Protein Structure: Key Themes

• Proteins are made of covalent backbones containing hundreds of individual bonds
• Specific chemical or structural functions suggest each protein has a unique 3D structure
• Protein structure is always malleable, with changes in structure being as important as the structure itself

Six Key Themes in Protein Structure:

• The amino acid sequence of a protein determines its three-dimensional structure.
• The function of a protein depends on its structure.
• Most isolated proteins exist in one, or a small number, of stable structural forms.
• Noncovalent interactions are the most important forces that stabilize a protein's specific structure.
• Structural patterns help in understanding protein architecture.
• Protein structures are not static and undergo conformational changes ranging from subtle to dramatic.
• Some proteins lack discernible structure, which is critical to their function

Overview of Protein Structure

• Conformation: The spatial arrangement of atoms in a protein or part of a protein
• Protein conformations include any structural state achievable without breaking covalent bonds
• Conformation can change by rotation about single bonds

Factors Affecting Protein Conformation

• Multiple stable conformations enable necessary changes for protein binding or catalysis
• Proteins in functional, folded conformations under specified conditions are termed native proteins
• Native proteins have the lowest Gibbs free energy (G), rendering them thermodynamically stable
• Intrinsically disordered protein segments or entire proteins have no discernible structure
• Weak interactions, including hydrogen and ionic bonds, and hydrophobic interactions, predominantly stabilize protein conformations

Stabilization by Noncovalent Interactions

• Native proteins are only marginally stable, requiring merely 20 to 65 kJ/mol to separate folded and unfolded states under physiological conditions
• A high degree of conformational entropy in the unfolded state and hydrogen-bonding with water tends to maintain the unfolded state of proteins
• Disulfide bonds and noncovalent interactions stabilize the native conformation
• Weak interactions, including ionic, hydrogen and hydrophobic, stabilize polypeptide chain folding into secondary and tertiary structures

Disulfide Bonds

• Disulfide bonds are much stronger than individual weak interactions (200 to 460 kJ/mol needed to break a single covalent bond, compared to 0.4–30 kJ/mol to disrupt a weak interaction)
• Disulfide bonds are not often found because the environment inside most cells is highly reducing due to high concentrations of reductants such as glutathione
• Disulfide bonds more likely to occur outside the cell since the environment is more oxidizing than the cell
• Eukaryotes primarly produce disulfide bonds in secreted, extracellular proteins
• Thermophilic bacteria and archaea have many disulfide bonds to stabilize proteins

The Role of Weak Interactions

• The protein conformation with the lowest free energy exhibits the maximum number of weak interactions
• Hydrophobic interactions generally predominate when forming stable protein structure
• Hydrogen bonds in proteins may not contribute to stability since they replace similar hydrogen bonds with water
• Nonpolar groups cluster to decrease the solvation layer and result in a favorable increase in entropy
• Hydrophobic amino acid side chains cluster in a protein's interior, away from water as a result of the increase in entropy of the surrounding water
• Formation of hydrogen bonds in a protein is driven by the same entropic effect that drives hydrophobic interactions, contributing to the protein-folding process
• Buried salt bridges provide significant stabilization to protein structures, especially in thermophilic organisms because the strength of a salt bridge increases as it moves to an environment of lower dielectric constant
• Hydrophobic residues are largely buried in the protein interior, away from water
• The number of hydrogen bonds and ionic interactions within the protein is maximized, thus reducing the number of hydrogen-bonding and ionic groups that are not paired with a suitable partner
• Van der Waals interactions within the tightly packed arrangements of protein confer structural flexibility and uniqueness
• Soluble intrinsically disordered protein segments have charged side chains of Arg, Lys, Glu or small side chains of Gly, Ala

Peptide Bonds

• Covalent bonds also constrain polypeptide conformation
• Linus Pauling's research showed the peptide bond is shorter and coplanar, indicating resonance or partial sharing of electrons between the carbonyl oxygen and the amide nitrogen
• The oxygen has a partial negative charge, and hydrogen has a net partial positive charge with the six atoms of the peptide group lie in a single plane with the oxygen atom of the carbonyl group trans to the hydrogen atom of the amide nitrogen
• Pauling and Corey concluded that peptide C-N bonds have hindered rotation because of their partial double-bond properties, although the N-Cα and Cα-C bonds can rotate
• Polypeptide chain backbones can be pictured as a series of rigid planes with consecutive planes sharing a point of rotation at Cα restricting range of conformations for the polypeptide chain
• Three dihedral angles (φ, ψ, ω) define peptide conformation showing rotation of the three repeating bonds of the peptide backbone
• All amino acids exist in either D or L form, referring to isomers
• D amino acids will distrupt regular structures consisting of L amino acids and vice versa

Ramachandran Plots

• Steric interference between atoms in the polypeptide backbone and amino acid side chains means φ and ψ cannot have any value but many values are prohibited
• A Ramachandran plot shows plots of φ versus ψ to reveal common steric clashes and allowed values