4.1-4.3

Protein Conformation:

—A protein conformation refer to its 3D structure it can achieve without breaking covalent bonds, however, rotation among single bonds is prevalent.

—In typical biological contexts, only a small set of conformations dominate, the ones with the lowest Gibbs energy.

—Most proteins must change conformation to function

: enzymes undergo conformational changes during catalysis.

: receptors shift during ligand binding.

: structural proteins deform under force.

: transport protein can cycle through shapes.

Protein stability is marginal

The free energy difference between folded and unfolded states is small: 5-65 kJ/mol.

—Marginal stability allows folded structures to persist, conformational changes to occur easy.

Unfolded state stabilized by: high conformational entropy (many shapes), excessive H bonding with water.

Folded state stabilized by: formation of internal interactions, burial of hydrophobic residues and optimized packing.

Hydrophobic effect:

Water forms a tightly bound hydrogen-bonded network. When non-polar side chains are exposed, water forms ordered solvation shells, decreasing entropy.

—When hydrophobic residues cluster in the protein interior,

the ordered water is released, entropy of water increases, free energy decreases.

CONSEQUENCES:

proteins have hydrophobic cores, polar/ charged residues face outward.

the core provides structural rigidity, folding occur spontaneously.

Hydrogen Bonds

Hydrogen Bonds form between backbone groups (secondary structure)

between side chains (tertiary structure).

—Each hydrogen bond forms inside the protein replaces one broken with water, net delta G= 0.

—Unsatisfied polar groups in the core are extremely destabilizing

—Hydrogen bond form cooperatively in helices and sheets.

—they guide folding pathways.

—Guide geometry in secondary structures.

Ionic Interactions (Salt bridge)

Salt bridges form between oppositely charged residues such as Lys and Glu.

their strength depends on the environment.

—Weak on surface, high dielectric, strong when buried, low dielectric.

Buried salt bridges: stabilize thermophilic proteins, limit flexibility, and give unique fold identity.

Van der Waals

Arise from transient or permanent dipoles

Properties:

—very weak individually

—operate at only close distances (0.3-0.6 nm).

—extremely numerous in folded proteins.

REQUIRE TIGHT PACKING

Proteins are dense, small mutations can destabilize folding, complimentary surfaces bind well.

Disulfide bonds

Covalent bond between cysteine residues

-rare inside cells as the cells are a reducing environment.

-Common in extracellular proteins

stabilize unfolded state by reducing entropy.

-common in thermophiles and secreted proteins.

Why weak interactions dominate over covalent bonds

A single covalent bond requires about 200 kJ/mol to break and weak interactions only require 0.4-30 kJ/mol to break. But because thousands of weak interactions form, they dominate overall stability.

Protein stability is emergent, not additive, it depends on the collective behavior, not individual bond strength.

Intrinsically disordered Proteins (IDP)

-Some protein segments (or whole proteins) lack stable structure yet remain functional.

—These regions are enriched in charged residues (Arg, Lys, Glu), and small residues (Gly, Ala), lack hydrophobic cores and remain flexible.

—Common in: signaling pathways, transcriptional regulation, protein-protein interaction hub.

Peptide bond planarity and backbone geometry

—The peptide bond has a partial double bond character due to resonance, making it planar, rigid, and unable to rotate.

—The six atoms of the peptide group lie in one plane, usually in the trans configuration. w=180.

Only two backbone bonds rotate:

• ϕ (phi): N–Cα

ψ (psi): Cα–C

Thus the backbone is a chain of rigid planes connected by rotating joints.

Dihedral Angles and Steric Constraints

ϕ and ψ angles define protein backbone geometry.

each angle ranges from -180 degrees to + 180 degrees

many values are forbidden due to steric clashes.

allowed regions define a helices and b sheets.

visualized in Ramachandran plots.

Secondary Structure (general)

-refers to the local, regular arrangement of the polypeptide backbone in a specific segment of a protein without regard for side-chain positions or interactions within other parts of the molecule with repeating values of phi and psi.

-Ina regular secondary structure, dihedral angles remain the same across the stretch of residues.

A helix

Predicted by Linus Pauling and Robert Corey based on: knowledge of peptide bond geometry and hydrogen bonding.

—Polypeptide backbone winds around an imaginary axis.

—R groups project outward from the helix.

—One full turn every 3.6 residues.

