Increase of RNase a N-terminus polarity or C-terminus apolarity changes the two domains' propensity to swap and form the two dimeric conformers of the protein.
Do the polarities of the N-terminus or the apolarity of the C-terminus of bovine RNase A influence the relative yields of its two 3D domain-swapped dimeric conformers, the N-dimer and C-dimer? We have addressed this question by substituting Ala-4 or Ala-5 with serine (A4S and A5S mutants) or Ser-123 with alanine (S123A mutant) through site-directed mutagenesis. Both the polarity of the N-terminus and the apolarity of the C-terminus of RNase A were, therefore, increased. CD spectra revealed no significant differences between the secondary structures of the mutants and native RNase A. According to thermal denaturation analyses, the A4S and A5S mutants are less stable, and the S123A mutant is more stable than wild type RNase A. By subjecting the mutants under mild or drastic denaturing conditions, side-by-side with native and recombinant RNase A, to a thermally induced oligomerization procedure, the following results were obtained. (i) The N-terminal mutants showed a higher propensity, with respect to the native protein, to form N-dimers under mild unfolding conditions. (ii) The C-terminal mutant showed a higher propensity to form the C-dimer under severely unfolding conditions. These results are discussed in light of the relative stabilities of the various RNase A species under different environmental conditions, and we conclude that the hydrophilic or hydrophobic character of the RNase N-terminus or C-terminus can be an important variable governing the oligomerization of RNase A and possibly other proteins through the 3D domain-swapping mechanism.
Gotte
Giovanni
G
Dipartimento di Scienze Morfologico-Biomediche, Sezione di Chimica Biologica, Facoltà di Medicina e Chirurgia, Università di Verona, Strada Le Grazie 8, I-37134 Verona, Italy.
Donadelli
Massimo
M
Laurents
Douglas V
DV
Vottariello
Francesca
F
Morbio
Manuela
M
Libonati
Massimo
M
D000409:Alanine / Q000737:chemistry; D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D019281:Dimerization; D004795:Enzyme Stability; D019791:Guanidine / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000737:chemistry
D011994:Recombinant Proteins; D012694:Serine; D012259:Ribonuclease, Pancreatic; D019791:Guanidine; D000409:Alanine