Protein Folding and Structure

Introduction to Protein Folding

• Moving from DNA to proteins.

• Focus on how proteins fold into their correct shape and how this controls their function.

• Protein shape = function.

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Protein Structure Levels

• Four levels of protein structure:

  • Primary.

  • Secondary.

  • Tertiary.

  • Quaternary.

• Understanding how each level influences the next aids understanding of disease resulting from amino acid sequence errors.

Learning Goals

• Detailed understanding of how each level of protein structure influences the next.

• Understanding of the importance of protein-protein interactions in regulating protein function.

• Understanding of post-translational modifications (PTMs): reversible modifications after protein synthesis.

  • Over 200 types of PTMs.

  • Examples: phosphorylation, glycosylation, methylation, acetylation, disulfide bonds.

  • Significance: alters activity and function of proteins.

Definitions

• Terms used in the course:

  • Proteins: versatile macromolecules involved in a range of processes (structural support, communication, etc.).

  • Peptide: two or more amino acids joined by a peptide bond.

  • Polypeptide: a longer chain of amino acids.

  • Protein: can contain one or more polypeptide chains.

• Terms peptide and polypeptide will be used interchangeably, as well as protein, except in the case of multimeric proteins.

• Multimeric proteins: contain two or more polypeptide chains.

• Proteins, peptides, and polypeptides are linear chains of amino acids linked by covalent peptide bonds.

The Importance of Protein Shape

• Protein structure determines function.

• Understanding levels of protein structure helps explain how changing one amino acid can change protein shape and function, leading to disease.

Four Levels of Protein Structure

• Primary Structure: amino acid sequence.

• Secondary Structure: areas of local folding, influenced by primary structure.

• Tertiary Structure: final three-dimensional shape, influenced by secondary structure.

• Quaternary Structure: arrangement of multiple polypeptide chains (multimeric proteins only).

Amino Acids

• 20 different amino acids in human proteins.

• Each amino acid has a unique chemical property.

• Primary structure contains all information needed for final protein structure.

• Every protein has a unique amino acid sequence; changing an amino acid changes the protein.

• Single amino acid change can result in a disease (e.g., sickle cell anemia, where a single change in one amino acid produces a dramatic change in the quaternary structure).

Amino Acid Structure

• Amino group bonded to a central alpha carbon.

• Carboxyl group bonded to the central alpha carbon.

• Hydrogen ion.

• R group (side chain) differs for each amino acid.

  • Side chains vary in size, shape, charge (negative, positive, or uncharged), and hydrophobicity (polar or nonpolar).

  • Side chains can be reactive and modified.

• Table of amino acids (not for memorization).

Reactive Side Chains and Post-Translational Modifications

• Side chains can be modified, forming the basis of post-translational modifications.

• Serines, threonines, and tyrosines can be phosphorylated (addition of a phosphate group, introducing a negative charge).

• Side chains can also be glycosylated (addition of an oligosaccharide).

  • Asparagines: N-linked glycosylation (associated with protein folding and stability).

  • Serine and threonine: O-linked glycosylation (associated with expression and stability).

• Methylation: addition of a methyl group to an arginine or a lysine amino acid.

• Lysine amino acids can also be acetylated.

• All of these reactivities mentioned so far are reversible and can be removed by the cell as well.

• Disulfide bonds: covalent bonds between cysteine amino acids.

  • Cysteines have a reactive sulfhydryl group that can form a covalent bond with another cysteine residue.

  • Act like atomic staples and stabilize protein structure.

Peptide Bond Formation

• Individual amino acids are bonded together via the formation of peptide bonds.

• Carboxyl group of one amino acid links to the amino group of the adjacent amino acid (condensation reaction, removal of water).

• Side chains are not used.

• Peptide bond is rigid and planar, producing a polypeptide backbone.

• Polypeptide backbone has side chains projecting out either side.

Polypeptide Backbone

• Residue: another term for an amino acid.

• Amino terminus (N terminus): free amino group at one end.

• Carboxyl terminus (C terminus): free carboxyl group at the other end.

• Proteins run in an N-terminal to a C-terminal direction.

• Angles of rotation around the central alpha carbon:

  • $\psi$: rotation around the alpha carbon-carbon bond.

  • $\phi$: rotation around the alpha carbon-nitrogen bond.

• Shape of a protein is determined by combinations of $\psi$ and $\phi$.

• Steric effects and other constraints limit possible $\psi$ and $\phi$ combinations, producing regular structures.

Non-Covalent Bonds in Protein Folding

• Proteins adopt shapes involving the least amount of effort.

