Protein: Structure, Function and Folding

What is the structure hierarchy of proteins?

1. Primary - chemical structure

2. Secondary - localstructural elements

3. Tertiary - long range interactions overall fold into single polypeptide chain

4. Quaternary - interactions between polypeptide chains

What is the configuration of the backbone?

- can be cis or trans (favoured)

- cis commonly seen for X-Pro seq

What does it mean that each plane shares one alpha-carbon?

- Each peptide bond is part of a relatively flat, rigid plane.

- Adjacent planes connect through the shared alpha-carbon

- The alpha-carbon is attached to the amino acid's R group, H, amino group, and carbonyl group.

What are the main rotational angles and the issue with them?

Allows for folding:

1. Phi - rotation around N and alpha-carbon

2. Psi - rotation around alpha-carbon and carbon

Issue

- causes steric hinderance

- unfavourable positions

- only specific combos allowed restricting folding

- sometimes glycine (due to small side chain) and proline (due to ring structure) form unfavourable positions

What are the key elements of the secondary structure?

1. Alpha helix

2. beta sheet

3. beta turns

4. loops

What are the key features of the alpha helix?

1. Shape: Right-handed helix (twists clockwise as it moves away from you).

2. Residues per turn: 3.6 amino acids.

3. Pitch: 5.4 Å (0.54 nm) per complete turn.

4. Hydrogen bonds: The C=O of residue (n) hydrogen-bonds with the N-H of residue (n+4).

- hydrogen bond connects amino acids that are 4 positions apart.

- ex: Residue 1: its C=O group forms a hydrogen bond with the N-H group of residue 5.

- ex: Residue 2: its C=O group forms a hydrogen bond with the N-H group of residue 6.

5. R groups: Point outward from the helix axis.

What are beta sheets?

- Two separate sections of extended polypeptide interact via H-bonds

- beta turn: 4 amino acids

Possible orientations:

1. Antiparallel strands: straight H-bonds (same direction)

2. parallel strands: The H-bonds are not straight (opp direction)

What are the key features of a beta sheet?

1. Hydrogen bonds: Hold neighboring β-strands together.

2. Orientation: Strands can be parallel (run in the same direction) or antiparallel (run in opposite directions). A sheet can contain both.

3. Puckered and twisted: The sheet is not perfectly flat; it has a folded, slightly twisted shape.

4. R groups: Point above and below the sheet, roughly at right angles to its plane.

5. β-bulges: Occur when amino acid residues don't line up perfectly between strands.

6. Turns: Can connect one β-strand to another via tight turns or large loops

What type of strands are connected by beta turns?

- beta turn: tight turns

- beta turns have pro at position 2

- type 2 beta turns have gly at position 3

How does sequence impact secondary structure?

- Different amino acids have different helical propensities: Some amino acids are more likely to form α-helices than others.

- Side-chain interactions: R groups can interact with each other and disrupt or distort the α-helix or β-sheet

Ex: Proline

- Rarely occurs in α-helices because its rigid structure restricts backbone movement

- it cannot donate the usual backbone N-H hydrogen bond.

- It can disrupt the helix.

How does sequence impact alpha helix stability?

- Not every amino acid sequence forms an α-helix. Some amino acids make helices more stable, while others disrupt them.

1. Alanine (Ala) and Leucine (Leu): Strong helix formers; they tend to support α-helix formation.

2. Proline (Pro): A helix breaker because its rigid ring restricts rotation around the N-Cα bond, limiting the backbone angles needed for an α-helix.

3. Glycine (Gly): A helix breaker because its tiny R group gives it more flexibility, allowing it to adopt other conformations.

What are two ways to determine or estimate how much α-helix, β-sheet, and random coil a protein contains?

1. Computer programs - predict secondary structure

2. Circular dichroism (CD) — measure secondary structure

How do computer programs predict secondary structure?

- Programs use statistical analysis of the amino acid sequence.

- They compare the sequence with known patterns showing which amino acids tend to occur in α-helices, β-sheets, or coils.

- They predict which secondary structures the protein is likely to form.

How does Circular Dichroism measure secondary structure?

- measures the difference in how a molecule absorbs left- versus right-circularly polarized light

- Why it works: Peptide bonds produce different CD signals depending on the protein's conformation.

What it tells us: The protein's CD spectrum can be analyzed to estimate the percentage of:

- α-helix

- β-sheet

- Random coil

- Chromophores: Light-absorbing groups in a chiral environment produce characteristic signals. Peptide bonds contribute to the secondary-structure signal, while aromatic amino acid residues can also absorb light.

How can you summarize secondary structure?

- Hydrogen bonds: Form between backbone carbonyl oxygens and amide hydrogens.

