Fast parallel proteolysis explained

Fast parallel proteolysis (FASTpp) is a method to determine the thermostability of proteins by measuring which fraction of protein resists rapid proteolytic digestion.^[1]

History and background

Proteolysis is widely used in biochemistry and cell biology to probe protein structure.^{[2] [3]} In "limited trypsin proteolysis", low amounts of protease digest both folded and unfolded protein but at largely different rates: unstructured proteins are cut more rapidly, while structured proteins are cut at a slower rate (sometimes by orders of magnitude). Recently, several other assays of protein stability based on proteolysis have been proposed, exploiting other proteases with high specificity for cleaving unfolded proteins. These include Pulse Proteolysis,^[4] Proteolytic Scanning Calorimetry ^[5] and FASTpp.

How it works

FASTpp measures the quantity of protein that resists digestion under various conditions. To this end, a thermostable protease is used, which cleaves specifically at exposed hydrophobic residues. The FASTpp assay combines the thermal unfolding, specificity of a thermostable protease for the unfolded fraction with the separation power of SDS-PAGE.^[6] Due to this combination, FASTpp can detect changes in the fraction folded over a large physico-chemical range of conditions including temperatures up to 85 °C, pH 6–9, presence or absence of the whole proteome. Applications range from biotechnology to study of point mutations and ligand binding assays.

Applications

FASTpp has been used to probe:^[1]

• Lysate effect on protein stability
• Thermal proteome stability ^[7]
• Coupled folding and binding ^[8]
• Ligand effects on fraction folded & stability ^[9]
• Effects of mutations on fraction folded & stability (e.g. point mutation/missense mutations^{[9] [10]})
• Kinetic protein stability ^[11]

Technology

First, a cell lysate is generated by glass beads beating, pressure homogenisation or chemical or physical lysis methods that do not denature the protein(s) of interest. (Optionally for targeted analysis) a protein of interest is purified out of this lysate by affinity methods based on intrinsically disordered tags ^[12] or other suitable purification strategies, often involving several orthogonal chromatographic steps.

This (total or purified) protein solution is aliquoted into several tubes of a PCR strip. All aliquots are exposed in parallel in a thermal gradient PCR cycler to different maximal temperatures in presence of the thermostable protease thermolysin (see figure). Automated temperature control is achieved in a thermal gradient cycler (commonly used for PCRs). Reaction products can be separated by SDS-PAGE or western blot.^[6] The protease thermolysin can be fully inactivated by EDTA. This feature of thermolysin makes FASTpp compatible with subsequent trypsin digestion e.g. for mass spectrometry.^{[13] [14] [7]}

Notes and References

1. Minde . D. P. . Maurice . M. M. . Rüdiger . S. G. D. . Uversky . Vladimir N . Determining Biophysical Protein Stability in Lysates by a Fast Proteolysis Assay, FASTpp . 10.1371/journal.pone.0046147 . PLOS ONE . 7 . 10 . e46147 . 2012 . 23056252 . 3463568 . 2012PLoSO...746147M . free .
2. Johnson . D. E. . Xue . B. . Sickmeier . M. D. . Meng . J. . Cortese . M. S. . Oldfield . C. J. . Le Gall . T. . Dunker . A. K. . Uversky . V. N. . 10.1016/j.jsb.2012.05.013 . High-throughput characterization of intrinsic disorder in proteins from the Protein Structure Initiative . Journal of Structural Biology . 180 . 1 . 201–215 . 2012 . 22651963 . 3578346 .
3. Hoelen . H. . Kleizen . B. . Schmidt . A. . Richardson . J. . Charitou . P. . Thomas . P. J. . Braakman . I. . Uversky . Vladimir N . The Primary Folding Defect and Rescue of ΔF508 CFTR Emerge during Translation of the Mutant Domain . 10.1371/journal.pone.0015458 . PLOS ONE . 5 . 11 . e15458 . 2010 . 21152102 . 2994901 . 2010PLoSO...515458H . free .
4. Park . C. . Marqusee . S. . 10.1038/nmeth740 . Pulse proteolysis: A simple method for quantitative determination of protein stability and ligand binding . Nature Methods . 2 . 3 . 207–212 . 2005 . 15782190 . 21364478 .
5. Tur-Arlandis . G. . Rodriguez-Larrea . D. . Ibarra-Molero . B. . Sanchez-Ruiz . J. M. . Proteolytic Scanning Calorimetry: A Novel Methodology that Probes the Fundamental Features of Protein Kinetic Stability . 10.1016/j.bpj.2009.11.028 . Biophysical Journal . 98 . 6 . L12–L14 . 2010 . 20303845 . 2849053 . 2010BpJ....98L..12T .
6. 10.1038/227680a0 . Laemmli . U. K. . Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature . 227 . 5259 . 680–685 . 1970 . 5432063. 1970Natur.227..680L . 3105149 .
7. 28232526 . 2017. Leuenberger. P. Ganscha. S. Kahraman. A. Cell-wide analysis of protein thermal unfolding reveals determinants of thermostability.. 10.1126/science.aai7825. Science. 355. 6327. eaai7825. 8432125.
8. Demarest . S. J. . Martinez-Yamout . M. . Chung . J. . Chen . H. . Xu . W. . Dyson . H. J. . Jane Dyson. Evans . R. M. . Wright . P. E. . 10.1038/415549a . Mutual synergistic folding in recruitment of CBP/p300 by p160 nuclear receptor coactivators . Nature . 415 . 6871 . 549–553 . 2002 . 11823864 . 4423920 .
9. 10.1016/j.bbapap.2015.01.004. Impaired tropomyosin–troponin interactions reduce activation of the actin thin filament. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 2015. Robaszkiewicz . K. . Ostrowska . Z. . Cyranka-Czaja . A. . Moraczewska . J. . 1854 . 5. 381–390 . 25603119.
10. Minde . D. P. . Anvarian . Z. . Rüdiger . S. G. . Maurice . M. M. . Messing up disorder: How do missense mutations in the tumor suppressor protein APC lead to cancer? . 10.1186/1476-4598-10-101 . Molecular Cancer . 10 . 101 . 2011 . 21859464. 3170638 . free .
11. Tur-Arlandis . G. . Rodriguez-Larrea . D. . Ibarra-Molero . B. . Sanchez-Ruiz . J. M. . Proteolytic Scanning Calorimetry: A Novel Methodology that Probes the Fundamental Features of Protein Kinetic Stability . 10.1016/j.bpj.2009.11.028 . Biophysical Journal . 98 . 6 . L12–L14 . 2010 . 20303845 . 2849053 . 2010BpJ....98L..12T .
12. 28516025. 2013. Minde. DP. Halff. EF. Tans. SJ. Designing disorder: Tales of the unexpected tails.. 10.4161/idp.26790. Intrinsically Disordered Proteins. 1. 1. 5424805. e26790.
13. 22733688. 2012. Chang. Y. Schlebach. JP. Verheul. RA. Park. C. Simplified proteomics approach to discover protein-ligand interactions. 21. 9. 1280–7. 10.1002/pro.2112. Protein Science. 3631357.
14. 15782190. 2005. Park. C. Marqusee. S. Pulse proteolysis: A simple method for quantitative determination of protein stability and ligand binding. 2. 3. 207–12. 10.1038/nmeth740. Nature Methods. 21364478.