—Rise of 5.4 Angstroms.

—Backbone atoms adopt characteristic psi and phi angles.

—Sometimes bend and kinks.

—Left handed helicies not possible in natural proteins.

What stabilizes the a helix?

Hydrogen bonding

—The carbonyl oxygen of residue i hydrogen bonds with the amide hydrogen of residue i +4.

—Nearly every peptide bond, except those near the ends.

At the ends of the helix several peptide bonds cannot participate in this pattern, they are either hydrogen bonded with water or capped with other protein parts.

Length of an a helix

because the a helix rises 1.5 Angstroms per residue, its length can be calculated directly from the number of residues.

80 residues x 1.5 angstrom = 120 Angstrom=12 nm

Amino acid sequence determines what ?

A helix stability.

Intrinsic Helix-Forming Propensity:

Each amino acid has a characteristic tendency to form an a helix.

Alanine has the highest helix-forming propensity.

Side-Chain Interactions:

Side chains separated by 3-4 residues interact in the helix.

Oppositely charged residues can form stabilizing ion pairs.

Aromatic residues spaced appropriately to prevent hydrophobic interactions.

Electrostatic Repulsion:

Adjacent Glu residues (neg) repel each other

Positively charged Lys or Arg residues repel each other.

Size and shape of R-groups:

Asn, Ser, Thr and Cys can destabilize helices is clustered.

Bulky side chains create steric strain.

Proline and Glycine disrupt helices:

Proline introduces a rigid kink and lacks a Hydrogen for H bonding.

Glycine is too flexible and favors other conformations.

The helix dipole

Each peptide bond has a small dipole, in an alpha helix, these dipoles align, producing a net helix dipole.

Partial positive charge near the amino terminus, partial negative charge near the carboxyl terminus.

Negatively charged residues stabalize the amino end and positively residues stabilize the carboxyl end.

Helix stability depends strongly on residue identity at helix ends.

Beta conformation and sheets

In the beta conformation, the polypeptide backbone is extended in a zigzag structure rather than a helix. A single segment is called a strand and multiple strands form a sheet.

Features

Back bone adopts characteristic psi and phi angles

hydrogen bonds form between adjacent strands.

Side chains alternate above and below the sheet.

Sheets have a pleated experience.

Sheets can be formed from those adjacent in sequence, distant in sequence, or on different polypeptide chains entirely.

Anti Parallel vs Parallel sheet

• Anti Parallel

• Adjacent strands run in opposite directions.

• Hydrogen are nearly linear

• Repeat distance ~ 7.0 Angstroms

• Twice as common in natural proteins.

Parallel Beta sheets:

—Adjacent strands run in the same direction

—Hydrogen bonds are angled and less optimal

—Repeat distance of 6.5 Angstroms.

Beta Turns & direction reversal

Proteins with compact globular structures require frequent direction changes. These changes occur in turns and loops, escpeically beta turns.

BETA Turns:

-180 degree turn involving four residues

-Hydrgen bond involving residue 1 and 4.

Central two residues do not hydrogen bond

Commonly connect antiparallel Beta strands.

Beta turns are usually surface exposed, allowing central residues to hydrogen bond.

A less common structure is the y turn, involving 3 residues with a hydrogen bond between residues 1 and 3.

Dihedral angle and Ramachandran plots

All secondary structures are defined by their psi and phi angles, which can be visualized using Ramachandran plots, which show sterically allowed conformations.

a helices and B sheets occupy distinct regions, and most residues fall in these regions.

Glycine appears outside these regions because of its small side chain.

CD spectroscopy

Measures the differential absorption of left and right handed circularly polarized light by chiral molecules.

The measurements are made in the far UV, (190-250 nm).

The chromophore is the peptide bond.

Signals only arise when peptide bond is in ordered chemical enviornments.

Uses:

Identify if a protein is folded, estimating a helix and B sheet content, monitor folding and unfolding transitions.

Tertiary V Quaternary Structure

—Tertiary structure is the overall three dimensional arrangement of all atoms in a protein. Unlike secondary structure, tertiary structure allows for the long interaction of amino acids that may be far apart in the primary sequence and in different secondary strcutures

—Tertiary structure is primarily stabalized by weak interactions of the polypeptide chain, involving hydrophobic effect, hydrogen bonds, ionic interactions, and van der waals interactions.