- α-helices: Hydrogen bonds form between nearby residues, usually n + 4, sometimes n + 3

- β-sheets: Hydrogen bonds connect strands that may be far apart in the amino acid sequence.

- Globular proteins: Secondary structure elements interact and fold together to form compact proteins.

- Fibrous proteins: Secondary structures help form long, structural proteins.

- Circular dichroism (CD) spectroscopy: Estimates the percentage of α-helix, β-sheet, and random coil in a protein.

What are the two major classes of tertiary structure?

1. fibrous proteins

- typically insoluble

- made from a single secondary structure long extended structures

- many polypeptides

- often structural function

2. globular proteins

- water-soluble globular proteins

- lipid-soluble membrane proteins

- roughly spherical in shape

- polypeptide folds back upon itself

- enzymes are globular

What are some key features of fibrous proteins?

- Much or most of the polypeptide chain is organized approximately parallel to a single axis

- Fibrous proteins are often mechanically strong

- Fibrous proteins are usually insoluble in water

- Fibrous proteins usually play a structural role in nature

What is alpha keratin?

- a fibrous protein found in mammalian hair, fingernails, claws, and horns.

- Structure: Contains α-helical rod segments of about 311-314 amino acid residues, with non-helical regions at the N- and C-termini.

- Coiled-coil: Two α-helices wrap around each other to form a coiled-coil with a left-handed twist.

- 3.5 residues per turn: Each α-helix in the coiled-coil has approximately 3.5 residues per turn.

- Seven-residue repeat: The sequence follows (a-b-c-d-e-f-g)n

Positions a and d contain nonpolar (hydrophobic) amino acids.

- These hydrophobic residues help the two helices associate with each other.

- Examples of nonpolar residues: Alanine (Ala), leucine (Leu), valine (Val), methionine (Met), and phenylalanine (Phe).

How does the coiled-coil of alpha keratin form?

The nonpolar residues at positions a and d face inward toward the other helix. Their hydrophobic interactions help hold the two α-helices together.

- has intra and inter strand ionic interactions that stabilize the coiled coil

- seen in dimerization domains of transcription factors

What is Silk Fibroin?

• Fibroin is the main protein in silk from moths and spiders

• Antiparallel b sheet structure

• Small side chains (Ala and Gly) allow the close packing of sheets

• Structure is stabilized by

- hydrogen bonding within sheets

- London dispersion interactions between sheets

- Hydrophobic interactions between alanine side chains

What is the structure of collagen?

- Three polypeptide chains wrap around each other to form a right-handed superhelix.

- Each individual chain forms a left-handed helix.

- Each chain has 3 amino acid residues per turn.

- Backbone angles: phi = -51, psi = +153

- No intrachain hydrogen bonds: Hydrogen bonds form between neighboring chains instead.

What is the amino acid sequence of collagen?

Collagen contains many repeats of Gly-X-Y, where:

- Gly (glycine): Every third residue; its small R group allows the chains to pack closely together.

- X: Often proline.

- Y: Often 4-hydroxyproline.

- Collagen also contains hydroxylysine.

- The chains are cross-linked near their N- and C-termini, helping strengthen the structure.

How does hydrogen bonding occur in collagen?

- Hydrogen bonds form between the three chains.

- The N-H group of glycine on one chain can hydrogen-bond with a carbonyl group on a neighboring chain.

- These bonds help stabilize the collagen triple helix.

Why is 4-hydroxyproline important for collagen?

- Increases collagen stability.

- Replacing 4-hydroxyproline with proline substantially decreases collagen's melting temperature.

- Its hydroxyl (-OH) group changes interactions and helps stabilize the structure.

- The enzyme that produces 4-hydroxyproline requires vitamin C as a cofactor.

What is globular tertiary structure?

- Tertiary structure = the overall 3D shape of a single protein formed when its secondary structures fold and interact.

- Globular proteins are generally compact, roughly spherical proteins.

How do globular proteins fold?

- Secondary structures form wherever possible because they are stabilized by many hydrogen bonds (H-bonds).

- Examples: α-helices and β-sheets.

- Helices and sheets pack closely together to form a compact structure.

- The backbone sections connecting secondary structures are usually short and direct, helping the protein fold efficiently.

How do proteins fold into an energetically favourable shape?

- Forming stabilizing interactions, including hydrogen bonds.

- Minimizing contact between hydrophobic (water-avoiding) residues and water.

What are some design principles of globular proteins?

1. most polar residues face the outside of the protein and interact with solvent

- exception: membrane proteins & subunit interfaces

2. most hydrophobic residues face interior of protein and interact w each other

- exception: membrane proteins & subunit interfaces

3. "Random coil" is not random

- occurs due to shape restrictions

4. structures are not static, acc dynamic

5. Various levels of protein flexibility

6. Has various kinds and rates of protein motion

What are the different kinds of protein motion and their rate?

