Lecture 4 - Tertiary Structure/Protein Purification

Tertiary Structure

Folding of secondary elements for a complete 3-D structure of one polypeptide, allowing distant amino acids to be closer

Interactions stabilizing tertiary structure

H-bonding, covalent bonds, salt bridges, LDFs, hydrophobic effect between backbone AND side-chains

What structure is most favorable?

Primary: highest entropy

What structure is least favorable?

Tertiary: lowest entropy due to folding

What is the strongest force in protein folding?

The hydrophobic effect (though it’s still not enough to make it favorable)

Hydrogen Bonding in Tertiary Structure

1. Side-chain to side-chain

2. Backbone to backbone

3. Side-chain to backbone

Salt bridges (ion-pairing)

Interaction between + and - charged amino acids

Positively charged amino acids

Lysine, histidine, arginine

Negatively charged amino acids

Aspartate, glutamate

van der Waals (LDF) Interactions

Weak electrostatic between fixed/induced dipoles; strong collectively

What creates van der Waals forces?

Attraction between electron-rich and poor regions of two molecules (electrons attract to positive nucleus)

Hydrophobic Effect

Increases entropy of water due to non-polar R-groups distributing to the interior of molecule

What increases entropy in protein folding?

The hydrophobic effect

What decreases enthalpy in protein folding?

Many van der Waals forces

Disulfide Bonds

Covalent bonds between cysteine residues (reversible)

Serum Albumin

Protein containing many disulfide bonds

Where is information needed to fold protein contained?

Primary structure (linear amino acid sequence)

How do we know primary sequence holds folding information?

If we denature protein with BME and Urea, the protein can refold IF done in the correct process of removing urea, then oxidizing

Role of BME

Denature protein by reduction (adding hydrogen)

Role of urea

Denatures protein by breaking H-bonds (unfolding)

Steps to mis-folded protein

1. Oxidize (reform disulfides randomly)

2. Remove urea (refolding, H-bonds)

Steps to correctly folded protein

1. Remove urea (H-bonds, refolding)

2. Oxidize (reform disulfides)

SPECIFIC steps, so that disulfides form in correct places

Temperature increase causes..

Unfolding of proteins (denaturing)

Temperature decreases causes..

Proteins to refold

Energy Landscape of Proteins

Like a funnel; denatured are high entropy, folded are low entropy with many different conformations

Fibrous Proteins

Rod-shaped proteins that provide structure, organized as high-order arrays of secondary structures (helices/sheets)

What shape are fibrous proteins?

Rods

What is the function of fibrous proteins?

Structure

Motifs

Repeating sequence units (often in fibrous proteins)

Examples of fibrous proteins

a-Keratins, fibroin, collagen

a-Keratins are found in…

Hair, wool, nails

Fibroin is found in…

Silk

Collagen is found in…

Connective tissues, bones, tendons, vessels, etc.

Diversity of Fibrous Proteins

Is limited because of motifs; often primarily 3 amino acids

a-Keratin Motif

7-residue repeat (abcdefg)_n where a and d are hydrophobic

Fibroin Motif

(Ser-Gly-Ala-Gly)_n

Tropocollagen Motif

(Gly-X-Y)_n

Structure of a-keratins

• All a-helical with pseudo-repeat motif

  • (abcdefg)

• Hydrophobic a/d positions form a sticky strip, creating a coil of coils

  • Higher order structure = VERY stable

Covalent bonding in a-keratins

Disulfide bonds can be broken and rearranged (reduced then oxidized); used in perms

What bond stabilizes a-keratins?

Disulfide bonds

Fibroin Structure

• Antiparallel beta-sheet

• Repeating motif: (Ser-Gly-Ala-Gly)_n

  • All small amino acids

• Side chains between adjacent sheets alternate between big and small

  • Interdigitate to allow for stable packing

Fibroin Packing

Antiparallel b-sheets are already extended and VERY strong; flexible because inter-sheet van der Waal forces are weak (slide between)

Tropocollagen Structure

• Left-handed helix (NOT alpha-helix) with 3 residues per turn

• Gly-X-Y motif

  • X = proline, Y = hydroxyproline

• H-bonding INTER-chain, NOT intra-chain

• Peptide bonds perpendicular to plane

Tropocollagen Superhelix

• 3 interwound helices twisted right-handed

  • H-bonds stabilize between amide hydrogens and carbonyl oxygens

• Glycine is in the central position

Post-translational Modifications

• Cross-linking

• Hydroxylation of proline

• Hydroxylation of lysine

ALL used to stabilize collagen

Cross-linking

Post-trans. modification requiring Cu²⁺ for lysyl oxidase activity to stabilize collagent

Cross-linking increases with…

Age - loss of elasticity and brittle bones

What two substances are needed for cross-linking?

Cu²⁺ and lysyl oxidase

Copper deficiency leads to..

Weakened collagen (affecting bone, tendons, muscles)

Hydroxylations

Post-trans. modifications that stabilize collagen by hydroxylating proline or lysine

Enzymes for hydroxylation

Proline hydroxylase, lysine hydroxylase

What other molecule is needed for hydroxylation?

Ascorbate (Vitamin C)

Vitamin C Deficiency (Scurvy)

Leads to weak collagen due to its role in hydroxylation