Biochemistry I Fall Term, 2000

Lecture 7: Protein Tertiary & Quaternary Structure

Assigned reading in Campbell: Chapter 4.4-4.5.

Take the Review Quiz on Lecture 7 concepts.

The CMU Chime Tutorial, covered previously in cluster sesssions, can also be accessed this week. Be sure you understand the operation of this visualization tool.
The Protein Architecture Tutorial introduces the key principles of protein 3-D structure using Chime-generated images of Protein G.
The "Alpha_bet Helix" shown in lecture today as an example of an ideal a-helix. But note the lack of one H-bond and the steric clash that is seen at the Pro residue.

4.4 Tertiary Structure of Proteins

Disulfide Bonds

The formation of a covalent disufide bond between two Cys residues can contribute to the stability of protein tertiary structure. The "S-S" bond covalently crosslinks two regions of the structure that may be distant in sequence, but nearby in the folded state.
Disulfide bonds are only found in proteins that function outside of the cell, e.g. extracellular enzymes, antibodies, plasma proteins, etc. The cysteine residues in intracellular proteins are kept in their reduced, -SH, state by an active enzyme pathway using glutathione as the reducing agent.

Noncovalent Forces in Protein Structure and the Hydrophobic Effect

Noncovalent energies are 2 to 3 orders of magnitude smaller than covalent bonds; they act at short-range; and they are exceedingly numerous. A key feature of protein structure is that the stability depends on the simultaneous presence of all of the noncovalent interactions of the native state. Thus, the interactions described below cooperate to produce the native structure.

1. Electrostatic Interactions
The free energy of bringing two charges together is:

DG = z₁z₂*e²/(e*r)
where,

z₁ and z₂ are the ionic valences;

e is the unit of charge;

r is the distance between the ions considered as point charges; and

e is the dielectric constant of the medium between them.

2. Hydrogen Bonds are due primarily to partial electrostatic charges. The energetics are determined, in part, by the values of the partial charges. The groups of interest in proteins include amide (N-H) and carbonyl (C=O) groups. Typical partial charge assignments for these groups are -0.3e and +0.3e for N and H, respectively; and -0.4e and +0.4e for O and C, respectively. Typical DG's are 1 - 4 kJ/mol per H-bond. H-bond donors in the core of a protein are (nearly) always paired with an acceptor. H-bond donors and acceptors on the surface of a protein can also H-bond with other residues, but are more frequently H-bonded to water.

3. van der Waals Forces
  a) Forces between atoms are attractive and occur between any pair of atoms at distances of 4-6 Å. Typical DG's are 0.5 - 1.0 kJ/mol per pair of atoms.
  b) The repulsion of the electronic shells occurs at distances < the sum of the van der Waals radii. The unfavorable DG's rise rapidly as two nonbonded atoms are forced to occupy the same space (referred to as "steric repulsion").

The Hydrophobic Effect
During protein folding, the transition from the countless unfolded states to a single native state is accompanied by the burial of solvated nonpolar side chains (and polar peptide units) into the nonsolvated core of the protein. The "hydophobic effect" or "hydophobic interaction" in protein structure is derived from the combined properties of H-bonds in water and van der Waals forces applied to amino acid residues with nonpolar side chains. A nonpolar side chain in water makes less favorable van der Waals interactions than if it were dissolved in an apolar solvent. In addition, the solvating water molecules cannot satisfy their four potential H-bonds while they surround an apolar solute. In contrast, a nonpolar side chain in the apolar core of a protein has gained favorable van der Waals interactions and has rid itself of the dissatisfied solvating water.
The interior of folded proteins is tightly packed. Proteins have very few cavities the size of a water molecule. The rule, in a phrase coined by Francis Crick, is that "Knobs fit into holes." In other words, each core side chain fits into a complementary space created by several (from 5-8) of the other core side chains.

Entropy disfavors the folded structure of proteins
The most significant energetic effect opposing all of the favorable interactions described above is the unfavorable entropy of forming a unique 3-D structure. If it were not in its native state, the protein could assume a huge number of conformations. To illustrate this point, consider the number of conformational states in peptides containing only alanine residues:

1. Ala-Ala-Ala: The tripeptide has two rigid peptide bonds that remain fixed in the trans conformation. However, the two Y and two F angles can each rotate to three positions. Thus, the number of different conformational states is 3²*3² or 3⁴. This result (81) while large is not too large to comprehend.
2. (Ala)₂₅: This peptide forms an a-helix in solution. Under denaturing conditions, it is a flexible polymer; the total number of conformational states results from 3² states for each peptide unit or 3^2*24. This result is comparable to Avogadro's number.

A typical small protein (say, 100 residues) will have many amino acids with more side chain degrees of rotational freeedom than do alanine peptides. On average there will be roughly 3⁵ conformational states for each peptide unit. Viewed as a flexible chain, the small protein has about 3^5*99 conformational states.
Without the favorable energetic contributions, the probability of a protein being found in the native state is nil. A conceptual way of combining the energetic factors that characterize native protein structure is to conclude that the numerous, albeit weak, favorable interactions acting together are sufficient to make the native state the most likely conformation.

4.5 Quaternary Structure of Proteins

Most proteins are monomers, consisting of a single polypeptide chain. There are also many examples of dimers and tetramers consisting of two and four polypeptide chains, respectively. There are only a few examples of trimeric and hexameric proteins. The oligomeric proteins (more than one subunit) are usually made up of identical subunits, but there are many examples of non-identical subunits that associate to form the dimer or tetramer, e.g. hemoglobin.
The association of monomer subunits to form higher oligomers uses all of the interactions described above under tertiary structure. Particularly prominent are hydrophobic interactions; these are often localized to well-defined surfaces at the monomer-monomer interfaces.
In some multimeric proteins, the function of one subunit can be affected by the others, e.g. cooperative O₂ binding in hemoglobin. In most cases, however, the subunits are non-interacting and the "reasons" for the observed quaternary structure is not known.

4.6 Muscle Proteins

Return to Home Page.