Lecture 5-8: Protein Sequence and Shape

What is the basic structure of a protein, and what is myoglobin?

• Proteins consist of long, linear chains of amino acids connected by peptide bonds.

• The specific sequence of amino acids forms the polypeptide chain.

• Myoglobin is an oxygen-binding protein found in muscle tissue.

• Myoglobin contains 153 amino acids in its polypeptide chain.

How does myoglobin's linear polypeptide chain form its three-dimensional structure?

• Myoglobin's linear chain of 153 amino acids folds into a compact, three-dimensional globular structure.

• A space-filling model provides a detailed representation of its structure.

• However, the high level of detail makes it difficult to visualize the protein's folding patterns.

• Simplified structural representations can make these patterns easier to understand.

What does the ribbon representation of myoglobin reveal about its structure?

• The ribbon traces the path of the polypeptide backbone without showing amino acid side chains (peptide bonds only!).

• The ribbon is colour-coded from blue at the N-terminus to red at the C-terminus.

• Myoglobin contains eight spiral-shaped (helical) segments.

• Amino acid side chains occupy the spaces surrounding the backbone, even though they are not shown.

• The structure also contains a heme ring.

What are the four levels of protein structure?

• Primary: Linear sequence of amino acids in a polypeptide chain.

• Secondary: Regular, repetitive folding patterns along short sections of the polypeptide (5–20 amino acids), such as helical sections in myoglobin.

• Tertiary: Overall three-dimensional folding of an entire polypeptide chain.

  • Ex. In myoglobin, 8 helical sections enclose a central cavity

• Quaternary: Arrangement of multiple polypeptide chains into a protein complex (e.g., 4 globin units in hemoglobin).

• Not all proteins possess quaternary structure.

Why is the three-dimensional folding of proteins important for their function?

• Protein function depends on the precise three-dimensional positioning of key amino acids.

• Amino acids that are far apart in the primary sequence may become close together through folding.

• The exact folding pattern is critical for proper protein function.

What is protein hydrolysis, and what methods can be used to hydrolyze peptide bonds?

• Protein hydrolysis breaks peptide bonds to release individual amino acids.

• Acid hydrolysis: 6 M HCl at 110°C for 24–72 hours.

• Base hydrolysis: 4 M NaOH at 110°C for 16 hours.

• Enzymatic hydrolysis: Uses proteases, which catalyze peptide bond hydrolysis.

• Some amino acids may be destroyed during acid or base hydrolysis.

How can acid or base hydrolysis alter amino acids, and how are amino acids analyzed afterward?

• Acid and base hydrolysis can destroy certain amino acids.

• Hydrolysis of amide groups converts Asn → Asp and Gln → Glu, releasing ammonia.

• After protein hydrolysis, chromatography can determine the amount of each amino acid present.

• This reveals amino acid composition, but not their original sequence.

What causes chemical reactivity, and what are nucleophiles and electrophiles?

• Chemical reactivity arises from an uneven distribution of valence electrons.

• C–C and C–H bonds share electrons relatively equally, making them nonpolar and relatively unreactive.

• Nucleophiles: Atoms with available lone pairs that donate electrons to form new bonds.

• Electrophiles: Electron-deficient atoms that accept electron pairs.

• Many biochemical reactions are initiated by nucleophiles

• Negatively charged oxygen is a stronger nucleophile than neutral oxygen

What are the three ways an atom can use its lone pair of electrons?

• Hydrogen bond acceptor: Uses its lone pair to attract an O–H or N–H group through hydrogen bonding.

• Base: Uses its lone pair to capture a proton (H⁺).

• Nucleophile: Shares its lone pair with an electron-deficient atom to form a new covalent bond.

• Key distinction: A base accepts H⁺, while a nucleophile forms a bond with an electrophile.

What is the difference between nucleophilic substitution and nucleophilic addition?

• Nucleophilic substitution: An incoming nucleophile attacks an electron-deficient atom and displaces a leaving group.

• The leaving group takes its bonding electrons when it departs.

• Nucleophilic addition: A nucleophile attacks an atom involved in a double bond.

• One bond of the double bond breaks, but the original group remains attached.

• Key distinction: Substitution replaces a group; addition does not remove a group.

How does peptide bond hydrolysis occur through nucleophilic displacement?

• The carbonyl carbon of the peptide bond is electron-deficient (electrophile) because oxygen attracts electrons.

• OH⁻ acts as a nucleophile and attacks the carbonyl carbon, forming a new C–O bond.

