Hydrodynamics Or How to study the structure of a protein you haven’t crystallized.

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.

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

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

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

Solution Behavior Solvent Factors governing solubility pH Isoelectric point pH pH where protein has no net charge. Point of lowest solubilty pH Optimum Functional limit Protein Denatures

Solution Behavior Solvent Factors governing solubili Salt LowHigh Solubility

Surface/shape effects on proteins in solution

Diffusion Molecules undergo Brownian motion Translational motion is Diffusion  c/  t= D(  2 C/  x) Integrated it looks like D= x 2 /2t distance is proportional to the square root of time

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

Diffusion Observed rates of diffusion are expressed as Einstien Sutherland eq. f=k b T/D Frictional ratio expressed as f/f o Always greater than unity because of hydration

Selected hydrodynamic data 1 ProteinDimensions in Å f/f o PTI29 x 19 x Cytochrome C25x25x Carbanic anhydrase47 X 41 X Alcohol dihydrogenase45 X 55 X Carboxypeptidase A50 X 42 X

Sedimentation analysis Hydrodynamic properties assessed by movement though a gravitational field. dr/dt = {[M w (1-νρ)]/N a f }ω 2 r Rearranged to focus on sedimentation we get the Svedberg eq. s= [M w (1-νρ)]/N a f = [M w (1-νρ)]/DRT S= s =Svedberg S is Mass, shape dependent as well as density.

Gel Filtration See chapter one for details

Rotation Very sensitive to shape Measured as relaxation time  R Correlation time + 1/3 relaxation Rotational difusion constant 1/(2  R )  R =3V    / k b T Primarily Measured by Fluorescence polarization Two phenomena measured NMR on smaller molecules

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

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

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

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

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 Side chainSolution/surface pK a Interior pK a His Asp Glu Ser>14≈ 8 in serine proteases

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. Serine proteasesser 195 LysosymeIodine mediated cross link of trio 108 to glu35 Ribolucliase AHis 12 and his 119 reaction with Iodoacetate

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