Chapter 7 Proteins TE Creighton
Proteins in Solutions and Membranes y The folded conformations of a native protein gives it
properties that are different from the unfolded polypeptide chain. y The properties are NOT only the sum of all properties of the single aminoacids y The compactness allows the proteins to rotate and diffuse rapidly. y Domains of proteins are relatively resistant to protease.
Proteins in Solutions and Membranes y Multidomain proteins can be separated by protease y y y y
treatment. The separated domains can be reconstituted to form a functional protein The folded conformation of proteins brings residues into close proximity They are being held in place by the fold Their local relative concentration is so high that reactions occur between them.
Proteins in Solutions and Membranes y Many of these properties are not evident in protein y y y y
crystals Appear in solution or membranes Proteins need a certain flexibility Protein conformation is largely unaltered when in a crystal Exceptions intrinsically flexible sidechains and surface loops
Proteins in Solutions and Membranes y The intermolecular forces of proteins in a crystal
y y y y y y y
lattice are similar to the intra molecular forces of a folded protein Crystallization conditions favor the folded proteins Exception to this Very small proteins They have the most mobile conformations Glucagon: 29 aa Diluted solution‐‐‐ random coil Concentrated solution ‐‐‐ trimeric helical structure
Proteins in Solutions and Membranes y Conformations of small peptides in crystal structures y y y y
need to be validated in solution Protein domains have only one compact folded structure Conformational changes in a protein are mainly used to rationalize unexpected protein behaviour Many conformational changes may involve localised alterations or changes in degree of flexibility. Structural rearrangements have been found only for quarternary structures
Aqueous solubility y Some proteins aree extremely soluble y Structural proteins are nearly insoluble y Proteins interact with the solvent with their surfaces y Globular proteins have charged and polar residues on
their surfaces y Solubility is goverend by their interactions with water y Structural proteins interact with other proteins more strongly than with water
Aqueous solubility y Solubility of a protein increases at pH values farther
away from pI y pI of a protein is the pH where the protein has zero net charge y At extreme pH values proteins unfold ‐‐‐affects solubility y Most proteins can be solubilised in aqueous solution by adding detergents or chaotropic ions (urea, GdnHCl)
Aqueous solubility
Preferential Hydration
Hydrodynamic Properties in Aqueous Solution Diffusion
Einstein‐Sutherland equation
Sedimentation analysis
The Svedberg Equation
Gel Filtration Rotation
Hydrodynamic properties
Spectral Properties ‐ fluorescence
Chemical Properties
Chemical Properties
Chemical Properties
Chemical Properties
Chemical Properties
Membrane Proteins
Side Chain Rotations
Reversible Unfolding transitions
Reversible Unfolding Transitions
Reversible Unfolding Transition
Reversible Unfolding Transition
Reversible Unfolding Transition
Reversible Unfolding Transition
Reversible Unfolding Transition
Reversible Unfolding Transition For a two‐state transition, the equilibrium constant between the N and V states can be measured directly from the average fraction of unfolding (a) in the transition region:
Where the value of a is significantly different from 0 or 1, the value of Keq is known. This gives the free energy of N relative to that of U, ΔGfold' under each set of conditions :
Reversible Unfolding Transition
Nature of the unfolded state y Some proteins have been found in a state that is neither folded y y y y y y y
nor unfolded. The molten globule state: 1) the overall dimensions of the protein aer much less then for the rndom coil and only slightly larger than for the folded state 2) the average content of secondary structure is similar tot he folded state 3) the side chains are in homogenous surrounding 4) Many amide groups exchange hydrogens much more rapidly than they do in the folded state 5) the enthalpy of the molten globule is nearly the same as for the fully unnfolded state Interconversion of the MG state with the folded state are slow and cooperative. MG – unfolded are rapid and non cooperaive
Nature of the unfolded state
Nature of the unfolded state
Nature of the unfolded state
Nature of the unfolded state
Thermodynamics of Unfolding
Thermodynamics of Unfolding
Thermodynamics of Unfolding
Thermodynamics of Unfolding
Thermodynamics of Unfolding
Thermodynamics of Unfolding
Thermodynamics of Unfolding
Thermodynamics of Unfolding
Thermodynamics of Unfolding
Thermodynamics of Unfolding
Thermodynamics of Unfolding
Thermodynamics of Unfolding
Thermodynamics of Unfolding
Thermodynamics of Unfolding
Thermodynamics of Unfolding
Thermodynamics of Unfolding
Thermodynamics of Unfolding
Thermodynamics of Unfolding
Thermodynamics of Unfolding
Thermodynamics of Unfolding
Thermodynamics of Unfolding
Biosynthetic folding y Molecular chaperones y Prolyl peptide isomerases y Protein disulfide isomerase