Order propensity of an intrinsically disordered protein, the cyclin-dependent-kinase inhibitor Sic1.
Intrinsically disordered proteins (IDPs) carry out important biological functions and offer an instructive model system for folding and binding studies. However, their structural characterization in the absence of interactors is hindered by their highly dynamic conformation. The cyclin-dependent-kinase inhibitor (Cki) Sic1 from Saccharomyces cerevisiae is a key regulator of the yeast cell cycle, which controls entrance into S phase and coordination between cell growth and proliferation. Its last 70 out of 284 residues display functional and structural homology to the inhibitory domain of mammalian p21 and p27. Sic1 has escaped systematic structural characterization until now. Here, complementary biophysical methods are applied to the study of conformational properties of pure Sic1 in solution. Based on sequence analysis, gel filtration, circular dichroism (CD), electrospray-ionization mass spectrometry (ESI-MS), and limited proteolysis, it can be concluded that the whole molecule exists in a highly disordered state and can, therefore, be classified as an IDP. However, the results of these experiments indicate, at the same time, that the protein displays some content in secondary and tertiary structure, having properties similar to those of molten globules or premolten globules. Proteolysis-hypersensitive sites cluster at the N-terminus and in the middle of the molecule, whereas the most structured region resides at the C-terminus, including part of the inhibitory domain and the casein-kinase-2 (CK2) phosphorylation target S201. The mutations S201A and S201E, which are known to affect Sic1 function, do not have significant effects on the conformational properties of the pure protein.
Brocca
Stefania
S
Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy.
Samalíková
Mária
M
Uversky
Vladimir N
VN
Lotti
Marina
M
Vanoni
Marco
M
Alberghina
Lilia
L
Grandori
Rita
R
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D050756:Cyclin-Dependent Kinase Inhibitor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017510:Protein Folding; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
D050756:Cyclin-Dependent Kinase Inhibitor Proteins; C086926:SIC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins