Section Week 3Junaid Malek, M.D.

Biological Polymers• DNA

• 4 monomers (building blocks), limited structure (double-helix)

• RNA

• 4 monomers, greater flexibility, multiple structures

• Proteins

• 20 Amino Acids, greatest variety of possible structures

Proteins

• 4 major functions

• Structural

• Enzymatic

• Carriers

• Regulatory

Amino Acids: Components

• Major components include amine group, carboxylate group and the side chain

• Remember that 19 of 20 amino acids are chiral

• Amine and carboxylate groups participate in peptide linkages

Amino Acids: Structure

• Important to memorize structures, 3-letter and 1-letter codes for the exam

The Peptide Bond

H3N O

OH3N O

O

OH

+

H3N NH

O

O

O

OH

H2O

alanine serine

peptide (amide) bond in alanylserine

H3N NH

O

O

O

OHOH2

Double bond property of a peptide bond

H2NNH

O

O

OH

OH

H2NHN

O

O

OH

OH

• The reason why a peptide bond is not capable of rotating

• Rotation also restricted by R groups

cis vs. trans nomenclature

H2NN

O

R

R'

O

OH

H2NN

O

R R' O

OHH

H

Trans: The α-carbonsare on opposite sides

H2N

N

O

R

R'

O

OH

H2N

N

O

RR'

O

OH

H

H

Cis: The α-carbonsare on the same side

Why is trans favored over cis?

• Trans configuration favored due to less steric hindrance between R side-chain and peptide backbone

N

O

ON

O

OH

H

Heavily favored

Trans Cis

Glycine• Only achiral amino acid

• Adds flexibility to any polypeptide chain

• Less steric hindrance

Proline

• The only exception to the cis vs. trans rule

• Because the nitrogen contains two substituents, both cis and trans contain steric strain. Therefore, they exist in nearly equal proportions.

Slightly favored

Trans Cis

NC

OON

O

CO

Cysteine

• Sulfur-containing amino acid

• Capable of undergoing reduction-oxidation reactions

• Don’t need to know specifics of red-ox reactions

• Just know that two distinct states exist that are not spontaneously interconvertable

• Disulfide bonds are important for protein folding

Histidine• Can act as an acid or a

base

• Note that pKa is near physiological pH

Draw the following oligopeptide at pH=7

• Lys-Phe-Met-Arg

H3N+

O

NH

+NH3

HN

O

S

NH

O

O-

O

NH

NH2

H2N+

Lys

Phe

Met

Arg

H3N+

O

NH

H2N

HN

O

NH

O

O-

O

OH

Gln

Ile

Glu

Thr

O

-O

O

Draw the following oligopeptide at pH=7

• Gln-Ile-Glu-Thr

H3N+

O

NH

+NH3

HN

O

S

NH

O

O-

O

NH

NH2

H2N+

Lys

Phe

Met

Arg

H3N+

O

NH

H2N

HN

O

NH

O

O-

O

OH

Gln

Ile

Glu

Thr

O

-O

O

Circle the Hydrophobic GroupsPut a square around the alcohol group

H3N+

O

NH

+NH3

HN

O

S

NH

O

O-

O

NH

NH2

H2N+

Lys

Phe

Met

Arg

H3N+

O

NH

H2N

HN

O

NH

O

O-

O

OH

Gln

Ile

Glu

Thr

O

-O

O

More Questions

H3N+

O

NH

+NH3

HN

O

S

NH

O

O-

O

NH

NH2

H2N+

Lys

Phe

Met

Arg

H3N+

O

NH

H2N

HN

O

NH

O

O-

O

OH

Gln

Ile

Glu

Thr

O

-O

O

• Are these oligopeptides chiral?

• Yes

• Which of the two peptides interacts best with DNA? Why?

• The peptide on the left, because it has two positively charged groups that can interact with negatively charged DNA

More Questions

• How many different pentapeptides can one make using the amino acids Gly, His, Cys, Ile, Try?

• 5!=5x4x3x2x1=120

• Which peptide would you expect to be more soluble in water, one rich in aspartate and lysine or one rich in valine and alanine?

• Aspartate and lysine have side-chains capable of H-bonding while valine and alanine do not

Protein Structure

• Primary Structure

• Secondary Structure

• Tertiary Structure

• Quaternary Structure

Primary Structure

• Linear sequence of amino acids bonded by peptide bonds

• No folding or side-chain interaction

• By convention, written from N-terminus to C-terminus

• As with DNA and RNA, directionality matters!

