Fast parallel proteolysis

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FASTpp (Fast parallel proteolysis). A protein mixture is aliquoted into several tubes, which are exposed in parallel to different temperatures and a thermostable protease. The remaining protein can be resolved on SDS-PAGE.

Fast parallel proteolysis (FASTpp) is a method to determine the thermostability of proteins that does not need previous purification and labelling.[1]

History and background[edit]

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[edit]

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 cytosolic proteome. Applications range from biotechnology to study of point mutations and ligand binding assays.

Applications[edit]

FASTpp has been used to probe:[1]

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

Technology[edit]

Principles[edit]

A protein mixture is aliquoted into several tubes, which are exposed in parallel to different temperatures and a thermostable protease (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.[11][12]

Importance of (non)-equilibrium[edit]

Many complementary protein stability assays (such as nuclear magnetic resonance, circular dichroism, fluorescence spectroscopy) use (near)equilibrium conditions, which allow for determining free energy differences of unfolding. FASTpp measures in (automatically controlled and therefore highly reproducible) non-equilibrium conditions: It constantly removes the unfolded fraction by cutting at exposed hydrophobic stretches. The proteasome might have a similar effect on unfolded protein in the living eukaryotic cell: Its 20S core can degrade unfolded protein without energy supply.[13]

See also[edit]

References[edit]

  1. ^ a b Minde, D. P.; Maurice, M. M.; Rüdiger, S. G. D. (2012). Uversky, Vladimir N, ed. "Determining Biophysical Protein Stability in Lysates by a Fast Proteolysis Assay, FASTpp". PLoS ONE 7 (10): e46147. doi:10.1371/journal.pone.0046147. PMC 3463568. PMID 23056252.  edit
  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. (2012). "High-throughput characterization of intrinsic disorder in proteins from the Protein Structure Initiative". Journal of Structural Biology 180 (1): 201–215. doi:10.1016/j.jsb.2012.05.013. PMC 3578346. PMID 22651963.  edit
  3. ^ Hoelen, H.; Kleizen, B.; Schmidt, A.; Richardson, J.; Charitou, P.; Thomas, P. J.; Braakman, I. (2010). Uversky, Vladimir N, ed. "The Primary Folding Defect and Rescue of ΔF508 CFTR Emerge during Translation of the Mutant Domain". PLoS ONE 5 (11): e15458. doi:10.1371/journal.pone.0015458. PMC 2994901. PMID 21152102.  edit
  4. ^ Park, C.; Marqusee, S. (2005). "Pulse proteolysis: A simple method for quantitative determination of protein stability and ligand binding". Nature Methods 2 (3): 207–212. doi:10.1038/nmeth740. PMID 15782190.  edit
  5. ^ Tur-Arlandis, G.; Rodriguez-Larrea, D.; Ibarra-Molero, B.; Sanchez-Ruiz, J. M. (2010). "Proteolytic Scanning Calorimetry: A Novel Methodology that Probes the Fundamental Features of Protein Kinetic Stability". Biophysical Journal 98 (6): L12–L14. doi:10.1016/j.bpj.2009.11.028. PMC 2849053. PMID 20303845.  edit
  6. ^ a b Laemmli, U. K. (1970). "Cleavage of structural proteins during the assembly of the head of bacteriophage T4". Nature 227 (5259): 680–685. doi:10.1038/227680a0. PMID 5432063.  edit
  7. ^ Demarest, S. J.; Martinez-Yamout, M.; Chung, J.; Chen, H.; Xu, W.; Dyson, H. J.; Evans, R. M.; Wright, P. E. (2002). "Mutual synergistic folding in recruitment of CBP/p300 by p160 nuclear receptor coactivators". Nature 415 (6871): 549–553. doi:10.1038/415549a. PMID 11823864.  edit
  8. ^ a b Robaszkiewicz, K.; Ostrowska, Z.; Cyranka-Czaja, A.; Moraczewska, J. (2015). "Impaired tropomyosin–troponin interactions reduce activation of the actin thin filament". Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. doi:10.1016/j.bbapap.2015.01.004.  edit
  9. ^ Minde, D. P.; Anvarian, Z.; Rüdiger, S. G.; Maurice, M. M. (2011). "Messing up disorder: How do missense mutations in the tumor suppressor protein APC lead to cancer?". Molecular Cancer 10: 101. doi:10.1186/1476-4598-10-101. PMC 3170638. PMID 21859464.  edit
  10. ^ Tur-Arlandis, G.; Rodriguez-Larrea, D.; Ibarra-Molero, B.; Sanchez-Ruiz, J. M. (2010). "Proteolytic Scanning Calorimetry: A Novel Methodology that Probes the Fundamental Features of Protein Kinetic Stability". Biophysical Journal 98 (6): L12–L14. doi:10.1016/j.bpj.2009.11.028. PMC 2849053. PMID 20303845.  edit
  11. ^ Chang, Y; Schlebach, JP; Verheul, RA; Park, C (2012). "Simplified proteomics approach to discover protein-ligand interactions". Protein science : a publication of the Protein Society 21 (9): 1280–7. doi:10.1002/pro.2112. PMID 22733688. 
  12. ^ Park, C; Marqusee, S (2005). "Pulse proteolysis: A simple method for quantitative determination of protein stability and ligand binding". Nature methods 2 (3): 207–12. doi:10.1038/nmeth740. PMID 15782190. 
  13. ^ Tsvetkov, P.; Myers, N.; Moscovitz, O.; Sharon, M.; Prilusky, J.; Shaul, Y. (2012). "Thermo-resistant intrinsically disordered proteins are efficient 20S proteasome substrates". Molecular BioSystems (Royal Society of Chemistry) 8 (1): 368–373. doi:10.1039/c1mb05283g. PMID 22027891.  edit