Lec 7: Protein Structure

Why study Proteins?

• what are proteins?

• where do they occur?

• how much of cellular dry weight do they constitute

• what are they product of?

• Proteins are the most abundant biological macromolecules

• Occur in all cells and all parts of cells

• Constitute >50% of cellular dry weight

• Thousands of different kinds in a single cell

• Products of genes → agents of biological function

3 Structural Classes

• describe their shape

• water soluble or insoluble

• roles

• proteins can be generally categorized by shape and solubility

1. Fibrous

2. Globular

3. Membrane

4 Levels of Protein structure

protein structure architecture includes four levels:

1. Primary (linear amino acid sequence): Determined by gene sequence

2. Secondary (α-helices and β-sheets): Stabilized by backbone H-bonds

3. Tertiary (3D folding of single peptide): Stabilized by multiple interactions

4. Quaternary (subunit organization): Assembly of multiple polypeptide subunits -Stabilized by same forces as tertiary

Protein Structure Image

Non-Covalent Forces and Folding

• what are secondary structures stabilized by?

• what about tertiary and quaternary?

• what does each level depend on?

• what is folding dictated by?

• Secondary structures are stabilized by H-bonds. Tertiary and quaternary structures are stabilized by H-bonds, ionic interactions, van der Waals forces, hydrophobic interactions, and disulfide bonds.

• Each level depends on the level below it.

• Folding is dictated by the order of AAs in the primary structure.

Seven Principles Linking Structure and Function (1-3)

1. FUNCTION DEPENDS ON STRUCTURE

• A protein's shape determines what it can do

2. STRUCTURE DEPENDS ON SEQUENCE + WEAK FORCES

• Primary sequence encodes the fold while non-covalent forces drive folding

3. THE NUMBER OF FOLDING PATTERNS IS LARGE BUT FINITE

• Only ~1,400 distinct folds exist, and new proteins typically adopt one of these established patterns.

Seven Principles Linking Structure and Function (4-6)

4. GLOBULAR PROTEINS ARE MARGINALLY STABLE

• Net ΔG of folding: only -20 to -40 kJ/mol

• Compare: single covalent bond ≈ -350 kJ/mol

5. MARGINAL STABILITY FACILITATES MOTION

• Proteins exhibit conformational dynamics, continuously transitioning between closely related structural states.

6. MOTION ENABLES FUNCTION

• Conformational changes are essential for enzyme catalysis, signal transduction and molecular transport

Non-Covalent Forces:

• name the 4 forces

• strength

• primary role

• what is secondary and tertiary structure made out of

• Net ΔG of folding?

• Secondary structure: primarily backbone H-bonds

• Tertiary structure: collective action of ALL forces

• Net ΔG of folding: -20 to -40 kJ/mol (marginally stable)

Hydrogen Bonds: The Most Versatile Force

• who do they form with

• function? how do they stabilize secondary structure and support tertiary structure?

• strength kj/mol

• surface:

• Hydrogen bonds form between donor (N–H) and acceptor (C=O) groups

• Backbone H-bonds stabilize secondary structure; side chains support tertiary structure

• Weak individually (~5–20 kJ/mol) but are collectively strong - Directional (strongest when linear)

• Surface: H-bonds with water

The Hydrophobic Effect: The Dominant Driver of Protein Folding

• what do non polar side chains cause?

• what does burying nonpolar residues in the protein core do?

• what is the primary driving force for protein folding?

• Nonpolar side chains cause ordered water “cages” that are entropically unfavorable.

• Burying nonpolar residues in the protein core releases ordered water, gives an entropy gain and forms the hydrophobic core

• Burial of hydrophobic residues is the primary driving force for protein folding

Ionic Interactions: Charged Partners

• where do they form?

• where are they found?

• what is unique about them?

• Ionic interactions form between oppositely charged side chains (salt bridges / ion pairs)

• Found on protein surfaces, since burying charges in the core is unfavorable

• pH-dependent: changes in pH can disrupt ionic interactions, causing conformational changes or denaturation

Van der Waals Forces: The Packing Force

• are the forces weak or strong? who are they between?

• arise from what?

• strength in kj/mol

• what is unique about them?

• function?

• who do they work together with?

• Weak attractive forces between ALL atoms

• Arise from transient dipoles (electron fluctuations)

• Strength: 0.4-4 kJ/mol per interaction

• Distance-dependent: optimal at ~3-4 Å

• Individually very weak but are collectively significant

• Contribute to close packing of hydrophobic core

• Work together with hydrophobic effect

Protein Folding: A Delicate Thermodynamic Balance

• what are 4 favorable and unfavorable contributions

This small net ΔG is a FEATURE, not a flaw:

• Allows conformational flexibility

• Enables regulation and function and allows controlled unfolding when needed

The Peptide Bond: Fundamental

• what restricts rotation?

• what is the peptide plane

• what configuration pre dominates?

• where does the backbone flexibility come from?