Quaternary Structure: Some proteins are composed of two or more polypeptide chains called subunits ( which may be identical or different), the three dimensional arrangement of these subunits in a functional complex is the protein quaternary structure.

Fibrous Proteins

Fibrous proteins are specialized for structural roles and share key properties- built from simple repeating secondary structure elements- insoluble in water- high hydrophobic residue content in the exterior and interior.

Hydrophobic residues are typically buried through tight packing of many polypeptide chains into supramolecular assemblies.

-usually dominated by a single secondary structure.

EX:

— A helix + disulfide cross link: tough protective structures —> a-keratin (hair, nails, etc).

—Beta conformation—> soft flexible filaments, silk fibronin.

collagen triple helix- high tensile strength without stretch. like bones and matrix.

Alpha keratin found where

— Found: In mammals and make up all the dry weight of hair, wool, nails, and claws, and much of the outer skin layer. Alpha keratins belong to a family called intermediate filament proteins, which are structural proteins in animal cytoskeletons

Alpha Keratin other characteristics

—Coiled Coil: the core secondary structure is a right handed alpha helix. Crick and Pauling proposed that keratin is a coiled coiled: - two alpha helical chains align in parallel (N→C direction)They wrap around each other in a left handed supertwist.

Kertain X ray differ from ideal a helix:

A helix: 5.4 angstroms per turn.

Keratin repeat: 5.15-5.2 Angstroms.

Discreptancy explained by additional twisting of a helices into a coiled coil.

Hydrophobic Packing in Coiled Cells:

The helix-helix contact surfaces are rich in hydrophobic residues whose R groups interlock in a regular pattern, allowing tight packing.

A keratin is therefore rich in: Ala, Val, Leu, Ile, Met, and Phe.

Higher order assembly:

Two chain coils assemble into: Protofilaments, protofibrils, and intermediate filaments.

An intermediate filament contains 32 keratin strands via protofibril bundling.

Covalent Reinforcement: Disulfide bond,

Strength is enhanced by covalent cross links: -in A keratin, these disulfide bonds between Cys residues. In very tough keratins, like horns, 18% of residues are cysteines in disulfide bonding.

Collagen

Found: In tendons, cartillage, cornea.

It is the most abundant protein in mammals, typically 25-35% of the total protein.

Collagen ke unique secondary structure:

Collagen forms a left handed helix

3 residues per turn

distinct from the a helix.

Collagens Quaternary/ Tertiary arrangement:

-Three seperate polypeptides called alpha chains twist around one another

-The triple helix is a coiled coil but the superhelical twist is right handed.

Opposite of A helices.

Rpeating sequence Gly-X-Y:

Collagen has a characteristic tripeptide repeat: Gly-X-Y-X, Pro-Y, and 4-Hyp.

Typical composition: 35% gly, 11% ala, 21% pro, 4-Hyp.

Gelatin is derived from collagen but is less nutritional value as collagen is low in several amino acids.

Glycine is required because at the tight junction where the three strands pack together, only glycine is small enough to fit.

Pro and 4-Hyp are enabled to conduct sharp twisting required by collagen’s helix geometry.

Vitamin C and Scurvy

Vitamin C is required for the hydroxylation of Pro and Lys residues in collagen, the critical collagen residue is 4-Hyp.

Why does hydroxyproline matter: Collagen stability requires the Y position of the Pro/ 4 Hyp residues to be in exo conformation, which only happens with hydroxylation.

Ascorbate is not required for the normal reaction, but can catalyze the reduction of iron back to the active state, allowing continued pro hydroxylation.

When Fe2+ becomes oxidized, the enzyme becomes inactive.

Humans cannot synthesize vitamin C because they lack the final enzyme in the pathway converting glucose to ascorbate.

Collagen Fibrils and Cross Linking:

The collagen molecules assemble into collagen fibrils, arranged in a staggered pattern and cross linked for strength.

Key Structural facts: Collagen molecules are rod shaped, 3000 ang long, 15 A thick. Each alpha chain has about 1000 residues. Fibril arrangement produces cross striations.

Covalent Cross Links: Collagen chains and fibrils are cross linked using unusual covalent chemistry involving Lys, HyLys, and His. These can create uncommon residues

With age, accumulated cross links increase rigidity and brittleness of connective tissue.

