Thermodynamic stability of the two isoforms of bovine seminal ribonuclease.
Bovine seminal ribonuclease (BS-RNase) is a dimeric protein with two identical subunits linked by two disulfide bridges, each subunit showing 80% of sequence identity with pancreatic RNase A. BS-RNase exists in two different quaternary conformations in solution: the MxM form, in which each subunit exchanges its alpha-helical N-terminal segment with its partner, and the M=M form with no exchange. By differential scanning microcalorimetry (DSC), the denaturation of the two dimeric forms of BS-RNase was found to be more complex than a simple two-state process. Monomeric derivatives of the dimeric protein follow instead a simple two-state mechanism, but are distinctly less stable than RNase A. The three-state N if I if D denaturation process of the two quaternary isoforms was interpreted by identifying in the dimers a central highly structured core, enclosing the covalently bonded subunit interface, which unfolds only after the periphery (mainly the N-terminal peptide) unfolds. Circular dichroism spectra of the two forms in the far-ultraviolet region show large differences between the secondary structure of the isoforms and that of the native BS-RNase mixture at equilibrium. This has been attributed to the presence in the equilibrium mixture of intermediate forms with displaced and disordered N-terminal alpha-helical segments.
Giancola
C
C
Department of Chemistry, University "Federico II" of Naples, Via Mezzocannone 4, 80134 Naples, Italy.
Del Vecchio
P
P
De Lorenzo
C
C
Barone
R
R
Piccoli
R
R
D'Alessio
G
G
Barone
G
G
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D019281:Dimerization; D004795:Enzyme Stability; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism*; D012661:Semen / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D007527:Isoenzymes; D012260:Ribonucleases