The Peptide Bond Is Rigid and Planar

• C=O ↔ C–N resonance gives ~40% double-bond character → restricts rotation

• Six atoms lie in a single plane: Cα–C–O–N–H–Cα (the peptide plane)

• Trans configuration predominates (less steric clash)

• Backbone flexibility comes only from rotation at φ (Cα–N) and ψ (Cα–C) bonds

Encoding of Folding Information in Primary Structure

• describe analogy in the image

• what does Φ (phi) represent

• what does ψ (psi) represent

• Analogy: rigid peptide planes connected by hinges at each alpha carbon

• The conformation shown here is Φ = 180o and ψ = 180^o

• The angle about the Cα-N bond is denoted Φ (phi)

• The angle about the Cα-C bond is denoted ψ (psi)

• Some values of Φ and ψ are more likely than others

Defining φ (Phi) and ψ (Psi) Angles

Steric Constraints on φ/ψ Angles

• what do most φ/ψ combinations cause?

• what percentage of f φ/ψ space sterically allowed?

• what does this restriction explain?

• Most φ/ψ combinations cause steric clashes between backbone atoms

• Only ~20% of φ/ψ space is sterically allowed

• This restriction is why proteins adopt specific, repeatable secondary structures (α-helix, β-sheet)

The Ramachandran Plot: A Map of Allowed Conformations

Secondary Structures: Local Folding Patterns

• 3 main types of folding patters?

• function?

DEFINITION: Regular, repeating conformations stabilized by BACKBONE hydrogen bonds. Main types:

1. α-HELIX

• Most common helix

2. β-SHEET

• Extended strands

• Inter-strand H-bonds

3. β-TURN

• Reverses chain direction

• 4 residues

a-Helix Architecture

The α-Helix: Most Common Secondary Structure

• which is the most common helix?

• what do side chains do?

• core structure?

• H bond structure?

• name the 6 parameters and their values

• Right-handed helix (most common)

• Side chains (R) project OUTWARD

• Core is tightly packed (van der Waals contact)

• H-bonds are nearly parallel to helix axis

α-Helix: The i → i+4 Hydrogen Bonding Pattern

• what residues do H bonds form with?

Each backbone C=O forms H-bond with N-H, four residues ahead:

1. Residue 1 (C=O) ──H-bond──→ Residue 5 (N-H)

2. Residue 2 (C=O) ──H-bond──→ Residue 6 (N-H)

3. Residue 3 (C=O) ──H-bond──→ Residue 7 (N-H)

4. ... and so on

The α-Helix Dipole

• what bonds points toward the N terminus and C terminus

• what does the create?

• what binds at the matching end?

The α-Helix Has a Dipole

• All N–H bonds point toward the N-terminus; all C=O bonds point toward the C-terminus

• Creates a net dipole: N-terminus is δ⁺, C-terminus is δ⁻

• Charged ligands bind at the matching end (e.g., negative ligands near the δ⁺ N-terminus)

α-Helix Architecture

Φ and ψ degrees for right handed helix?

Helices May be Polar, Nonpolar or Amphiphilic

• what kind of faces do α-helices have?

• what reveals this segregation?

• 3 functional roles?

• Many α-helices have distinct faces: one hydrophobic, one hydrophilic

• A helical wheel (end-on view) reveals this segregation

Functional roles:

• Membrane-spanning helices

• Surface helices on globular proteins

• Antimicrobial peptides that disrupt membranes

Another Amphipathic Helix

AAs can be classified as helix -formers or breakers

• name 5 helix former AA

• 2 helix breakers AA

• context dependent

HELIX FORMERS (favor helix):

• Alanine (Ala) - small, fits well

• Leucine (Leu) - hydrophobic, good packing

• Methionine (Met) - flexible hydrophobic

• Glutamate (Glu) - can form salt bridges

• Lysine (Lys) - can form salt bridges

HELIX BREAKERS (disfavor helix):

• Proline (Pro) - RIGID, causes kink, no N -H for H -bond

• Glycine (Gly) - TOO FLEXIBLE, destabilizes helix

CONTEXT -DEPENDENT:

• Most other amino acids

• Depends on neighboring residues and environment

The B-Pleated Sheet

• describe structure: what is it composed of?, how are strands connected?, what does it form?

• what are the 2 arrangements?

STRUCTURE:

• Composed of β-STRANDS in extended conformation

• Strands connected by INTER-STRAND H-bonds

• Forms a pleated (corrugated) surface

β-Sheet: Key Parameters

• phi and psi angles in anti parallel and parallel B sheet

The b-Pleated Sheet: Side Chain Arrangement

• where are the side chains located on the sheet plane?

• what do the side chain create?

• what does it allow?

SIDE CHAIN ARRANGEMENT:

• Side chains alternate ABOVE and BELOW the sheet plane

• Creates a "pleated" appearance

• Allows for amphipathic sheets: - One face hydrophobic - Other face hydrophilic

β-Turns: Reversing Chain Direction

• function?

• what are the short turns (4 residues) stabilized by?

• where do adjacent strands connect?

• what is it enriched with?

• where is it commonly found? and involved it what?

• FUNCTION: Allow polypeptide chain to reverse direction

• Short turns (4 residues) stabilized by an i → i+3 hydrogen bond

• Connect adjacent strands in antiparallel β-strands

• Enriched in Proline (rigid, φ-angle constraint) and Glycine (small, flexible it fits tight turns)

• Commonly found on the protein surface, exposed to solvent and often involved in protein–protein interactions

Secondary Structure Summary

1. levels of protein structure

2. forces driving folding

3. backbone conformation

4. a-helix

5. B-Sheet