Biochemistry Exam #1 Chapter 4

Amino acid

Monomer (building block) of proteins

Oligopeptide

Short chain of amino acids (typically 2-20 residues

Polypeptide

Long chain of amino acids linked by peptide bonds

Protein

One or more folded polypeptides that perform a biological function

What must be memorized for the 20 standard amino acids?

• Structure

• 3-letter code

• 1-letter code

• Side-chain properties

what are the properties of amino acids?

• Nonpolar (hydrophobic) → Avoids water

• Polar uncharged → Hydrogen bond, no charge

• Acidic → Negatively charged at physiological pH

• Basic → Positively charged at physiological pH

• Aromatic → Contains aromatic rings, absorbs UV light

What happens during amino acid ionization?

Amino acids gain or lose H+ depending on pH, changing their charge

Draw the structure of glycine at pH 7

(zwitterion form)

What is the isoelectric point (pI) ?

The pH at which an amino acid has a net charge of 0

How do you calculate pI for an amino acid with no ionizable side chain (ex. glycine)?

• Average the two pKa values surrounding the zwitterion

• pI = ½ (pKj + pKj)

Practice: Ionization and pI calculation for glutamate (slide 53)

Answer on module 2 handout

What is a zwitterion?

A molecule with both a positive and negative charge but an overall net charge of 0

What is the overall charge when pH> pI for amino acids/ peptide/ protein?

• pH < pI → +

• pH = pI → Neutral (0)

• pH > pI → -

What is cystine?

Two cysteine amino acids join by a disulfide bond

What type of reaction forms cystine?

• Oxidation reaction

• Cystine is the oxidized form where a disulfide bridge connects the two residues

Cysteine is the only amino acid that can be oxidized and reduced. True or false

True

What reaction converts cystine back into two cysteines?

Reduction reaction

What happens during oxidation vs reduction of cysteine?

• Oxidation → Forms disulfide bond (S-S)

• Reduction → Breaks disulfide bond into two SH groups

What is a disulfide bond?

A covalent bond formed between two sulfur atoms of cysteine residues

How is a peptide bond formed?

By a condensation (dehydration) reaction between two amino acids

What molecule is released during the peptide bond formation?

Water (H2O)

What is a peptide bond?

A covalent bond between the carboxyl group of one amino acid and the amino group of another amino acid

What are the properties of peptide bonds?

• Planar

• Partial double-bond character

• Rigid

• Usually trans

What is Ψ (psi) bond rotation?

Rotation around the N-Cα bond

What is Φ ( phi) Bond Rotation?

Rotation around the Cα-C bond

Why can Ψ and Φ rotate?

They are single bonds

Can the peptide bond rotate?

No. The peptide bond has partial double-bond character and is rigid

What is another name for peptide bond?

An amide bond

What are amino acids called once they are incorporated into a peptide?

Residues

What enzyme catalyzes peptide hydrolysis?

Proteases

What are the two unique ends of every peptide?

N-terminus (amino terminus) ad C-terminus (carboxyl terminus)

How are peptide residues numbered?

Start at the N-terminal residue (1) and count toward the C-terminus

What is the difference between a peptide backbone and side chains?

• A backbone (main chain) = all atoms not in R groups

• Side chains = R groups

How do you determine the structure (draw) of a peptide at a given pH?

1. Identify ionizable groups

2. Compare pH to Pka

3. Assign charges

How do you determine the overall charge of a peptide at a given pH?

1. Identify all ionizable groups (N-terminus, C-terminus, and ionizable side chains).

2. Compare the pH to each group's pKa:

• pH < pKa → protonated

• pH > pKa → deprotonated

3. Assign charges to each group.

4. Add all charges together to get the net charge.

What is the overall charge of a peptide relative to its pI?

• pH < pI → net positive (+)

• pH = pI → net zero (0)

• pH > pI → net negative (−)

What are the types of proteins?

• Globular

• Fibrbous

• Membrane

• Motor

What are globular proteins?

Compact, roughly spherical proteins that are usually water-soluble and perform functions such as catalysis, transport, and regulation

What are fibrous proteins?

• Long, extended proteins that provide structural support and strength.

• Examples: Collagen, keratin

What are membrane proteins?

Proteins associated with cell membranes that function in transport, signaling, and cell recognition

What are motor proteins?

• Proteins that convert chemical energy (ATP) into mechanical movement

• Examples: Myosin, kinesin, dynein

Compare the four major types of proteins

• Globular: Compact, soluble, functional

• Fibrous: Structural support

• Membrane: Embedded in membranes; transport/signaling

• Motor: Movement using ATP

What is a conjugated protein?

A protein that contains a non-protein component required for its function

What is a prosthetic group?

A non-protein component that is tightly bound to a protein and is necessary for its function

What prosthetic group does a glycoprotein contain?

A carbohydrate prosthetic group

What is the difference between a protein and a conjugated protein?

