Biochemistry I Fall Term, 2004

September 15, 2004

Lecture 7: Protein Tertiary & Quaternary Structure

Assigned reading in Campbell: Chapter 4.4-4.5. Key Terms: Denaturation & refolding Conformational states Disulfide bonds Heme group Electrostatic interactions X-ray crystallography Hydrogen bonds NMR spectroscopy van der Waals forces Dimer, trimer & tetramer "Hydrophobic effect" Oligomer (Topics relating to O2 binding by myoglobin and hemoglobin will be covered later in the course.)

Links : (I) Review Quiz on Lecture 7 concepts (I) 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.

(I) The Protein Architecture Tutorial introduces the basic principles of protein 3-D structure
using Chime images of Protein G.

(S) The "Alpha_bet Helix" shown in lecture today is an example of an ideal -helix.
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 shortrange; 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: G = z1z2*e2/(*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 is the dielectric constant of the medium between them. For charges of +1 and -1; in water ( = 80); at a distance, r = 4; we calculate G = -4 kJ/mol. In the hydrophobic core of a protein, the value of is estimated to be on the order of 2 - 4 (e.g. benzene). Thus, if a charge is buried in the core of a protein, there is a large energetic advantage in burying a group of opposite charge nearby (or a corresponding penalty for leaving it unpaired). On the surface of a protein, the advantage and the penalty are both much smaller because of the presence of water, which shields both charges. 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 G'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 . Typical G's are 0.5 - 1.0 kJ/mol per pair of atoms. b) The repulsion of the electronic shells occur at distances < the sum of the van der Waals radii. The unfavorable G's rise rapidly as two nonbonded atoms are forced to occupy the same space (referred to as "steric repulsion"). 4. Metal Ion Coordination This is another electrostatic interaction. The separate category is justified by the fact that certain proteins have very specific requirements for metal ions. For example, Zn2+ is required to stabilize the tertiary folding of the zinc finger domain in many DNA-binding proteins.

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 the 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 on the order of 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. However, the two and two 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 forms 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 32*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 35*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 nonidentical 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 "reason" for the observed quaternary structure is not known. 9.7.04