1. Atomic vibrations

- displacement: 0.01-1 Ǻ

- time: 10^-15 to 10^-13 s

- source: Kinetic, thermal

2. Collective motions

- Fast: ring flips, methyl rotation

- Slow: Hinge bending between domains

- displacement: 0.01-5 Ǻ

- time:10^-12 to 10^-3 s

- source: Kinetic, thermal

3. Triggered conformation changes

- enzyme

- displacement: 0.5-10Ǻ or more

- time: 10-9 to 103 s

- source: Interactions with ligand

Why is folding important for protein function?

- Individual secondary structures, such as helices and sheets, are not usually active on their own.

- They must fold into the correct tertiary structure to create the protein's functional shape.

- This 3D arrangement brings important amino acid residues together to form binding sites or active sites.

- aka: structure determines function

What is empty space?

- packing of residues is close

- But due to van Der Waals volume to total volume is only 0.72 to 0.77 leading to empty space

- the empty space exists as small cavities

What are the structural families of globular proteins?

1. all alpha

2. all beta

3. a + b secondary structure elements separate in sequence, antiparallel beta strands

4. a/b mixed in the sequence, parallel strands in beta sheets

Why are protein structures repeated in nature?

- proteisn have similar structural patterns that repeat indiff proteins

- such shapes are useful cus they perform certain functions

- key terms: motifs and domains

What is a structural motif?

- small, recurring arrangement secondary structures within a protein

Ex: Rossmann fold

- in nucleotide binding proteins

- helps proteins bind nucleotides like NAD and NADP

- in ~ 56 protein families

Key sequences:

- ADP/NAD-binding proteins : Gly-X-Gly-X-X-Gly

- NADP-binding proteins : Gly-X-Gly-X-X-Ala

What is a domain?

- A domain is a distinct region of a protein that can fold into a stable 3D structure, often independently of the rest of the protein.

- Domains can have specific functions, such as binding a molecule or catalyzing a reaction.

- Some domains can retain their structure and function when separated from the larger protein.

Ex: SH2 (Src homology)

- in proteins involved in tyrosine kinase signalling pathways.

- Their function is to bind phosphorylated tyrosine residues.

What is a kringle domain?

- Kringle domains are approximately 80 amino acids long.

- They form looped structures stabilized by 3 disulfide bonds per domain.

- Disulfide bonds are strong covalent bonds between cysteine residues that help stabilize protein structure.

Kringle domains are found in:

- Proteases involved in the blood-clotting cascade.

-Growth factors.

What are the different structural families?

- Similar sequence similar structure

- Similar function similar structure

- Similar function or structure does not imply similar sequence

What is quaternary structure?

The arrangement of two or more polypeptide chains (subunits) that join together to form a functional protein.

What are some advantages/features of quaternary structure?

1. Stability: Interactions increase the stability of the subunit.

2. Activity: The active site is created at the subunit interface.

3. Regulation of Activity: Cooperativity of substrate binding improves biding efficiency.

4. Distal binding site for activators and enhancers increase sensitivity to such molecules.

5. Genetic Fidelity: Each chain in an homo oligomer is coded for by a different gene. If one is wrong the other compensate.

6. Genetic efficiency: One gene many subunits

How is protein stability maintained in 3* and 4* structure?

- by different interactions between and within amino acids

Types:

1. Electrostatic forces

- ion-ion

- close range

- salt bridges

2. Van der Waal forces

- LDF

3. Hydrogen bonds

- do not contribute much to the overall stability of the protein because there is not much difference in energy between an intramolecular H bond and one with w

4. Hydrophobic Interactions

Main: covalent bonds

- includes all bonds in the amino acids, plus peptide bonds and disulfide bonds these are clearly the strongest, 200-300 kJ/mo

What holds proteins together

1. Hydrophobic effect

- mainly in large proteins

2. Hydrogen bonds

3. Buried salt bridges

How does the microenviornment effect properties of amino acids?

1. Charged side chains buried in hydrophobic pockets

- Negative side chains (−): Prefer to become uncharged in a hydrophobic pocket. Their pKa increases.

- Positive side chains (+): Also tend to become uncharged. Their pKa decreases.

2. Buried Salt Bridges: attraction between oppositely charged side chains.

- Negative side chain (−): pKa decreases.

- Positive side chain (+): pKa increases.

What is the relation between Ka and pKa?

Which form is more stable?

1. A- (deprotonated) -> Ka inc and pka dec

2. HA (protonated) -> Ka dec and pKa inc

How can mutations affect proteins?

- Mutations change the amino acid sequence, which can change protein structure and function.

