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Protein Pressure Unfolding High-Pressure SAXS Nozomi Ando Jan. 30, 2003 Gruner Group Journal Club Cornell University,

Protein Pressure Unfolding High-Pressure SAXS Nozomi Ando Jan. 30, 2003 Gruner Group Journal Club Cornell University, Ithaca, NY 14853. Proteins. Membrane proteins Globular proteins: enzymes, antibodies, etc. Fibrous and Structural proteins: fibers, etc.

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Protein Pressure Unfolding High-Pressure SAXS Nozomi Ando Jan. 30, 2003 Gruner Group Journal Club Cornell University,

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  1. Protein Pressure Unfolding High-Pressure SAXS Nozomi Ando Jan. 30, 2003 Gruner Group Journal ClubCornell University, Ithaca, NY 14853

  2. Proteins • Membrane proteins • Globular proteins: enzymes, antibodies, etc. • Fibrous and Structural proteins: fibers, etc.

  3. special sequences function conformation Protein Structure Peptide chain, 20 amino acids

  4. Globular Protein: Folded State • Amino acid and solvent interactions (electrostatic, hydrophobic, hydrogen, S-S) determine globular conformation. • Liquid-Hydrocarbon model: hydrophobic core stabilizes globular protein. Hydrophobic core of myoglobin (cross-sectional view); polar amino acids are green non-polar amino acids are red. http://www.chembio.uoguelph.ca/educmat/chm730/d730.htm

  5. Distinguish salmon roe from imitation salmon roe by dropping into hot tea. • Mackerel is pickled in vinegar for preservation. Protein Unfolding: Sushi Restaurant When foods with proteins are exposed to heat and certain chemicals (such as vinegar), they turn white.

  6. Protein Unfolding: Pressure? • 1895 Royer discovered that high hydrostatic pressure kills bacteria. • 1899 Hite uses pressure for milk preservation. • 1914 Bridgman notices that egg white looks ‘cooked’ after pressure treatment. Though it isn’t intuitive, proteins also unfold with pressure.

  7. Temperature and Chemical Effects Temperature: • Thermal excitations disrupt interactions • Water penetrates as result of protein unfolding. Unfolds first then water enters. Chemicals: • Chemical denaturants disrupt balanced interactions • Additional solute in picture • Acids break salt bridges • Urea (reducing agent – breaks H bonds, S-S bonds)

  8. Kauzmann Paradox Pressure increases the density - more water in same volume. Q: Kauzmann Paradox: if core is hydrophobic, why does more water cause it to blow up? A: With pressure, the energy loss for bringing water into contact with the hydrophobic core is much less than the energy gained from minimizing the total volume.

  9. Hummer Model • Free energy landscape of two methane-like solutes in water. (Consider volume of solutes and hydrophobic interactions). • Pressure reduces preference for methane-methane contact • At high pressure, water molecule in between two methanes is energetically more favorable. • Amino acids are much larger than methane, so we can expect a negative DW at a lower pressure.

  10. Minimization of Volume atmospheric P hydrophobic packing? unfolding? More efficient packing is accomplished when small water molecules penetrate the hydrophobic core. (10 basket balls and 1000 golf balls – pack the basket balls clustered or separated. Which takes up less space?)

  11. Pressure Effects: Negative DV Ambient conditions: globule is (relatively) loosely packed with cavities and hydrophobic core. High pressure: By adding more water into same volume, ‘efficient packing’ becomes necessary. Water penetrates the protein interior. Conclusion: • DV is negative because water molecules go into protein (hydrophobic groups don’t come out into water). • Proteins unfold as a result of water penetration. • Protein becomes more soluble in water.

  12. Water as the solvent What doesn’t unfold with pressure? • Dry stuff • Bacterial spores • Proteins in glycerol (by extrapolation) What happens to hydrophobic molecules under pressure? • They dissolve in water. Interesting thoughts on Hydration: • Protein pressure unfolding is related to the fact that water is the solvent?? • Hydration and water packing?

  13. Protein states: elastic region Assumes 2 states: folded (native) and unfolded Folden <-> unfolded Elliptical phase diagram: -reversible, elastic -cold denaturation Snase, Rnase A

  14. Protein states: plastic region Often times, there are more than 2 states Disassociated state, molten globule state (partially unfolded, secondary structures in tact), aggregate state Transthyretin (Quintas)

  15. Protein states: order of events Simplified visualization of the order of events in terms of water packing and the volumes of structures. unfolded (1o) oligomer (4o) molten globule (2o) effects on covalent bonds (0o) monomer (3o) 3 –5 kbar 1 –2 kbar > 10 kbar >30 kbar atm P aggregation pressure *note: water turns into ice above 10 kbar at room temp.

  16. Probing Proteins in Solution High-resolution techniques (local): • FTIR • Flourescence • NMR • UV absorption Low-resolution techniques: • SAXS • DLS

  17. High-Pressure NMR (J. Jonas) NMR: external magnetic field, detect chemical shifts of atomic nuclei with nonzero spin. Detect local changes.

