Physical and Chemical Properties of Proteins in Solution

1
Physical and Chemical Properties of Proteins in
Solution

• Hydrodynamics
• Or
• How to study the structure of a protein you
  havent crystallized.

2
Solution Behavior

• Crystal structure is solution structure
• We had hoped so
• Proofs
• NMR data
• Protein crystals are mostly solvent
• Random coil loops the exception.
• -Localized by crystallization.

3
Solute

• To be soluble the solute must interact with the
  solution more favorable than its self.
• Charge

4
Solution Behavior

• Solubility
• Varies tremendously
• From insoluble to 350 mg/ml
• Solubility in Aqueous media
• Depends on surface charge
• pH
• Salts
• Often used in purification
• Co-solvents
• May also be used in purification

5
Behavior in Aqueous solutions

• Solubility in Aqueous media
• Depends on surface charge
• pH
• Salts
• Often used in purification
• Co-solvents
• May also be used in purification

6
Solution Behavior

• Solvent Factors governing solubility
• pH
• Isoelectric point

pH where protein has no net charge. Point of
lowest solubilty
1
14
7
pH
pH Optimum
Protein Denatures
Protein Denatures
Functional limit
Functional limit
7
Solution Behavior

• Solvent Factors governing solubili

Solubility
Low
High

• Salt

8
Surface/shape effects on proteins in solution
9
Diffusion

• Molecules undergo Brownian motion
• Translational motion is Diffusion
• dc/dt D(d2C/dx)
• Integrated it looks like
• D x2/2t
• distance is proportional to the square root of
  time

10
Diffusion

• Rate of motion is dependent on
• Size
• Shape
• Spherical ab
• Oblate spheroids agtb Axis of rotation is b
• Prolate Spheroids agtb axis of rotation is a
• solvent protein interactions
• Values for diffusion are for Hydrated spheres

11
Diffusion

• Observed rates of diffusion are expressed as
  Einstien Sutherland eq.
• fkbT/D
• Frictional ratio expressed as
• f/fo
• Always greater than unity because of hydration.
• 1.05-1.38

12
Selected hydrodynamic data1
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Sedimentation analysis

• Hydrodynamic properties assessed by movement
  though a gravitational field.
• dr/dt Mw(1-??)/Naf ?2r
• Rearranged to focus on sedimentation we get the
  Svedberg eq.
• s Mw(1-??)/Naf Mw(1-??)/DRT
• S10-13 s Svedberg
• S is Mass, shape dependent as well as density.

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Gel Filtration

• See chapter one for details

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Rotation

• Very sensitive to shape
• Measured as relaxation time tR
• Correlation time 1/3 relaxation
• Rotational difusion constant 1/(2 tR)
• tR3Vh0 /kbT
• Primarily Measured by Fluorescence polarization
• Two phenomena measured
• NMR on smaller molecules

16
Spectral Properties

• Absorbance
• Phe Tyr and Trp
• l max is environmentally dependent
• use cosolvents to change enviroment
• protected groups dont shift.
• Average of the whole molecule

17
Spectral Properties

• Fluorescence
• Phe Tyr and Trp
• l max is environmentally dependent
• Trp exposed vs buried
• Tyr not seen unless no Trp
• Phe not seen unless no Trp and Tyr

18
Spectral Properties

• Circular dichroism (CD) and Optical Rotary
  dispersion (ORD).
• sensitive to conformation
• Strong signals indicate Alpha helix and Beta
  sheet.
• interfering signals from disulfides and aromatic
  residues
• Reasonable probe of changes to environment of
  those residues

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• Short peptides Sum of the amino acids
• Proteins ? Sum of the amino acids
• Compact folding
• Resist protease degradation
• Stable with breaks in the peptide change
• One Primary fold
• Shifts in structure occur mostly in quatanary
  structure or domain structure.
• Why Chapter 7

20
Ionization of side chains

• ionizable side chains on the surface of a protein
  behave as those in free solution
• Ionizable side chains in the interior of a
  protein may have radically shifted pKas

21
Chemical Propreties

• Principle actor in this case is the principle of
  effective concentration.
• Proteins holds groups in positions that result in
  hyperactivity of the groups.

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Definition

• Domain Region of a protein that folds to a
  stable structure mostly independent of other
  structure in the protein (other domains).