A conjugated protein contains a prosthetic group (non-protein component) in addition to amino acids

Conjugated protein vs prosthetic group?

• Conjugated protein = protein + non-protein component

• Prosthetic group = the non-protein component itself

What is the primary (1°) structure of a protein?

The linear amino acid sequence of a polypeptide.

What is the secondary (2°) structure of a protein?

• Local folding patterns stabilized by hydrogen bonds.

Examples:

• α-helix

• β-sheet

• β-turn

What is an α-helix?

A right-handed coiled secondary structure stabilized by hydrogen bonds.

What interactions stabilize protein tertiary structure?

• Hydrogen bonds

• Salt bridges (ionic interactions)

• Van der Waals interactions

• Hydrophobic interactions

• Disulfide bonds

What stabilizes an α-helix?

Hydrogen bonds between backbone C=O and N-H groups.

Which amino acids tend to disrupt α-helices?

Proline and glycine.

What is a β-sheet?

A secondary structure made of extended strands connected by hydrogen bonds.

What are the two types of β-sheets?

Parallel and antiparallel.

What stabilizes β-sheets?

Hydrogen bonds between adjacent strands.

What is a β-turn?

A short turn that reverses the direction of a polypeptide chain.

What stabilizes β-turns?

Hydrogen bonds.

What are super secondary structures (motifs)?

Combinations of α-helices and β-sheets that form recurring patterns.

What is the purpose of motifs?

• Bury hydrophobic amino acids

• Increase protein stability

• Create common folding patterns

What are examples of structures that can form motifs?

• α-helices

• β-sheets

• β-turns

Tertiary (3°) Structure

• Overall 3D shape of a single polypeptide

• Stabilized by:

  • Hydrogen bonds

  • Salt bridges

  • Van der Waals interactions

  • Hydrophobic effect

  • Disulfide bonds

• Contains domains

• Hydrophobic effect is the major driving force for folding

Fibrous vs. Globular Proteins

Fibrous

• Long and rope-like

• Structural support

• Usually insoluble

• Examples: collagen, keratin

Globular

• Compact and spherical

• Functional proteins

• Usually soluble

• Examples: enzymes, hemoglobin

What contributes to protein folding?

• ΔH (Bond Energy)

• ΔS (Entropy)

What interactions contribute to ΔH (bond energy) during protein folding?

• Covalent interactions

• Non-covalent interactions

What is a covalent interaction involved in protein folding?

Disulfide bonds between cysteines.

What non-covalent interactions stabilize proteins?

• Electrostatic interactions (salt bridges)

• Hydrogen bonds

• Van der Waals interactions

What are electrostatic interactions?

Attractions between opposite charges (salt bridges/ion pairs).

What are hydrogen bonds?

Attractions between H attached to an electronegative atom and another electronegative atom.

What are van der Waals (London dispersion) forces?

Weak attractions between nearby atoms.

What contributes to ΔS (entropy) during protein folding?

The hydrophobic effect.

What is the hydrophobic effect?

Nonpolar amino acids move to the protein interior away from water, helping drive folding.

What is quaternary (4°) structure?

The arrangement of 2 or more polypeptide subunits into one functional protein.

What is a subunit?

An individual polypeptide chain within a multi-subunit protein.

What is a domain?

An independently folding and functional region within a protein.

Subunit vs Domain

Subunit

• Entire polypeptide chain

• Found in quaternary structure

Domain

• Part of a polypeptide chain

• Independently folds and functions

What is protein denaturation?

Unfolding of a protein that disrupts its native structure and function.

What reagents promote protein denaturation (unfolding)?

• Heat

• Extreme pH

• Detergents (SDS)

• Organic solvents (alcohols)

• Reducing agents (β-mercaptoethanol, DTT)

What are the effects of denaturing reagents?

They disrupt the interactions that stabilize protein structure:

• Hydrogen bonds

• Salt bridges

• Hydrophobic interactions

• Disulfide bonds (reducing agents)

Result: protein unfolds and loses function.

What does each denaturing reagent disrupt?

• Heat: hydrogen bonds & hydrophobic interactions

• Extreme pH: ionic interactions (salt bridges)

• Detergents: hydrophobic interactions

• Reducing agents: disulfide bonds

Chromatography separates proteins based on what 3 properties?

• Size

• Charge

• Binding affinity

How does Size Exclusion Chromatography work?

Separates proteins based on size.

• Large proteins elute first

• Small proteins elute last

Memory Trick: Big = Bypass pores = First out

How does Ion Exchange Chromatography work?

Separates proteins based on charge.

• Opposite charges attract

• Proteins bind to a charged column and are eluted later

How does Affinity Chromatography work?

Separates proteins based on specific binding interactions.

• Protein binds a ligand on the column

• Other proteins wash away

• Desired protein is eluted

Compare the three types of chromatography.

Type	Separates By
Size Exclusion	Size
Ion Exchange	Charge
Affinity	Specific binding