Dissecting the effect of trifluoroethanol on ribonuclease A. Subtle structural changes detected by nonspecific proteases.
With the aim to distinguish between local and global conformational changes induced by trifluoroethanol in RNase A, spectroscopic and activity measurements in combination with proteolysis by unspecific proteases have been exploited for probing structural transitions of RNase A as a function of trifluoroethanol concentration. At > 30% (v/v) trifluoroethanol (pH 8.0; 25 degrees C), circular dichroism and fluorescence spectroscopy indicate a cooperative collapse of the tertiary structure of RNase A coinciding with the loss of its enzymatic activity. In contrast to the denaturation by guanidine hydrochloride, urea or temperature, the breakdown of the tertiary structure in trifluoroethanol is accompanied by an induction of secondary structure as detected by far-UV circular dichroism spectroscopy. Proteolysis with the nonspecific proteases subtilisin Carlsberg or proteinase K, both of which attack native RNase A at the Ala20-Ser21 peptide bond, yields refined information on conformational changes, particularly in the pretransition region. While trifluoroethanol at concentrations > 40% results in a strong increase of the rate of proteolysis and new primary cleavage sites (Tyr76-Ser77, Met79-Ser80) were identified, the rate of proteolysis at trifluoroethanol concentrations < 40% (v/v) is much smaller (up to two orders of magnitude) than that of the native RNase A. The proteolysis data point to a decreased flexibility in the surrounding of the Ala20-Ser21 peptide bond, which we attribute to subtle conformational changes of the ribonuclease A molecule. These changes, however, are too marginal to alter the overall catalytic and spectroscopic properties of ribonuclease A.
Köditz
Jens
J
Department of Biochemistry/Biotechnology, Martin-Luther University Halle-Wittenberg, Halle, Germany.
Arnold
Ulrich
U
Ulbrich-Hofmann
Renate
R
D002942:Circular Dichroism; D019286:Endopeptidase K / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013381:Subtilisins / Q000737:chemistry / Q000378:metabolism; D014270:Trifluoroethanol / Q000737:chemistry* / Q000494:pharmacology
D012996:Solutions; D014270:Trifluoroethanol; D012259:Ribonuclease, Pancreatic; D013381:Subtilisins; D019286:Endopeptidase K