PROBING PROTEIN-STRUCTURE USING BIOCHEMICAL AND BIOPHYSICAL METHODS -PROTEOLYSIS, MATRIX-ASSISTED LASER DESORPTION IONIZATION MASS-SPECTROMETRY, HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND SIZE-EXCLUSION CHROMATOGRAPHY OF P21(WAF1/CIP1/SDI1)/

The cyclin-dependent kinase (Cdk) inhibitor p21(Waf1/Cip1/Sdi1), impor tant for p53 tumor suppressor-dependent cell growth control in humans and other organisms, mediates G(1)/S-phase arrest through inhibition o f cyclin-dependent kinases (Cdks). The enzymatic activity of these kin ases is essential for progress through the cell division cycle and one level of cell cycle regulation is exerted through inhibition of Cdks by a family of small proteins, including p21. Cdk inhibition requires a sequence of approximately 60 amino acids within the p21 NH2-terminus . Using proteolytic mapping, matrix-assisted laser desorption/ionizati on (MALDI) mass spectrometry, HPLC and size-exclusion chromatography, we show that p21, active as a Cdk inhibitor, exists in an extended, no n-globular conformation in the absence of its biological target and th at p21 lacks the hallmarks of stable secondary and tertiary structure. We have developed an efficient approach to obtain detailed proteolyti c maps that takes advantage of the high accuracy and sensitivity of MA LDI mass spectrometry. Our method allows a proteolytic map to be obtai ned from a single mass spectrum for fragments produced from a single p roteolytic reaction. (C) 1997 Elsevier Science B.V.