Se on MBP.proteolysis in lysate up to 59uC (Fig. 6C

Se on MBP.proteolysis in lysate up to 59uC (Fig. 6C). From 61uC to 70uC, the apo MBP band intensity was nearly lost. In contrast, MBP’s proteolytic resistance persisted up to 70uC in presence of 5 mM maltose ligand (Fig. 6D). Both for purified protein and lysate samples, maltose addition increases the unfolding temperature by more than 10uC. Interestingly, when compared with purified MBP, the apo MBP lysate displays a sudden unfolding transition between 59uC and 61uC while purified MBP has a much broader unfolding range between 50uC and 58uC. Lysate stabilised MBP without addition of maltose ligand. Since lysates are complex mixtures we assume that the balance of all (presumably mostly weak and transient) interactions determines the differences between biophysical stability of protein in lysates compared to experiments with purified proteins in more diluted solutions of isolated proteins [14,17?1]. We conclude that FASTpp is suitable to monitor stability changes in whole cell lysates.BSA thermostability is not affected by maltoseTo exclude un58543-16-1 web specific protein stabilisation by maltose, we monitored the stability of the non-maltose-binding protein BSA in the presence and absence of maltose. Maltose did not change the thermal unfolding transition of BSA in a buffer with reducing redox potential between 4uC and 59uC (Fig. 7A, B). This corroborates our conclusion that FASTpp detects specific ligand stabilisation effects.FASTpp determines protein stability in lysateTo test whether FASTpp is also suitable to assay protein stability in lysates, we compared the in vitro stability of MBP to the ex vivo stability of E. coli lysate overexpressing MBP. MBP resistsFast Proteolysis Assay FASTppFigure 6. FASTpp can detect ligand effect on purified protein and in complex mixtures. A, FASTpp of purified MBP. Unfolding was observed above 58uC. B, FASTpp of purified MBP plus 5 mM maltose. Unfolding was not observed up to 70uC. C, FASTpp proteolysis of MBP 166518-60-1 overexpression lysate. Unfolding was observed above 59uC. D, FASTpp of MBP overexpression lysate plus 5 mM maltose. Unfolding was not observed up to 70uC. E, Fluorescence melting curves of MBP. MBP melted in absence of a ligand between 30uC and 40uC. In presence of 5 mM maltose, unfolding was observed between 40uC and 50uC. doi:10.1371/journal.pone.0046147.gLarge proteins assemblies can be analysed with FASTppTo investigate if FASTpp is also applicable to larger proteins, we tested the 240 kDa, tetrameric Pyruvate Kinase (PK). We used a temperature range from 55 to 65uC. The protein becomes susceptible to proteolysis at 59uC (Fig. 8A, B). Surprisingly, approximately 10 of the initial PK band intensity remains at higher temperatures up to 65uC. We suspect, therefore, that thermal aggregation competes with protein cleavage above this temperature. Also, another cleavage-resistant 34 kDa fragment appears above 60uC, which might be either a more stable domain or a rapidly aggregating domain, which was protected from cleavage. We conclude that FASTpp is applicable to large multiprotein assemblies.FASTpp detects stability differences of point mutantsAs a test case for stability discrimination of point mutants, we compared three evolved Sortase A variants that have been selected for enhanced transpeptidase kinetics: Sortase A triplemutant (36M), Sortase A tetramutant (46M), Sortase A pentamutant (56M) [22].Figure 7. Maltose does not stabilise a control substrate that has no known maltose-binding activity. A, FAST.Se on MBP.proteolysis in lysate up to 59uC (Fig. 6C). From 61uC to 70uC, the apo MBP band intensity was nearly lost. In contrast, MBP’s proteolytic resistance persisted up to 70uC in presence of 5 mM maltose ligand (Fig. 6D). Both for purified protein and lysate samples, maltose addition increases the unfolding temperature by more than 10uC. Interestingly, when compared with purified MBP, the apo MBP lysate displays a sudden unfolding transition between 59uC and 61uC while purified MBP has a much broader unfolding range between 50uC and 58uC. Lysate stabilised MBP without addition of maltose ligand. Since lysates are complex mixtures we assume that the balance of all (presumably mostly weak and transient) interactions determines the differences between biophysical stability of protein in lysates compared to experiments with purified proteins in more diluted solutions of isolated proteins [14,17?1]. We conclude that FASTpp is suitable to monitor stability changes in whole cell lysates.BSA thermostability is not affected by maltoseTo exclude unspecific protein stabilisation by maltose, we monitored the stability of the non-maltose-binding protein BSA in the presence and absence of maltose. Maltose did not change the thermal unfolding transition of BSA in a buffer with reducing redox potential between 4uC and 59uC (Fig. 7A, B). This corroborates our conclusion that FASTpp detects specific ligand stabilisation effects.FASTpp determines protein stability in lysateTo test whether FASTpp is also suitable to assay protein stability in lysates, we compared the in vitro stability of MBP to the ex vivo stability of E. coli lysate overexpressing MBP. MBP resistsFast Proteolysis Assay FASTppFigure 6. FASTpp can detect ligand effect on purified protein and in complex mixtures. A, FASTpp of purified MBP. Unfolding was observed above 58uC. B, FASTpp of purified MBP plus 5 mM maltose. Unfolding was not observed up to 70uC. C, FASTpp proteolysis of MBP overexpression lysate. Unfolding was observed above 59uC. D, FASTpp of MBP overexpression lysate plus 5 mM maltose. Unfolding was not observed up to 70uC. E, Fluorescence melting curves of MBP. MBP melted in absence of a ligand between 30uC and 40uC. In presence of 5 mM maltose, unfolding was observed between 40uC and 50uC. doi:10.1371/journal.pone.0046147.gLarge proteins assemblies can be analysed with FASTppTo investigate if FASTpp is also applicable to larger proteins, we tested the 240 kDa, tetrameric Pyruvate Kinase (PK). We used a temperature range from 55 to 65uC. The protein becomes susceptible to proteolysis at 59uC (Fig. 8A, B). Surprisingly, approximately 10 of the initial PK band intensity remains at higher temperatures up to 65uC. We suspect, therefore, that thermal aggregation competes with protein cleavage above this temperature. Also, another cleavage-resistant 34 kDa fragment appears above 60uC, which might be either a more stable domain or a rapidly aggregating domain, which was protected from cleavage. We conclude that FASTpp is applicable to large multiprotein assemblies.FASTpp detects stability differences of point mutantsAs a test case for stability discrimination of point mutants, we compared three evolved Sortase A variants that have been selected for enhanced transpeptidase kinetics: Sortase A triplemutant (36M), Sortase A tetramutant (46M), Sortase A pentamutant (56M) [22].Figure 7. Maltose does not stabilise a control substrate that has no known maltose-binding activity. A, FAST.