From ribonuclease A toward bovine seminal ribonuclease: a step by step thermodynamic analysis.
A proline, a leucine, and two cysteine residues, introduced at positions 19, 28, 31, and 32 of bovine pancreatic RNase A, i.e. the positions occupied by these residues in the subunit of bovine seminal RNase, the only dimeric RNase of the pancreatic-type superfamily, transform monomeric RNase A into a dimeric RNase, endowed with the same ability of BS-RNase of swapping its N-terminal segments. The thermodynamic consequences of the progressive introduction of these four residues into RNase A polypeptide chain have been studied by comparing the temperature- and urea-induced denaturation of three mutants of RNase A with that of a stable monomeric derivative of BS-RNase. The denaturation processes proved reversible for all proteins, and well represented by the two-state N<-->D transition model. The progressive introduction of the four residues into RNase A led to a gradual shift of the protein stability toward that characteristic of monomeric BS-RNase, which, in turn, is markedly less stable than RNase A with respect to both temperature- and urea-induced denaturation. On the other hand, the thermal stability of a dimeric active mutant of RNase A is found to approach that of wild-type seminal RNase.
Catanzano
F
F
Dipartimento di Chimica, Università di Napoli Federico II, Italy.
Graziano
G
G
Cafaro
V
V
D'Alessio
G
G
Di Donato
A
A
Barone
G
G
D000818:Animals; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D002942:Circular Dichroism; D003545:Cysteine; D004722:Endoribonucleases / Q000737:chemistry* / Q000378:metabolism*; D007930:Leucine; D008297:Male; D008956:Models, Chemical; D016254:Mutagenesis, Insertional; D011392:Proline; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism*; D012661:Semen / Q000201:enzymology; D013816:Thermodynamics
D011994:Recombinant Proteins; D011392:Proline; D004722:Endoribonucleases; C032523:ribonuclease SPL; D012259:Ribonuclease, Pancreatic; D007930:Leucine; D003545:Cysteine