• Combinations of weak non-covalent bonds constrain folding.

  • Electrostatic attractions: positively and negatively charged atoms and side chains influence the shape.

  • Van der Waals attractions: fluctuating polarizations and interactions between molecules.

  • Hydrogen bonds: attractive interaction of a hydrogen ion with an electronegative ion.

• Polarity of side chains influences tertiary shape (hydrophobic inside, hydrophilic outside; keeps the protein soluble).

Exam Question Example

• How are amino acids linked in peptide bonds?

• Correct Answer: D, carboxyl group of one amino acid is linked to the amino group of another.

Secondary Structure

• Areas of local folding.

• Conformation are hysterically allowed.

• Over 60% of polypeptide chain folds into alpha helices or beta sheets.

• Alpha helices and beta sheets are major supportive elements.

• Loops, turns, and random coils as well.

Alpha Helix

• Regular spiral or helical shape formed by hydrogen bonds.

• Carbonyl oxygen of each amino acid forms a hydrogen bond with the amide hydrogen of the amino acid four residues towards the C terminus, between amino acids that are four separated, produces a regular sprial effect.

• 3. 6 amino acids per turn.

• Side chains point outwards.

• Alpha helix can be hydrophobic, hydrophilic, or amphipathic (both).

• Hydrophobic alpha helices are common in membrane-spanning domains.

Beta Sheet

• Made up of laterally packed beta strands.

• Beta strand: 5 to 8 amino acid polypeptide chain that's nearly fully extended, not coiled.

• Formed by hydrogen bonding between backbone atoms in adjacent beta strands.

• Side chains point both upwards and downwards.

• Beta sheets can be parallel or anti-parallel.

• Proteins made of beta sheets are soft yet flexible filaments (e.g., fibroin in silk).

Loops and Turns

• Loops: unstructured areas formed in many different ways (intrinsically disordered regions).

• Turns: loop back onto each other (U shape), hydrogen bonds between arms (glycine and proline residues).

Motifs (Supersecondary Structures)

• Combinations of secondary structures:

  • Coiled coil: two coils wrapped around each other.

  • Helix-loop-helix: two helices joined together with a loop.

  • Zinc finger: alpha helix and two beta strands held together by a zinc ion.

• Zinc finger motif is often found in transcription factors.

• Motifs are classified as secondary structures that form a bridge between secondary and tertiary structures and influence the folding.

Exam Question Example

• Which of the following characteristics describing a beta sheet is correct?

• Correct Answer: D, side chains can point either upwards or downwards.

Tertiary Structure

• Final three-dimensional shape.

• Influenced by polarity, van der Waals, electrostatic attractions, hydrogen bonds.

• Hydrophobic chains in core, polar/charged chains on surface (aqueous environment).

• Disulfide bonds (cysteine residues) act like atomic staples and stabilize the structure.

Protein Domains

• Distinct regions within a larger protein (SH3, SH2, ATP-binding domain).

• Modular units from which larger proteins can be built.

• Compactly folded region of a polypeptide.

• Can be characterized by structural feature or function/activity (DNA-binding domain).

Quaternary Structure

• Only multimeric proteins have a quaternary structure.

• Stabilized by van der Waals, electrostatic attractions, and hydrogen bonds.

• Hydrophobic effect is the major factor stabilizing the aggregation of protein subunits.

• Each chain is called a subunit or polypeptide chain.

• Described by the number of chains and their arrangement (symmetrical, identical).

Examples of Quaternary Structures

• Symmetrical dimer: two identical subunits.

• Trimers, tetramers, hexamers, dodecamers.

• Filaments: long chains of identical protein molecules wrapped around each other (e.g., actin filaments).

Intrinsically Disordered Regions (IDRs)

• Largely unstructured regions that allow proteins to have a variety of functions.

• Can tightly wrap around molecules (binding).

• Can be post-translationally modified to change shape.

• Can act as diffusion barriers.

Protein Function

• Shape of a protein determines function.

• Proteins with similar functions often have similar shapes, domains, or motifs.

• Proteins perform their function because they bind to other molecules.

Protein Machines

• Proteins work together through shape changes to produce highly specific activity and complex function (spliceosome).

Disrupting Protein-Protein Interactions

• Can be used to treat diseases (e.g., blocking cell surface ligand-receptor interactions).

Post-Translational Modifications (PTMs) and Protein Shape

• Protein shape can change with reversible modifications.

• PTMs occur after translation, modifying side chains.

• Can alter shape, reveal/hide binding sites, change activity/location.

Examples of PTMs

• Phosphorylation: kinase adds phosphate to serine, threonine, or tyrosine (reversible by phosphatases).

