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 haven�t 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

7. Solution Behavior Solvent Factors governing solubili

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 a=b Oblate spheroids a>b Axis of rotation is b Prolate Spheroids a>b 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. f=kbT/D Frictional ratio expressed as f/fo Always greater than unity because of hydration. 1.05-1.38

12. Selected hydrodynamic data1

13. 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 S=10-13 s =Svedberg S is Mass, shape dependent as well as density.

14. Gel Filtration See chapter one for details

15. Rotation Very sensitive to shape Measured as relaxation time tR Correlation time + 1/3 relaxation Rotational difusion constant 1/(2 tR) tR=3Vh0 /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 don�t 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

19. 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 pKa�s

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.

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