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doi: 10.1371/journal.pone.0046147. Epub 2012 Oct 3.

Determining biophysical protein stability in lysates by a fast proteolysis assay, FASTpp

Affiliations

Determining biophysical protein stability in lysates by a fast proteolysis assay, FASTpp

David P Minde et al. PLoS One.2012.

Abstract

The biophysical stability is an important parameter for protein activity both in vivo and in vitro. Here we propose a method to analyse thermal melting of protein domains in lysates: Fast parallel proteolysis (FASTpp). Combining unfolding by a temperature gradient in a thermal cycler with simultaneous proteolytic cleavage of the unfolded state, we probed stability of single domains in lysates. We validated FASTpp on proteins from 10 kDa to 240 kDa and monitored stabilisation and coupled folding and binding upon interaction with small-molecule ligands. Within a total reaction time of approximately 1 min, we probed subtle stability differences of point mutations with high sensitivity and in agreement with data obtained by intrinsic protein fluorescence. We anticipate a wide range of applications of FASTpp in biomedicine and protein engineering as it requires only standard laboratory equipment.

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Conflict of interest statement

Competing Interests:The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. FASTpp combines automated temperature control and quantitatively characterised proteolysis to unveal protein interactions and stability.
A, Protein stability can be probed by measuring the thermal unfolding transition in the presence of a protease. The folded state resists protease digestion while the unfolded state is readily digested on the same timescale. The thermal unfolding transition of a protein may be shifted to higher temperatures by addition of a ligand of the folded state. A shift to lower transition temperatures may occur upon destabilisation of the protein by, for instance, cancer mutations.B, Temperatures are controlled automatically using a standard gradient PCR setup. A mastermix of sample and protease is prepared on ice or in a cold room at 4°C and subsequently aliquoted to a PCR strip that is simultaneously heated up during the heating timeth to a range of melting temperatures that are kept for a variable melting timetm. Subsequently simultaneous cooling (cooling time,tc ) brings all aliquots back to 4°C and the reaction is quenched by addition of EDTA.C, Scheme of all seven processing steps of the FASTpp assay. The representation of the termocycler indicates the automated steps of the FASTpp protocol, the gel indicates the final analysis by SDS-PAGE (T, temperature; ΔT, change of temperature; x–y°C, melting temperature gradient).
Figure 2
Figure 2. Thermolysin is active from 20°C to 80°C.
Thermal dependence of intrinsic rates of proteolysis of TL from 20°C to 80°C. After extensive thermal pre-equilibration, we determined kinetic traces of cleavage of an unstructured model substrate of TL, in triplicate for each temperature. The obtained rates were extrapolated based on empirically derived formulas to final concentrations of 0.1 g/L and on top 0.01 g/L of TL .
Figure 3
Figure 3. TL preferentially cleaves unfolded protein.
A, Thermal TL resistance ofapo Cytochrome C (CytC). Only in absence of protease at 4°C,apo CytC was detected on the gel. Upon addition of TL, cleavage already occured on ice and no protease resistant protein was observed after additional incubation at higher temperatures.B, Thermal protease resistance of CytC in presence of the bound heme ligand. A strong band of CytC is visible in absence and presence of TL from temperatures from 4°C to 60°C.
Figure 4
Figure 4. FASTpp is robust over 3 orders of magnitude of TL concentration changes.
A, Thermal TL resistance of MBP using limiting TL concentration of 0.001 g/L. Over the entire temperature range from 50°C to 70°C, MBP remains intact.B, Thermal protease resistance of MBP using a TL concentration of 0.01 g/L. At this TL concentration, a clear thermal unfolding transition becomes apparent between 50°C and 60°C. Likely due to kinetic competition between irreversible aggregation and proteolytic cleavage of the unfolded state, some MBP is not digested at 69 and 70°C.C, Thermal protease resistance of MBP using limiting TL concentration of 0.1 g/L. A similar unfolding transition of MBP as in B is observed.D, Thermal protease resistance of MBP using limiting TL concentration of 1 g/L. A similar thermal unfolding transition of MBP is observed as in B.
Figure 5
Figure 5. FASTpp can monitor kinetic stability of proteins by change of tm.
A, Thermal TL resistance of MBP using 6 stm. MBP was increasingly cleaved from 40°C to 60°C.B, Thermal TL resistance of MBP using 60 stm. MBP was increasingly accessible to digestion from 40°C to 53°C. Above 53°C, no MBP was detected.C, Thermal TL resistance of MBP using 600 s tm. MBP was increasingly accessible to digestion from 40°C to 49°C. Above 49°C temperature, no MBP was detected.
Figure 6
Figure 6. FASTpp can detect ligand effect on purified protein and in complex mixtures.
A, FASTpp of purified MBP. Unfolding was observed above 58°C.B, FASTpp of purified MBP plus 5 mM maltose. Unfolding was not observed up to 70°C.C, FASTpp proteolysis of MBP overexpression lysate. Unfolding was observed above 59°C.D, FASTpp of MBP overexpression lysate plus 5 mM maltose. Unfolding was not observed up to 70°C.E, Fluorescence melting curves of MBP. MBP melted in absence of a ligand between 30°C and 40°C. In presence of 5 mM maltose, unfolding was observed between 40°C and 50°C.
Figure 7
Figure 7. Maltose does not stabilise a control substrate that has no known maltose-binding activity.
A, FASTpp of BSA in absence of maltose. BSA was completely digested at temperatures from 62°C to 70°C after a gradual unfolding transition over a range of temperatures from 51 to 59°C.B, FASTpp of BSA in presence of 5 mM maltose. BSA was completely digested at temperatures from 62°C to 70°C after a gradual unfolding transition over a range of temperatures from 51 to 59°C.
Figure 8
Figure 8. Ligand-dependent stability of a 240 kDa protein can be probed by FASTpp.
A, Pyruvat kinase (PK) FASTpp. PK was resistant from 4°C to 58°C. 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 4°C to 59.6°C. Already at 60.4°C, nearly complete digestion was observed.
Figure 9
Figure 9. Missense mutation effects on protein stability can be probed by FASTpp.
A, Intrinsic fluorescence temperature depence of three Sortase A variants. 3×M is triplemutant, 4×M is tetramutant, 5×M is pentamutant.B, FASTpp of the same three Sortase A variants as in A.
Figure 10
Figure 10. FASTpp is suitable for a wide range of substrates.
Representative snapshots from crystallographic studies on the used model proteins. BSA is α-helically folded (pdb identifier 1E7I), MBP has some β-sheets (pdb identifier 1JWY, 1ANF), PK contains more β-sheets (pdb identifier 1F3W), Sortase A mostly β-sheets (pdb identifier 1T2O) and folded Cytochrome C in presence of heme contains extended loops (pdb identifier 1AKK) , , , . The PONDR-FIT predictions are shown in black frames in a simplified view with black indicating a score for intrinsic disorder above 0.5 and background color scores from 0 to 0.5.
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