• Methylation: addition of methyl group to arginine or lysine.

• Acetylation: addition of acetyl group on lysine (e.g., alters microtubule formation).

• Glycosylation: oligosaccharides added to serine, threonine, or asparagine (helps protein folding).

• Ubiquitination: addition of ubiquitin onto lysine (targets protein for degradation).

Exam Question Example

• Which of these statements about quaternary structures are incorrect?

• Correct Answer: C, only membrane-spanning proteins have one.

Consolidation of Lecture

• Four levels of structure: primary, secondary, tertiary, quaternary and how each influences the next.

• Amino acid sequence determines the final shape of a protein, proteins spontaneously fold into correct shape.

• Non-covalent forces, polarity, and steric effects impact shape and influence areas of folding.

• Protein interactions determine the function of a protein, and proteins can work together to perform more complicated functions in machines.

• Proteins can be modified post-translationally and are reversible, produced by enzymes.

Helpers in Protein Folding

• Translation isn't the end, proteins have to fold.

• Proteins will fold into their secondary structures as they're being translated.

• There are membrane bound ribosomes and free ribosomes.

• The endoplasmic reticulum is a important cellular structure.

• Proteins still need a little bit of help to fold or molecular chaperones.

Ribosomes and Protein Synthesis

• Two types of ribosomes from commmon source.

  • Membrane bound ribosomes. *Free Ribosomes.

    • SRP Cycle.

The Endoplasmic Reticulum

• Labyrinth of tubules and flattened sacs, interconnected and contiguous with the outer nuclear membrane.

• Important role in lipid and protein biosynthesis.

• Two types of endoplasmic reticulum:

  • Rough ER: has membrane-bound ribosomes for synthesis, folding, and PTM of proteins to be secreted, in membranes, or in specific organelles.

  • Smooth ER: lipid synthesis, detoxification, calcium storage, transport of proteins from ER to Golgi.

• ER Membrane:

  • Site of production of transmembrane and organelle proteins and proteins that will be secreted outside the cell

• ER lumen:

  • Secreted in some of those organelle proteins are initially delivered there and they undergo some processing before they move to their next location

Molecular Chaperones

• Help proteins to fold correctly; accelerate protein folding.

• Located in every cellular compartment.

• Two main types:

  • Molecular chaperones: e.g., heat shock protein 70 (HSP70) family.

    • Bind and stabilize unfolded or partially folded proteins.

    • Recognize hydrophobic patches on the surface of proteins (prevent aggregation).

  • Chaperonins: e.g., HSP60-like proteins.

    • Form isolation chambers lined with hydrophilic surfaces.

    • Surround misfolded proteins, directly helping them fold correctly.

  • Some proteins require multiple rounds of chaperone interactions to fold correctly.

Disulfide Bonds

• Slide missing from the slide, the illustration of what they are

Differentiating Newly Synthesized vs. Misfolded Proteins

• Number of glucoses on precursor oligosaccharide indicates how long the protein has been in the ER lumen.

• ER glucosidase trims glucose residues over time.

• Calnexin (lectin) binds to the glucose moiety on the precursor oligosaccharide when only one glucose remains, keeping the protein in the ER for more time to fold.

• Glucosidase cleaves the remaining glucose, releasing the protein.

• If folded correctly, it exits the ER to continue the biosynthetic secretory pathway journey.

• If still unfolded, glucosyl transferase adds another glucose, which then will be bound by the lectin again for another round of quality control.

Retrotranslocation

• If the protein cannot fold correctly after multiple rounds of lectin binding, it gets transported from the ER back to the cytoplasm.

• Tagged with polyubiquitin chains and destroyed by the proteasome.

Unfolded Protein Response (UPR)

• Activated in response to an accumulation of misfolded proteins in the ER lumen.

• Three parallel pathways (IRE1, PERK, ATF6) triggered by the dissociation of a chaperone called BiP.

• Activate the transcription of genes encoding proteins that can help with protein folding (e.g., chaperones).

• Eventually, if misfolded proteins persist, these pathways trigger apoptosis.

Cellular Response to Misfolded Proteins

*Accumulation activates the unfolded protein response(UPR)

*Three pathways, 1LE, PERK, and ATF6(not the expected level of detail)

*Genes that get up-regulated are those that help the protein struggling to fold.

Proteolytic Machinery

• Cells contain machinery to degrade misfolded or damaged proteins (proteolytic machinery).

• Hydrophobic regions on the surface of proteins can interact, leading to protein aggregation.

• Aggregates can be damaging to the cell.