7/27/2019 Biochem- Lec 07

1/4

1

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

Disulfide bonds

Electrostatic interactions

Hydrogen bonds

van der Waals forces

"Hydrophobic effect"

Conformational states

Heme group

X-ray crystallography

NMR spectroscopy

Dimer, trimer & tetramer

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 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.

7/27/2019 Biochem- Lec 07

2/4

2

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 ofis 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 aredetermined, 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 CoordinationThis 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.

7/27/2019 Biochem- Lec 07

3/4

3

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.

7/27/2019 Biochem- Lec 07

4/4

4

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 "reason" for the observed quaternary structure is not known.

9.7.04