MBIO 2700 / Topic 5a: Proteins

What is a peptide bond?

A special name to the amide bond covalently joining two AAs by condensation of the amino group on one AA and the carboxyl group of another AA.

Differentiate the following: peptides, polypeptides, and proteins.

• Peptides: Short chains of AAs.

• Polypeptides: Long chains of AAs.

• Proteins: Aggregates of polypeptides.

What are the two components of a polypeptide?

• Backbone: Polypeptide chain w/o R groups.

• Side chains: R groups only.

• What are the pK_a and pI of the chains and backbone termini similar to?

• Which is the only ionizable stuff in the backbone?

• pK_a and pI of free AAs.

• The termini.

+ When covalent peptide bonds are formed, α-amino and α-carboxyl groups of each AA comprising the peptide are now non-ionizable.

• What are the AA units in a peptide or protein also called?

• How is the amino acid sequence of peptides and proteins written?

• AA units are called residues.

• Sequence is written as N-terminus beginning on the left and C-terminus on the right.

Protein folding contributes to an increase in ______ and decrease of __, contributing overall to disorder and spontaneity.

Protein folding contributes to an increase in entropy and decrease of ∆G, contributing overall to disorder and spontaneity.

Polypeptide chains are very _______, so the way they fold to form a 3D structure is essential to understanding how proteins are assembled.

Polypeptide chains are very flexible, so the way they fold to form a 3D structure (that we will soon known as the structure of a protein) is essential to understanding how proteins are made.

What bonds in a polypeptide makes polypeptides so flexible? What bonds do not make it flexible?

Flexible via:

• α-carbon and α-hydrogen.

• α-carbon and R.

• α-carbon and carboxyl.

• α-carbon and amino.

Inflexible via:

• Carbonyl carbon and amine.

Why is the bond between the carbonyl carbon and amine inflexible?

> N lone pair delocalizes into carbonyl.

> Resonance between C=O and C-N.

> C-N peptide bond = shorter, stiffer, and planar → no free rotation like single bond.

Do you expect proteins to be localized to one part of a body?

> Proteins made every single second of day and moves from one place to another in seconds.

> No.

What are the seven primary functions of a protein?

Catalysis, transport, structure, contractile, nutrient, defence, and regulatory.

Are proteins usually small? Large? Light? Heavy?

Can be small to large and light to heavy.

+ Very diverse.

Differentiate the following: glycoproteins, lipoproteins, cofactors, prosthetic groups, and coenzymes.

• Glycoproteins: Proteins conjugated with sugars.

• Lipoproteins: Proteins conjugated with lipids.

• Cofactors: Non-protein molecules that assist in protein activity.

• Prosthetic groups: Cofactors that are permanently bound to the protein.

• Coenzymes: Cofactors for enzymes specifically.

+ Cofactors an be organic (e.g. vitamins) or inorganic (e.g. metal ions). Can be covalently bonded to protein or non-covalently bonded to protein.

Differentiate the four levels of protein structure.

• Primary structure: Linear sequence of AAs.

• Secondary structure: Atoms (on the backbone of a single, same polypeptide) bond via δ⁺ and δ^- in an intermolecular-bonding, intramolecular-appearing fashion, folding the polypeptide.

• Tertiary structure: Atoms (on the side chains of the folded polypeptide) bond in a variety of intramolecular and intermolecular fashions, crumpling the now globular polypeptide.

• Quaternary structure: Two or more tertiary structures form a protein.

When it comes to protein folding, in what way should you take into account sterics and entropy?

You have to find a way to fold a protein that decreases steric hinderance but at the same time increases disorder.

On a 1º structure, where does the folding happen between 1º and 2º? Why does it happen in these places?

Hydrogen bonding. (Shown in picture)

> What are you specifically trying to maximize in secondary folding?

> Why can’t you make too many 2º structure H-bonds?

> You’re trying to max out on H-bonds.

> More H-bonds → more twisting → more strain.

• What are the two main types of 2º structure?

• Can you link these two main types of 2º structures? How so, if ever?

> The two types of 2º are α-helix + β-sheet.

> They are linkable via linkers. α-helix-linker-β-sheet.

What’s the one AA that cannot fold to a 2º?

> Two H-atoms in H₂N⁺ will be used for peptide condensation.

> No H-atoms for H-bonding → no folding to 2º.

In the folding between 1º and 2º, what is the relationship between number of hydrogen bonds, disorder, free energy, and enthalpy?

↑ Hydrogen bonds ↑ Disorder ↓ Free energy (G) ↓ Enthalpy (H)

• In one turn of an α-helix, how many residues are there on average?

• How long is one turn, typically?

• Residues: 3.6 residues.

• Turn: 5.4 Å (= 1E-8cm).

