Biochemistry: pKa Values and Dielectric Constants

The effect of structure on pKa values

• The pKa value of a residue, which reflects its propensity to be protonated or deprotonated, can differ significantly within a protein compared to its solution value.
• The pKa shift arises from local interactions within the folded protein, which can favor a specific residue to be either neutral or charged, altering the energetics of protonation.

Mobile Dipoles and Dielectric Constant

• The dielectric constant of a medium influences the strength of electrostatic interactions, with higher dielectric constants attenuating these interactions.
• Water has a high dielectric constant due to its strong dipole moment and mobility, while non-polar solvents have low dielectric constants due to their weaker dipoles.
• The dielectric constant within a protein varies depending on location, with the protein interior having a lower dielectric constant than the surface.

Charge Interactions in Proteins

• The protein surface is typically dominated by charged residues, which interact with the solvent and dissolved ions.
• These charge-charge interactions are weakened by competition from dissolved ions.
• Charged residues contribute to protein solubility, but often do not form stable pairs.
• The low dielectric constant of protein interiors disfavors burial of charged residues, requiring stabilization by multiple hydrogen bonds.

Salt Bridges

• A salt bridge is a stabilizing interaction between oppositely charged residues, driven by electrostatic and hydrogen bonding forces.
• They contribute significantly to the stabilization of proteins, especially in less dielectric environments.
• Due to their temperature independence, salt bridges are crucial for stabilizing thermophilic proteins, which function at high temperatures.

Cation-Pi Interactions and Pi-Pi Stacking

• Aromatic residues, due to their delocalized pi electrons, can form favorable interactions with positive charges (cation-pi) and with each other(pi-pi stacking).
• This interaction strength is around 12 kJ/mol.

Entropic Contributions to Protein Energetics

• Entropy, reflecting the number of available states for a system, influences protein folding.
• Changes in protein conformation involve changes in the entropy of the protein itself (conformational entropy) and the entropy of surrounding water (hydrophobic effect).

Folding a Protein Entails Loss of Conformational Entropy

• Protein chains are highly flexible and sample a vast number of conformations in solution.
• This conformational entropy is significantly reduced upon folding into a defined structure, representing an entropic cost.

Conformational Entropy and Amino Acid Architecture

• Hydrophobic amino acids, with fewer rotatable bonds, minimize the entropic cost of burial.
• Many hydrophilic amino acids, with greater flexibility, tend to be surface exposed.

Liquid Water Structure

• The structure of liquid water is based on extensive hydrogen bonding, involving a continuous dynamic network.

The Hydrophobic Effect

• Non-polar solutes disrupt the water network, forcing rearrangement to maintain hydrogen bond integrity, creating cage-like structures.
• These cages restrict water molecule mobility, resulting in an entropic penalty for dissolving non-polar molecules.
• However, association of non-polar solutes releases some "caged" water molecules, leading to a net entropic gain and driving interactions between them.

Measuring Hydropathy

• Hydropathy is an empirical measure of the preference of a molecule for an aqueous or nonpolar environment.
• It can be measured as a partition coefficient between water and a nonpolar solvent, with a negative free energy of transfer indicating a preference for a non-aqueous environment.

Hydrophobic Interactions of Amino Acid Residues

• The free energy of transfer for non-polar amino acids is proportional to their surface area.
• At 25°C, the hydrophobic interaction contributes approximately 0.1 kJ/mol/Å2 buried non-polar surface area.

The Lipid Bilayer

• The lipid bilayer provides a non-aqueous environment with a hydrophobic core.
• Membrane proteins embedded in the bilayer typically have hydrophobic regions spanning this central core.

Membrane Embedded Proteins

• The region of membrane proteins spanning the bilayer is dominated by hydrophobic residues. Aromatic residues with partial hydrophilic character are concentrated at the interface with the aqueous environment.

Bound Lipid Molecules

• Membrane protein structures often show bound lipid molecules, associated with the protein even after detergent extraction.
• These lipids typically bind in grooves between helices, forming a well-ordered layer analogous to the water layer surrounding soluble proteins.

Amino Acid Diversity

• The chemical diversity of proteins arises from the polymerization of 20 different amino acids, with additional diversity created by covalent modifications after translation.

Interactions Within the Backbone: Peptide Bonds

• Peptide bonds form amide groups, which participate in hydrogen bonding, driving the formation of secondary structure.

Non-Polar Amino Acids

• Gly, Ala, Val, Leu, Ile, Met, Pro, Cys, and Phe are non-polar and tend to be buried within the protein interior.
• Their preference for burial is proportionate to their surface area.
• They can also be found on the surface, particularly in ligand binding sites.

Glycine

• Glycine is the smallest amino acid, flexible and can be both buried or surface exposed.
• Its achiral nature allows greater conformational flexibility than other amino acids.

Proline

• Proline's rigid structure, containing an imine ring, limits its rotational flexibility and prohibits it from acting as a hydrogen bond donor.

Smaller Hydrophobic Amino Acids

• Ala, Val, Leu, and Ile are small hydrophobic residues, preferring burial with varying degrees.
• Their differences in volume and shape contribute to packing within the protein interior.
• Methionine, with a sulfur atom, is non-polar and flexible.

Aromatic Amino Acids: Phenylalanine, Tyrosine, and Tryptophan

• These amino acids have aromatic rings, contributing to hydrophobicity.
• Tyrosine's hydroxyl group can form hydrogen bonds, while Tryptophan's indole ring contains a nitrogen capable of hydrogen bonding.