Pp of BSA in absence of maltose. BSA was completely digested at temperatures from 62uC to 70uC after a gradual unfolding CAL-120 chemical information MedChemExpress (-)-Indolactam V transition over a range of temperatures from 51 to 59uC. B, FASTpp of BSA in presence of 5 mM maltose. BSA was completely digested at temperatures from 62uC to 70uC after a gradual unfolding transition over a range of temperatures from 51 to 59uC. doi:10.1371/journal.pone.0046147.gFast Proteolysis Assay FASTppFigure 8. Ligand-dependent stability of a 240 kDa protein can be probed by FASTpp. A, Pyruvat kinase (PK) FASTpp. PK was resistant from 4uC to 58uC. A gradual decrease in the band intensity at higher temperatures indicates unfolding. Over a broad range of even higher temperatures, a small fraction of protease-resistant species persists (that likely represent aggregates formed rapidly upon unfolding). B, FASTpp of PK in presence of 5 mM ATP. PK was resistant against TL digestion from 4uC to 59.6uC. Already at 60.4uC, nearly complete digestion was observed. doi:10.1371/journal.pone.0046147.gFirst, we analysed these variants by FASTpp. To achieve an accurate relative quantification, we made use of the strong infrared fluorescence enhancement of Coommassie dyes upon protein binding [23]. Upon quantification, we obtained the following order of stability: 36M and 46M are equally stable with a transition starting above 40uC; the 56M variant displayed a less cooperative thermal unfolding transition consistent with an entropically broadened transition (Fig. 9B). Significantly more residual protein remained above 50uC for this protein variant. Second, we used intrinsic fluorescence to probe stability differences. The variants 36M and 46M behaved very similar in this assay with non-linear fluorescence decay above a Tu of 40uC, while 56M appeared to be slightly more stable with linear decrease continuing up to a Tu of 43uC (Fig. 9A). We can only achieve a qualitative validation of our FASTpp data by comparison to 
fluorescence data due to several physical differences between the two assays: 1. Heating times (hours in fluorescence, minutes in FASTpp) 2. Fluorescence measures in equilibrium until unfolding and aggregation start while FASTpp constantly removes unfolded protein from the equilibrium ?an effect that increases with tm. The results of FASTpp agree qualitatively with intrinsic fluorescence analysis of Sortase A variants. We conclude that FASTpp is sufficiently sensitive to detect subtle stability differences caused by point mutations.Figure 9. Missense mutation effects on protein stability can be probed by FASTpp. A, Intrinsic fluorescence temperature depence of three Sortase A variants. 36M is triplemutant, 46M is tetramutant, 56M is pentamutant. B, FASTpp of the same three Sortase A variants as in A. doi:10.1371/journal.pone.0046147.gFASTpp 23727046 is applicable to a wide range of protein foldsTo reconcile our data in structural terms, we assessed 
the structure elements of the proteins analysed by FASTpp andcompare these with our metapredictions of structural disorder using the PONDR-Fit algorithm in a simplified dichotomic representation discriminating well-structured/ordered and disordered regions (Fig. 10) [24]. A broad range of folds compatible with the assay: all a-helical, a/b and mostly b-sheet [25?8]. 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 lig.Pp of BSA in absence of maltose. BSA was completely digested at temperatures from 62uC to 70uC after a gradual unfolding transition over a range of temperatures from 51 to 59uC. B, FASTpp of BSA in presence of 5 mM maltose. BSA was completely digested at temperatures from 62uC to 70uC after a gradual unfolding transition over a range of temperatures from 51 to 59uC. doi:10.1371/journal.pone.0046147.gFast Proteolysis Assay FASTppFigure 8. Ligand-dependent stability of a 240 kDa protein can be probed by FASTpp. A, Pyruvat kinase (PK) FASTpp. PK was resistant from 4uC to 58uC. A gradual decrease in the band intensity at higher temperatures indicates unfolding. Over a broad range of even higher temperatures, a small fraction of protease-resistant species persists (that likely represent aggregates formed rapidly upon unfolding). B, FASTpp of PK in presence of 5 mM ATP. PK was resistant against TL digestion from 4uC to 59.6uC. Already at 60.4uC, nearly complete digestion was observed. doi:10.1371/journal.pone.0046147.gFirst, we analysed these variants by FASTpp. To achieve an accurate relative quantification, we made use of the strong infrared fluorescence enhancement of Coommassie dyes upon protein binding [23]. Upon quantification, we obtained the following order of stability: 36M and 46M are equally stable with a transition starting above 40uC; the 56M variant displayed a less cooperative thermal unfolding transition consistent with an entropically broadened transition (Fig. 9B). Significantly more residual protein remained above 50uC for this protein variant. Second, we used intrinsic fluorescence to probe stability differences. The variants 36M and 46M behaved very similar in this assay with non-linear fluorescence decay above a Tu of 40uC, while 56M appeared to be slightly more stable with linear decrease continuing up to a Tu of 43uC (Fig. 9A). We can only achieve a qualitative validation of our FASTpp data by comparison to fluorescence data due to several physical differences between the two assays: 1. Heating times (hours in fluorescence, minutes in FASTpp) 2. Fluorescence measures in equilibrium until unfolding and aggregation start while FASTpp constantly removes unfolded protein from the equilibrium ?an effect that increases with tm. The results of FASTpp agree qualitatively with intrinsic fluorescence analysis of Sortase A variants. We conclude that FASTpp is sufficiently sensitive to detect subtle stability differences caused by point mutations.Figure 9. Missense mutation effects on protein stability can be probed by FASTpp. A, Intrinsic fluorescence temperature depence of three Sortase A variants. 36M is triplemutant, 46M is tetramutant, 56M is pentamutant. B, FASTpp of the same three Sortase A variants as in A. doi:10.1371/journal.pone.0046147.gFASTpp 23727046 is applicable to a wide range of protein foldsTo reconcile our data in structural terms, we assessed the structure elements of the proteins analysed by FASTpp andcompare these with our metapredictions of structural disorder using the PONDR-Fit algorithm in a simplified dichotomic representation discriminating well-structured/ordered and disordered regions (Fig. 10) [24]. A broad range of folds compatible with the assay: all a-helical, a/b and mostly b-sheet [25?8]. 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 lig.