Based on the data presented in this paper and previously published results, we assume that the coordinated acetylation and phosphorylation of Rb2/p130 is important for its role in cell cycle regulation. The fact that HPV 16-E7 inhibits phosphoryla tion of Rb2/p130 although it enhances acetylation is in line with this assumption; HPV 16-E7 leads to heavily acetylated p130 molecules that are not hyperphosphorylated, thereby creating a modification status that does not represent the physiological condition. The unphysiological modification status of Rb2/p130 caused by the E7 protein thus contributes to cell cycle deregulation by human papilloma virus. Protein function and activity depends on their structure and stability. Protein structure and stability are affected by various factors, such as the specific cellular environment or binding to particular ligands. For instance, some proteins need the presence of specific metals or small-molecule or protein ligands to get sufficiently stabilised to perform their biological function. Binding proteins may induce structure in proteins that lack structure in isolation such as intrinsically disRegorafenib VEGFR/PDGFR inhibitor ordered proteins. Various powerful assays probe structure and stability of proteins. In vitro methods using purified protein include spectroscopic methods such as Circular Dichroism for secondary structure analysis, intrinsic fluorescence for tertiary structure analysis and NMR for residue-specific information. Thermal methods such as Differential Scanning Calorimetry and Isothermal Titration Calorimetry quantitatively determine protein stability and interactions by monitoring changes of enthalpy and entropy. Several strategies probe biophysical parameters in vivo or ex vivo, such as in vivo folding sensors using fluorescent proteins or fluorescent small-molecule tags or ex vivo pulse proteolysis. Inspired by the versatility of proteolysis as a label-free method, we aimed at developing a fast and broadly applicable proteolytic assay that probes thermal protein melting ex vivo using common laboratory equipment. We used the thermostable protease Thermolysin which preferentially cleaves near the hydrophobic residues Phe, Leu, Ile, Val. TL showed sufficient specificity for unfolded states to probe protein stability in lysates within seconds. We applied the Fast parallel proteolysis assay to monitor thermal unfolding of proteins ranging from 10 to 240 kDa and varying in secondary to quarternary structure. FASTpp detected stability alterations due to ligand binding and point mutations. Moreover, FASTpp can probe biophysical protein stability in cell lysates for biomedical screenings without genetic manipulation. To reconcile our data in structural terms, we assessed the structure elements of the proteins analysed by FASTpp and compare these with our metapredictions of structural disorder using the PONDR-Fit algorithm in a simplified dichotomic representation discriminating well-structured/ordered and disordered regions. A broad range of folds compatible with the assay: all a-helical, a/b and mostly b-sheet. BSA is an example for a mostly a-helical protein containing multiple disulfide bonds. Also cytochrome C in the presence of heme as well as MBP contain a large a-helical fraction while cytochrome C in the absence of ligand was previously reported to be largely devoid of structure. Pyruvate kinase forms a 240 kDa complex with somewhat higher b-sheet content. The mostly b-sheet Sortase A protein was amenable to FASTpp analysis as well. This comparison of folds suggests that most folded domains without large internal disordered linkers may be amenable.