• The primary sequence of amino acids will determine how the protein folds

Secondary Structure• Local structures adopted by contiguous amino acids

• α-helix

• Helical corkscrew structure that is typically right-handed due to chirality of amino acids

• Carbonyl groups all point in same direction

• β-pleated sheet

• Form sheet-like structures represented by arrows. Origin of arrow indicates amino terminus while arrowhead indicates carboxyl terminus.

• Carbonyl groups point in alternating directions

α-helix: Myoglobin

β-sheet: Superoxide Dismutase

α-helix and β-sheet: Acetylcholinesterase

Tertiary Structure

• Folding of a single polypeptide chain

• Forms due to interactions among R-groups and secondary structures

• Allows for amino acids far away on the primary chain to lie in close proximity to one another

• All information allowing a protein to form tertiary structure can be found in the primary sequence

Quaternary structure

• Interaction between multiple tertiary structures (from distinct polypeptide chains)

• Classic example is hemoglobin

Why does a protein fold?

• Anfinsen experiment: does a primary structure determine protein folding?

• A protein (Ribonuclease A) was denatured using urea and reduced to break disulfide bonds between cysteine residues

• Observation made that when urea was removed, the denatured polypeptide chain folded back into a compact structure

• When protein oxidized, regained 90% of original function

Protein folding

• In a second experiment, the denatured polypeptide chain was first oxidized before removing urea

• Result was a non-functional protein trapped in a non-native conformation by incorrect pairing of disulfide bonds

• These bonds prevented the protein from achieving its native structure

The Anfinsen Experiment: Conclusion

• Given that we prevent premature oxidation (and hence incorrect disulfide bridging), the protein will “find” its lowest energy conformation

• Thus, all information for proper protein folding can be found in its primary sequence!

• This is in turn determined by RNA sequence, which is determined by DNA sequence

• We can therefore conclude that all complex protein folding can be determined by the sequence of just 4 different base pairs!!!

What drives protein folding?

• Thermodynamics!

• Hydrogen bonding along the backbone

• Hydrogen bonds of the R groups with each other or with the backbone

• Ionic interactions between the R groups

• Van der Waal’s interactions between R groups

• Disulfide bridges between cysteine residues

The Hydrophobic Effect• Tendency for non-polar substance to interact with

each other rather than with water

• This leads to burial of non-polar sidechains within the interior of a protein as it “collapses” to form a globular structure

• Driven by the energetically unfavorable situation of having water molecules organized and surrounding a non-polar molecule

• Driven also to a lesser extent by Van der Waal’s forces

Question: Protein Folding

• Segments of proteins that connect successive regions of secondary structures are referred to as “turns” or “bends”. These are often rich in glycine and proline residues. Why?

• Glycine is found in bends because of its small size

• Proline constrains the conformation a polypeptide chain can adopt, thus it is often the initiator of bends

Question: H-bonding and Secondary structure

• Indicate whether the following amino acids can form H-bonds to participate in the formation of α-helices or β-sheets

• Arginine

• Glutamate

• Glycine

• Phenlyalanine

• Proline

• Threonine

Question: Amino acid interactions

• Indicate the strongest interaction that can form between the side chains of the following amino acid pairs:

• Pro-Leu

• Van der Waal’s

• Arg-Thr

• Hydrogen bonding

• Ile-Val

• Van der Waal’s

Question: Amino acid interactions

• More amino acid pairs:

• Tyr-Phe

• Van der Waal’s

• Arg-Asp

• Ionic

• Cys-Met

• Van der Waal’s

Question: Amino acid interactions

• More amino acid pairs:

• Gly-Ala

• Van der Waal’s

• His-Glu

• Ionic

• Cys-Cys

• Disulfide bond

Gibbs Free Energy

Gibbs Free Energy• If ΔG is negative, the process is favored (energy is

released)

• Two components of free energy are enthalpy (H) and entropy (S)

• If ΔH is negative, the reaction (or system) is exothermic

• If ΔH is positive, the reaction is endothermic

• Remember your Second Law of Thermodynamics (the entropy of the universe is always increasing)

What can we say about ΔH and ΔS?

ΔH ΔS Reaction

(-) (+) Spontaneous

(+) (-) Non-Spontaneous

(-) (-) Temperature dependent

(+) (+) Temperature dependent

Gibbs Free Energy

• We can relate Keq and free energy using the equation ΔG°=-RTlnKeq

• R=Equilibrium constant

• T=Temp (Kelvin)

• At equilibrium, there is a relationship between the chemical equilibrium and the change in free energy (ΔG) that occurs as a result of the chemical reaction. The change in free energy is related to the natural log of the equilibrium constant.

• If ΔG is negative, the process is favored (energy is released)