Conservative Mutations:

- Replace an amino acid with one that has similar properties.

- Usually cause smaller changes.

- Exception: Can have a big effect if the mutation occurs in an important location.

Nonconservative Mutations:

- Replace an amino acid with one that has different properties.

- Usually cause bigger changes.

- Exception: May have little effect if the mutation occurs on an unimportant surface region.

What are some methods used to determine protein structure?

1. Circular dichroism (CD): Estimates how much α-helix, β-sheet, and random coil a protein contains.

2. X-ray crystallography: Uses X-rays passing through protein crystals to determine the protein's structure.

3. NMR spectroscopy: Uses magnetic fields to study atoms in proteins and determine their structure.

4. Cryo-electron microscopy (Cryo-EM): Uses electron microscopy to determine protein structures by imaging frozen samples.

5. Modelling: Uses computers to predict protein structure

What is NMR Spectroscopy?

1. Which nuclei are used?

- Hydrogen-1

- Carbon-13

- Nitrogen-15

- These nuclei have a magnetic moment, meaning they behave like tiny magnets.

2. What happens in a magnetic field?

- The nuclei are placed in a static magnetic field.

- They precess, meaning their magnetic orientation rotates around the direction of the magnetic field.

3. What determines their frequency?

The precession frequency depends on:

- The type of nucleus (H, C, or N).

- Its chemical environment — the surrounding atoms and bonds affect the magnetic field experienced by the nucleus.

How does protein folding occur?

- Take folded proteins and unfold them and watch how they fold back up again

- Denature and renature

- Levinthal's paradox: various possible shapes that dont get tested, instead protein folds into favourable pathways

- retains partially folded intermediates -> nucleation condensation model

Why is protein folding considered to be hierarchichal?

1. Hydrophobic collapse: Hydrophobic amino acids move toward the interior, causing the protein chain to collapse into a compact shape.

2. Secondary structure (2°): α-helices and β-sheets form.

3. Tertiary structure (3°): Secondary structures fold and pack together into a 3D shape.

4. Final conformation: The protein reaches its correct, stable, functional shape.

Disulfide bonds:

- Disulfide bonds form between cysteine residues.

- During folding, these bonds may break and reform until the correct bonds stabilize the final structure.

What are some accessory proteins that assist in protein folding?

1. Protein disulfide isomerase: An enzyme that reduces disulfides and then helps reform new disulfide bonds

2. Molecular Chaperones: Heat shock proteins,GroEL GroES

What are GroEL and GroES?

What are they?

- GroEL: A protein made of two rings, each containing 7 subunits, that forms a folding chamber.

- GroES: A cap made of 7 subunits that closes the chamber.

- They help misfolded proteins fold correctly.

How does the cage work?

1. ATP binds to GroEL.

2. A misfolded protein binds to the opening of the chamber.

3. GroES caps the chamber, enclosing the protein.

4. ATP is hydrolyzed, providing energy for the process and allowing time for the protein to fold.

5. ATP and a new substrate protein bind to the opposite ring.

The first protein and ADP are released, allowing the cycle to repeat.

What is the Ribonuclease Refolding Experiment?

- Ribonuclease is a small protein that contains 8 cysteines linked viafour disulfide bonds

- Urea in the presence of 2-mercaptoethanol fully denatures ribonuclease

- When urea and 2-mercaptoethanol are removed slowly, the proteinspontaneously refolds, and the correct disulfide bonds are reformed

- The sequence alone determines the native conformation

- Quite "simple" experiment, but so important it earned Chris Anfinsenthe 1972 Chemistry Nobel Prize

What is the relation between the native structure and denaturation?

- native structure = the biologically relevant 3D structure of a protein.

- denaturation = disrupting the forces holding thenative structure together

How can denaturation of proteins be monitored and occur?

Monitored via:

- CD spectroscopy

- Activity assays

- NMR spectroscopy

Proteins can be denatured by

- heat or cold

- pH extremes

- organic solvents

- chaotropic agents: urea and guanidinium hydrochloride

What are some diseases that occur due to incorrect folding?

1. Alzheimer's

- Misfolded peptides form β-sheets and aggregate

- Consequence: Plaques form in the brain

2. Cancer involving p53 mutations

- p53 becomes unstable or unfolds

- consequence: Loss of tumour-suppressing function

3. Cystic fibrosis

- Mutant CFTR misfolds and is degraded

- consequence: Too little functional chloride channel reaches the membrane

What are some therapies for diseases due to incorrect protein folding?

1. Chemical chaperones (TMAO, glycerol)

- Stabilize protein structure and may improve folding

2. Inhibitors of specific chaperones

- May reduce degradation of mutant CFTR, allowing more protein to reach the membrane