In an α-helix backbone loop, why do the side chains protrude in space?

> If side chains pointed inward/straight down axis → bump into each other → clashes.

> If side chains protrude out in space → minimize spatial interference w/ each other.

If there are high concentrations of charged, bulky, or small residues on an α-helix, what will happen?

Helix disruption.

Across a peptide bond, why must α-carbons be trans to each other?

Minimize steric clashing.

What are the two defining characteristics of an α-helix?

• H-bonds form between every fourth amino acid.

• Side chains tend to point away from the helix.

What are the four disrupting characteristics of an α-helix?

• Electronics clash (e.g. two negatively charged groups).

• Sterics clash (e.g. two bulky groups).

• Small residues wanting to convert to β-conformation.

• Proline due to its (a) inability to form hydrogen bonds + (b) nitrogen’s (comprising the amide) lack of planarity (affecting flexibility).

What is the defining characteristic of a β-sheet?

β-sheets form as overlapping segments of the polypeptide chain when there are side chains spaced out evenly so as to not clash with each other and allow for H-bonds.

Differentiate each way a β-sheet can exist.

• Parallel: Overlapping segments all run in the same direction from C-terminus to N-terminus.

• Antiparallel: Overlapping segments run in different directions, with one sheet going from the C- to the N-terminus, and the adjacent sheet going N- to C- and so on.

Why are H-bonds in one category of β-sheets more stable than in the other?

> Antiparallel sheets have more linear H-bonds → more energy released → lower in energy in the end + stabilized.

> Parallel sheets have crooked H-bonds → higher in energy in the end → weaker + destabilized.

What are the usual side chains that make up β-sheets?

Gly, Ala, and Ser.

What happens when a polypeptide with α-helices is heated then cooled?

> Heating disrupts H-bonds in α-helices → unfolding α-helices.

> Upon cooling, unfolded polypeptide refold into β-sheets.

+ This is temporary. β-sheets will revert back to α-helices The formation of α-helices is a spontaneous and entropically stable reaction. The disruption and conversion of α-helices into β-sheets is the opposite, hence why we need to put in energy - heat.

> What if you had a tertiary structure with a ton of residues containing non-polar side chains? Hint: Where will these residues go within the space of a tertiary structure?

> What if you had a tertiary structure with a ton of residues containing polar side chains? Hint: Where will these residues go within the space of a tertiary structure?

> From lectures past, what is this event called?

> Non-polar side chains buried in protein interior.

> Polar side chains exposed on protein exterior.

> Hydrophobic effect.

What are electrostatic interactions within protein structures?

• Ionic bonds between acidic + basic residues.

• H-bonds between residues w/ partial positive + partial negative ends.

What are the four defining characteristics of a tertiary structure? How does each keep the protein happy?

• Hydrophobic effect: Favourable hydrophilic and hydrophobic interactions.

• Disulphide bonds: Maintenance of globular structure.

• Electrostatic interactions: Maintenance of globular structure.

• Metal chelation: Two functional groups that hate each other together by a mediator - the metal.

• What happens when oxidizing agents are present around cysteine?

• What happens when the pH of the solution is greater than the pK_a of cysteine?

• Oxidizes thiol → disulphide bonds are possible.

• pH of solution deprotonates thiols → negatively charged “thiols” → clashing bec of negative charges.

What are the two defining characteristics of 3º structures?

> 3º structures form to minimize unfavourable interactions + maximize favourable ones.

> Compact w/ few cavities + small amt of H₂O.

What are the factors that put together a quaternary structure?

The same R-group interactions present in tertiary structures.

On a quaternary structure, do you expect full positives and full negatives to be specifically meant for each other?

Nope.

What are the four different states of a protein? Differentiate each.

• Folding intermediate: Partially folded structure.

• Aggregates: Misfolded, clumpy structure.

• Native proteins: Folded, functional, and correct structure.

• Chaperone proteins: Specialized proteins assisting other proteins in folding correctly, preventing misfolding and aggregation.

+ An aggregate often leads to loss of protein function and diseases.

What is protein denaturation?

Folded/native form → unfolded form.

Do you expect protein denaturation to be costly, energy-wise?

> ∆G between native and unfolded form = ∆G = ~5–10kcal/mol.

> Change in solvent/apply heat → easy denature.

> No, not costly.

• Do you expect denatured proteins to refold spontaneously?

• Do you expect denatured proteins to stay in its denatured appearance? Or does it do something spontaneously?

• What could expect to be great protein denaturants, keeping proteins in denatured form?

• Happens rarely in few, wherein chaperones are usually required.

• Most denatured proteins, other than the rare case of refolding, aggregate together and precipitate.

• High concentrations of solutes that disrupt the H-bonding system of water, e.g. 8M urea, 6M guanidine hydrochloride