Lecture #05: The Peptide Bond - Formation & Properties

Question 1

Peptide Bond

Answer 1

• The linkage joining amino acids in a protein is called a peptide bond (or amide bond). The formation of a dipeptide from two amino acids is accompanied by the loss of a water molecule.
○ The equilibrium of this reaction lies on side of hydrolysis (left side with two amino acids) rather than synthesis under most conditions. This is because this reaction requires an input of free energy. Also fusing two molecules into one results in a decrease in entropy.
○ This reaction is carried out by a catalytic RNA located in the ribosome.
○ Peptide bonds are quite stable kinetically since the rate of hydrolysis is extremely slow.

Question 2

Polypeptide Directionality

Answer 2

• A polypeptide chain has directionality because its ends are different. An alpha-amino group is at one end, and an alpha-carboxyl group is at the other. The amino end is the beginning of the chain, so in amino acid sequences, the first one is the amino-terminal residue (amino acid unit in a polypeptide) and the last one is the carboxyl-terminal residue.

Question 3

Polypeptide Chain Composition

Answer 3

• A polypeptide chain has a regularly repeating part, called the main chain or backbone, and a variable part, comprising the distinctive side chains.
○ The backbone is rich in hydrogen-bonding potential. Each residue has a carbonyl group (CO), which is a good hydrogen-bond acceptor. Each residue also has proline, an amino group (NH) which is a good hydrogen bond donor. These groups interact with each other and with functional groups of side chains to stabilize particular structures.

Question 4

Cross Links in Polypeptide

Answer 4

• Some proteins have a linear polypeptide chain that’s covalently cross-linked. Most common ones are disulfide bonds, formed by oxidation of a pair of cysteine residues. The resulting unit of two linked cysteines is called cystine.

Question 5

Amino Acid Sequence

Answer 5

• A protein has a precisely defined amino acid sequence.
○ Amino acid sequences determine 3D structures of proteins.
○ Knowledge of the sequence of a protein is usually key to figuring out the mechanism of action.
○ Sequence determination is a component of molecular pathology, a rapidly growing area of medicine. Alterations in amino acid sequences can produce abnormal function and disease.

Question 6

Peptide Bond is Planar

Answer 6

• The peptide bond is planar. So, for a pair of amino acids linked by a peptide bond, six atoms lie in the same plane: the alpha carbon and CO group of the first amino acid and the NH group and alpha-carbon of the second amino acid. Also rigid.

Question 7

Peptide Bond Has Double Bond Character

Answer 7

• The peptide bond has considerable double bond character due to resonance structures. The electrons resonate between a pure single bond and a pure double bond.
○ Because of this partial double bond, rotation about this bond is prohibited and thus constrains conformation of the peptide backbone.

Question 8

Peptide Bond Charge

Answer 8

• The peptide bond is uncharged, so polymers of amino acids linked by peptide bonds can form tightly packed globular structures that would otherwise by inhibited by charge repulsion.

Question 9

Peptide Bond Conformation

Answer 9

• In peptide bonds, two conformations are possible, trans and cis. But, almost all peptide bonds in proteins are trans, because there is steric hindrance in the cis conformation.

Question 10

Bonds On Side of Peptide Bond

Answer 10

• In contrast with the peptide bond, the bonds between the amino group and the alpha-C atom and between the alpha-C atom and the carbonyl group are pure single bonds. The two adjacent rigid peptide units may rotate about these bonds to make various orientations. This freedom of rotation about two bonds of each amino acid allows proteins to fold in many different ways.

Question 11

Phi vs. Psi

Answer 11

○ Rotation about these bonds can be specified by torsion angles.
§ The angle of rotation about the bond between the nitrogen atom and the alpha-C atom is called Phi.
§ The angle of rotation about the bond between carbonyl carbon atom and the alpha-C atom is called Psi.
§ A clockwise rotation about either bond as viewed toward the alpha-C atom corresponds to a positive value.

Question 12

What Rotation About Bonds Permits

Answer 12

○ Rotation about these bonds permits proteins to fold in different ways–but not all combos are possible due to steric hindrance. Suggests that steric hindrance is a factor in protein folding, and restricted set of phi and psi angles limits number of possible structures.

Question 13

Ribonuclease

Answer 13

○ Ribonuclease is a single polypeptide chain that has 124 amino acid residues cross linked by four disulfide bonds.

Question 14

Christian A’s Experiment

Answer 14

○ Anfinsen’s plan was to destroy the enzyme’s 3D structure and to determine the conditions required to restore the tertiary structure.

○ Chaotropic agents, like urea, which disrupt all of the noncovalent bonds in a protein, were added to a solution of the ribonuclease. The disulfide bonds were then cleaved reversibly with a sulfhydrul reagent B-mercaptoethanol. In the presence of large excess of this reagent, the disulfides are fully converted into sulfhydruls (cysteines).

○ So when ribonuclease was treated with B-mercaptoethanol in 8 M urea, the product was a randomly coiled polypeptide chain devoid of enzymatic activity, essentially denatured. Anfinsen them made the critical observation that when the denatured ribonuclease is freed of urea and -mercaptoethanol by dialysis, it slowly regained enzymatic activity.
§ The significance of this chance finding is the enzyme spontaneously refolded into a catalytically active form with all of the correct disulfide bonds re-forming. This shows that the info needed to specify the catalytically active 3D structure of ribonuclease is contained in its amino acid sequence. SEQUENCE SPECIFIES CONFORMATION.

○ B-mercaptoethanol at very low concentrations can correct cystiene bonds that were bonded wrongly, and leave the correct ones alone.

Question 15

Factors That Drive Protein Folding

Answer 15

• Factors That Drive Folding
○ Amino acid sequence.
§ Satisfy constraints: phi and psi angles, disulfide bonds
§ Ribonuclease Experiment
○ Hydrophobic Effect
§ “Bury” the hydrophobic side chains, minimize contact with water.
§ Most polar residues face the outside of the protein and interact with solvent.
○ Retention of partially folded correct intermediates-related to the free energy.

Question 16

Can Proteins Self-Fold?

Answer 16

• It is not possible for all proteins to fold on their own. Some are too large and made of more than one polypeptide chain.

Question 17

Primary Structure

Answer 17

○ Primary refers to the linear sequence of amino acids in a protein; more generally, the linear sequence of units that form a polymer.

Question 18

Secondary Structure

Answer 18

○ Secondary refers to the spatial arrangement of amino acid residues that are near one another in the linear sequence; the alpha helix and the beta strand are both elements of the structure. R grounds do not participate.

Question 19

Tertiary Structure

Answer 19

○ Tertiary refers to the spatial arrangement of amino acid residues that are far from each other in the linear sequence, as well as the pattern of disulfide bonds. Involves R groups, and disulfide bonds in proteins that have them.

Question 20

Quaternary Structure

Answer 20

○ Quaternary refers to containing more than one polypeptide chain, the spatial arrangements of those chains and the nature of contacts among them.

Question 21

What Major Forces Primarily Determines Tertiary Structure?

Answer 21

Hydrophobic Effect