Collagen mutations & Disease

-Single residue substitutions can be devastating: Osteogenesis imperfecta: abnormal bone formation and Ehlers Danlos: loose joints.

Many disease variants result from substituting from alarger residue Cys or Ser for Gly in a collagen alpha chain.

Fibronin:

Beta sheets for strong flexible silk.

Composition and packing: Rich in Ala and Gly, permits tight Beta packing, and R groups interlock between sheet.

Stabilizing forces: Extensive hydrogen bonding between peptide linkages within B sheets.

Optimized van der waal forces between sheets.

Mechanical properties:

Silk does not stretch because beta strands are already extended.

Silk remains flexible because sheets are held by weak interactions not covalent cross links.

Globular Proteins

—Globular proteins fold into compact shapes because the polypeptide segments fold back on themselves.

—This compact folding produces structural diversity supporting many functions- enzymes, transport, proteins, motor proteins, regulatory protiens, and immunoglobins.

Many types of secondary struc

Myoglobin (ex of globular structure)

The first globular structure solved by x-ray diffraction was myoglobin: Kendrew (1950s).

Functional role: Oxygen binding protein in muscle, stores O2 and facilitates diffusion in actively contracting tissue.

Facts: Single chain, 153 residues, one heme group.

Secondary Structure content: 8 a helical right handed segments. (70% of residues in a helices)

Packing principles revealed by myoglobin: Hydrophobic side chains largely buried in the interior, polar side chains on the surface and hydrated.

Its close packing density trengthens the van der waal interactions.

PROTECTING HEME: Heme lies in a pocket with limited solvent access. This is crucial since free heme in oxygenated solution is rapidly oxidized.

Motifs and Folds and domains

A motif is a recognizable folding pattern involving two or more secondary structure elements and connections between them.

Motifs can be small (B-a-B loop) or large like a B barrel.

A motif may or may not be independently stable. A protein can sometimes largely be a motif.

Ex: A coiled coil in alpha keratin or a globin fold in an eight helix arrangement.

MOTIFS ARE NOT A STRICT INTERMEDIATE HIERARCHIAL LEVEL BETWEEN SECONDARY AND TERTIARY STRUCTURE, THEY ARE SIMPLY REOCCURING PATTERNS.

Domain: Is a part of a polypeptide chain that is independently stable or can move as a single unit relative to the rest of the protein.

Proteins with a few hundred residues may have multiple domains, as these domains may retain structure when isolated (after proteolysis). Different proteins have different functions like binding catalysis and regulation.

Folding Rules and Constraints n Globular Proteins

1. Hydrophobic effect determine stability

2. Burial of hydrophobic R group require at least 2 layers of secondary structure

3. Simple motifs like B-a-B can create these layers.

4. A helices and B sheets usually occupy different layers.

5. B backbone cannot bind to adajcent a helix.

6. Sequence adjacency predicts spatial adjacency.

7. Beta sheet are sightly right hand twisted.

8. Connection between parallel beta strands are usually right handed.

Intrinsically Disordered Proteins

Many proteins lack a stable structure in solution.

Properties: Lack a hydrophobic core, high density of charged reisudes, Lys, Glu, Arg. High proline content.

Functional Role: Spacers, insulators, and linkers, scavengers/ reservoirs for ions or small molecules.

Functional Promiscuity: Disorder can allow one protein to interact with many partners.

P27- wraps around and inhibits many protein kinases, lower p27 related to lower cancer prognosis.

P53- which has disordered regions, can interact with many.

Protein Structural Classification

SCOP2 data vase organizes protein info into categories including relationships, structural classes, types and revolutionary events.

Protein family: proteins with sequence or structural similarity and similar function.

Protein superfamily: protein with little structural similarity but major structural motifs and functional similarities.

Quaternary Structure

Why form them:

Regulation: small molecule binding changes subunit interactions

Division of labor: one might do catalysis the other may do regulation.

Structural roles: fibrous proteins and viral coats.

Multi-step reaction platforms, like ribosomes.

Multisubunit proteins are called oligomers. If different subunits then unsymmertrical and complex.

Protomer: Repeating structural unit of oligomer.

HEMOGLOBIN: 2 ALPHA AND 2 BETA SUBUNITS

-Subunits are arranged as symmetric pairs, each pair is an AB protomer.

Thus hemoglobin is described as a tetramer of protomers.