P
authors.initials: P
authors.lastname: Polverino de Laureto
authors.forename: A
authors.initials: A
authors.lastname: Fontana
mesh_terms: D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000621:Aminoisobutyric Acids / Q000138:chemical synthesis; D002942:Circular Dichroism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013696:Temperature; D013820:Thermolysin / Q000138:chemical synthesis / Q000737:chemistry
chemical_list: D000621:Aminoisobutyric Acids; D010446:Peptide Fragments; C100049:2-aminoisobutyric acid; D013820:Thermolysin; D000409:Alanine