Major concepts:

1. Why is protein structure important?
2. What determines protein structure?
3. What is the main advantage of Edman degradation?
4. What are the types of regular secondary protein structure, and why are they important?
5. What levels of protein structure are involved in a protein's native conformation?
6. How can both the native conformation of a protein and denaturation be thermodynamically favorable?

Core knowledge:

1. What are the different levels of protein structure, and how is each structure stabilized?
2. What's the difference between an amino acid residue and a protein subunit?
*3. What treatments are required prior to Edman degradation in order to sequence a large pure protein with no disulfide bonds? What is required for one with disulfide bonds?
4. What are the characteristics of an α-helix?
5. What are the characteristics of β-pleated sheets and β-bends?
6. What are motifs and domains, and how are they related to levels of protein structure?
7. What are the various denaturing agents, and how does each cause denaturation?
*8. Why is the ribonuclease renaturation experiment important, and why is it significant that ribonuclease was chosen rather than another enzyme?
*9. What are the functions of protein disulfide isomerase and protein chaperones?

Levels of protein structure
1. Primary = the sequence of amino acid residues (free α-amine = residue 1)
    determines the other levels of structure and therefore protein function
     stabilized by peptide (covalent) bonds
2. Secondary = the local arrangement of the backbone
3. Tertiary = three-dimensional arrangement of amino acid residues, including non-adjacent residues
4. Quaternary = arrangement of protein subunits, for proteins with more than one chain

Primary protein structure = the sequence of the amino acid residues
Residue 1 = free amine terminal residue
Primary structure determines all other levels of structure.
Primary structure therefore determines protein function.

Determining protein sequence
1. Requires pure protein
2. Amine terminal amino acid can be identified using dansyl chloride or other
     chemicals followed by acid hydrolysis
3. Acid hydrolysis and chromatography → identification of amino acid composition
4. Disulfide bonds must be broken prior to determining protein sequence. Fig. 3-26
      a. Oxidation → cysteic acid residues
      b. Reduction and acetylation → acetylated cysteine residues
5. Large proteins must be broken into manageable polypeptides (~ 50 residues)
     Treatment with a protease → smaller polypeptides. Table 3-7
6. Sequencing with the Edman sequenator

Edman degradation Fig. 3-25
1. React amine terminus with PITC (phenylisothiocyanate) → PTC adduct
2. Treat with trifluoroacetic acid to cut the N-terminal peptide bond
3. Extract the amine-terminus derivative (soluble in organic solvent)
4. Convert it to the stable PTH (phenylthiohydantoin) derivative
5. Identify the derivative by chromatography
6. Repeat 1-5 for the remaining water-soluble polypeptide chain.

Ordering polypeptides   Fig. 3-27
1. Treatment with different proteases → different polypeptides
2. Logical analysis → sequence

top of page

Peptide bond structure
Partial double bond character = planar structure of the peptide bond - Fig. 4-2
     partial charges on = O and - NH - create a dipole for the peptide bond also
Promotes trans configuration of the O and H
Rotation around the α-carbon occurs (except for proline)
     α-C to N rotation defines the angle φ
     α-C to C=O rotation defines the angle ψ
     Ramachandran plot: ψ plotted as a function of φ, showing angles that are allowed
         for a particular amino acid
Backbone character is therefore polar; N-H and C=O tend to form hydrogen bonds.

Once the primary structure of a protein is known, how are the remaining levels of structure determined? Box 4-4, Fig. 1
Protein crystals are x-rayed, and the x-ray diffraction patterns are analyzed.
Based upon electron density patterns, three-dimensional structures are calculated.

Secondary level of protein structure = local arrangement of the backbone
Stabilized by hydrogen bonds between backbone atoms ( α-amine and α-carbonyl)
Repeated regular structures = α-helix and β-sheet

α-helix = a coiled backbone, with all side chains on the outside of the coil - Fig. 4-4
Each α-amine has a hydrogen bond to the α-carbonyl of the fourth residue from it.
One complete turn = 3.6 residues, with a rise = 5.4 Angstroms.
Dipole of the helix created by repeated hydrogen bonds: amino terminus has δ+
     carboxyl terminus has δ-. Fig. 4-6
Side chain-side chain interactions affect α-helix stability as follows:
     1. ionic interactions between adjacent side chains (repulsion or attraction) - Fig. 4-5
     2. steric hindrance when adjacent bulky side chains interact
     3. Pro and Gly are destabilizing (too rigid or not rigid enough)
     4. side chains 3-4 amino acids apart interact (steric, hydrophobic, ionic)
     5. charged side chains stabilize or de-stabilize the ends of the helix

β-pleated sheet = extended backbones lying next to each other, with side chains
     above and below the plane of the sheet - Fig. 4-6
antiparallel: opposite orientation (N → C and C → N); more stable
parallel: same orientation (both N → C); diagonal hydrogen bonds, less stable

β-bend = tight turn that connects two strands of a β-sheet - Fig. 4-8
1^st α-carbonyl is hydrogen-bonded to the 4^th α-amine. Often include Pro and Gly.

Other secondary structures = random coil
Means that the structure is not a recognized regular structure.

Tertiary structure = the 3-dimensional arrangement of the protein - Fig. 4-16
Stabilized by interactions between side chains, backbone atoms, and water.
Also by disulfide bonds in some proteins

Quaternary structure = the arrangement of multiple protein chains (subunits)
Stabilized like tertiary structure
Highly variable because multi-subunit proteins may have 2 identical chains,
     2 different chains, 3 chains, 4 chains (2 pairs), 4 chains (all different), etc.
Organization is frequently symmetrical.

Fibrous proteins: structural, frequently consist of primarily secondary structures

Globular proteins: enormous variety, including all α, all β, and combinations.
Motifs (folds) = repeated combinations of secondary structures - Fig. 4-20
      used for classifying proteins--grouping by combinations (SCOP)
Domains = structures associated with a specific function - Fig. 4-19
     can fold independently

Protein structure determines protein function
   the reason tertiary structure is more strongly conserved than primary structure
   loss of structure ≡ denaturation ≠ loss of primary structure

Denaturing agents
   1. heat: frequently change occurs over a narrow temperature range
   2. pH extremes
   3. some solvents that mix with water (ethanol, acetone)
   4. some solutes (urea, guanidine hydrochloride)
   5. detergents

Renaturation and ribonuclease - Fig. 4-27
   Denaturation by urea (interacts with nonpolar side chains → increased water solubility)
     + reducing agent (–S–S– → 2 –SH for 4 disulfide bonds) = loss of function
   Renaturation: removing urea and the reducing agent → correct renaturation with function

Note : 1. Ribonuclease experiment showed that primary structure determines conformation.
   2. Most proteins are denatured irreversibly in vitro .

Why is native conformation thermodynamically favorable?
    Primarily because hydrophobic groups are separated from water,
       so water molecules have greater entropy.
    Intermediate states/alternative conformations exist for some proteins. - Fig. 4-29

top of page