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. Study holds ProTherm entries: 8307, 8308 Extra Details: disulfide bridges; dimeric forms; secondary structure;,alpha-helical segments
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:35 p.m.
|Number of data points||4|
|Proteins||Seminal ribonuclease ; Seminal ribonuclease|
|Assays/Quantities/Protocols||Experimental Assay: Tm pH:8.4 ; Experimental Assay: dHvH pH:8.4 ; Experimental Assay: Tm pH:5.0 ; Experimental Assay: dHvH pH:5.0|
|Libraries||Mutations for sequence KESAAAKFERQHMDSGNSPSSSSNYCNLMMCCRKMTQGKCKPVNTFVHESLADVKAVCSQKKVTCKNGQTNCYQSKSTMRITDCRETGSSKYPNCAYKTTQVEKHIIVACGGKPSVPVHFDASV|