Proteins under Pressure High-Pressure SAXS

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
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.c
a/educmat/chm730/d730.htm
5
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 isnt 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 dont come out into
  water).
• Proteins unfold as a result of water penetration.
  
• Protein becomes more soluble in water.

12
Water as the solvent

• What doesnt 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 lt-gt 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
gt 10 kbar
gt30 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 IIo exp(-Rg2/3)
• Detect global size changes.
• -gt 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).