  18. High-Pressure Fluorescence Phosphorescence/Fluorescence: shine light of one wavelength, excite fluorophores or fluorescent dyes, get emission spectra. • Fluorescent dye: Bis-ANS binds to hydrophobic regions. • Fluorophore: Trp Detect local changes.

  19. High-Pressure SAXS Study SAXS: shine X-ray on sample, look at scattering intensity vs. scattering angle. • Guinier approximation: I~Io exp(-Rg2/3) Detect global size changes. -> for pressure studies, this may give the most relevant information.

  20. Interesting SAXS Problems (1 of 2) Protein refolding: • In elastic region, we can study the protein refolding process. Pressure has the potential for this, while temp/chemical denaturation disrupts secondary features. Hydration • Solvation: the specificity of water. Water under pressure (packing, water-water interactions)? Water-amino acid interactions? Further testing with other solvents. Bacterial Spores • Spores – efficient packing?

  21. Interesting SAXS Problems (2 of 2) Fibril Formation: • In plastic region, we can study interesting things that happen to proteins once they unfold, such as fibril formation. Solubility of Aggregates: • Can they be dissolved (Hummer theory) and refolded? Multiple Domains • Multiple-domained proteins (such as actin): what happens to domains, when?

  22. References (1 of 2) • Claude Balny, Patrick Masson and Karel Heremans, High pressure effects on biological macromolecules: from structural changes to alteration of cellular processes, BBA - Prot Struc Mol Enz, 1595, 2002, p. 3-10. • Lazlo Smeller, Pressure-temperature phase diagrams of biomolecules, BBA - Prot Struc Mol Enz, 1595, 2002, p. 11-29. • Jack A. Kornblatt and M. Judith Kornblatt, The effects of osmotic and hydrostatic pressures on macromolecular systems, BBA - Prot Struc Mol Enz, 1595, 2002, p. 30-47. • Kangcheng Ruan and Claude Balny, High pressure static fluorescence to study macromolecular structure-function, BBA - Prot Struc Mol Enz, 1595, 2002, p. 94-102. • Patrizia Cioni and Giovanni B. Strambini, Tryptophan phosphorescence and pressure effects on protein structure, BBA - Prot Struc Mol Enz, 1595, 2002, p. 116-130. • Wojciech Dzwolak, Minoru Kato and YoshihiroTaniguchi, Fourier transform infrared spectroscopy in high-pressure studies on proteins, BBA - Prot Struc Mol Enz, 1595, 2002, p. 131-144. • Jiri Jonas, High-resolution nuclear magnetic resonance studies of proteins, BBA - Prot Struc Mol Enz, 1595, 2002, p. 145-159. • Roland Winter, Synchrotron X-ray and neutron small-angle scattering of lyotropic lipid mesophases, model biomembranes and proteins in solution at high pressure, BBA - Prot Struc Mol Enz, 1595, 2002, p. 160-184. • Catherine A. Royer, Revisiting changes in pressure-induced protein unfolding, BBA - Prot Struc Mol Enz, 1595, 2002, p. 201-209. • Theodore W. Randolph, Matthew Seefeldt and John F. Carpenter, High hydrostatic pressure as a tool to study protein aggregation and amyloidosis, BBA - Prot Struc Mol Enz, 1595, 2002, p. 224-234.

  23. References (2 of 2) • Boonchai B. Boonyaratanakornkit, Chan Beum Park and Douglas S. Clark, Pressure effects on intra- and intermolecular interactions within proteins, BBA - Prot Struc Mol Enz, 1595, 2002, p. 235-249. • Horst Ludwig, Cell biology and high pressure: applications and risks, BBA - Prot Struc Mol Enz, 1595, 2002, p. 390-391. • Rikimaru Hayashi, High pressure in bioscience and biotechnology: pure science encompassed in pursuit of value, BBA - Prot Struc Mol Enz, 1595, 2002, p. 397-399. • Quintas, A., Saraiva, M.J.M., Brito, R.M.M., JBC, 274, p. 32943-32949 (1999). • Hillson, N., Onuchic, J.N., Garcia A.E., PNAS, 96, 14848-14853 (1999). • Hummer, G., Garde, S., Garcia, A.E., Paulaitis, M.E., Pratt, L.R., Phys. Chem. B, 102, 10469-10482 (1998). • Hummer, G., Garde, S., Garcia, A.E., Paulaitis, M.E., Pratt, L.R., 95, 1552-1555 (1998). • Woenckhaus, J., Kohling, R., Thiyagarajan, P., Littrell, K.C., Royer, C.A., Winter, R., Biophysical Journal, 80, 1518-1523 (2000). • Ferrao-Gonzales, Astria D.; Souto, Sandra O.; Foguel, Debora, PNAS, 97, 6445-6450 (2000).

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