• Electrons from the C=O double bond move toward oxygen, forming a negatively charged intermediate (transition state).

• The C=O double bond reforms, and the C–N peptide bond breaks.

• The amino nitrogen acts as the leaving group.

• Curly arrows represent the movement of electron pairs during the reaction.

How does Sanger's method identify the N-terminal amino acid of a protein?

• At high pH, the free α-amino group at the N-terminus deprotonates and acts as a nucleophile (NH2-).

• The N-terminal amino acid reacts with fluorodinitrobenzene (FDNB), attaching a bright yellow tag.

• Complete hydrolysis releases the individual amino acids.

• The yellow-labelled N-terminal amino acid is identified using chromatography.

• Limitation: Hydrolysis destroys the remaining polypeptide chain, so only the first amino acid can be identified.

What is Edman degradation, and how does it improve upon Sanger's method?

• Edman degradation determines the amino acid sequence of a protein, starting at the N-terminus.

• The N-terminal amino acid is chemically labelled, removed, and identified.

• Unlike Sanger's method, the remaining polypeptide chain is not destroyed.

• The process can be repeated to identify successive amino acids.

• Key advantage: Multiple amino acids can be sequenced rather than identifying only the first residue.

What are the two steps of Edman degradation?

• Coupling (basic conditions/requires base): Phenyl isothiocyanate (PITC) labels the N-terminal amino acid.

  • The coupling reaction must finish before cyclization begins.

• Cyclization (acidic conditions/requires acid): The first peptide bond is cleaved, removing the labelled N-terminal amino acid.

  • The removed amino acid is identified, leaving a shortened polypeptide.

• The cycle can be repeated up to approximately 50 times using automated sequencers.

What are the cleavage specificities of trypsin, chymotrypsin, and cyanogen bromide?

• Trypsin: Cleaves peptide bonds on the C-terminal side of Lys (K) and Arg (R).

• Chymotrypsin: Primarily cleaves on the C-terminal side of aromatic amino acids Phe (F), Tyr (Y), and Trp (W).

• These reagents produce smaller peptide fragments that can be individually sequenced.

• Key concept: Cleavage occurs AFTER the recognized amino acid, toward its C-terminus.

• Selective hydrolysis cuts the polypeptide at specific locations, to yield a limited number of oligopeptides of definite size

How does proline affect peptide cleavage by trypsin and chymotrypsin?

• Trypsin: Normally cleaves after Lys (K) and Arg (R).

• Exception: No cleavage occurs if Lys or Arg is immediately followed by Pro (P).

• Proline has the wrong shape to fit into the enzyme's binding position.

• Chymotrypsin: The lecture diagram also shows no cleavage when Phe is followed by Pro.

• Key concept: Always check whether Pro follows a potential cleavage site!

How does cyanogen bromide (CNBr) cleave polypeptides?

• CNBr is a chemical cleavage reagent, not an enzyme.

• Specifically cleaves peptide bonds on the C-terminal side of methionine (Met, M).

• Unlike trypsin and chymotrypsin, CNBr can cleave even when the next amino acid is proline (Met–Pro).

• During cleavage, methionine is converted into homoserine (Hse).

• Key concept: CNBr cleaves AFTER Met, including Met–Pro bonds.

• For a linear peptide, the number of fragments equals the number of cleavage sites + 1.

• Exception: A Met at the very end of the peptide does not create an additional fragment because no peptide bond follows it.

What is peptide mass fingerprinting, and how is it used to identify proteins?

• Proteins digested with enzymes like trypsin yield characteristic patterns of
  fragments of different molar mass (fingerprint)

• Mass spectrometry determines the masses of the resulting fragments.

• The fingerprint is compared with those of known proteins to identify the original protein.

• For unknown proteins: Oligopeptide Fragments can be separated using chromatography and sequenced using Edman degradation.

How are overlapping peptide fragments used to reconstruct a protein's amino acid sequence?

• Two samples of the same protein are digested using different cleavage reagents (hydrolysis methods).

• Each reagent produces peptide fragments with different cleavage sites.

• The fragments are separated and individually sequenced.

• Overlapping amino acid sequences are identified between fragments from the two digestions.

• These overlapping regions establish the correct order of fragments.

• Key concept: Different cleavage patterns allow the original protein sequence to be reconstructed.

How are most protein amino acid sequences determined today?

• Most protein sequences are predicted from DNA sequences stored in genome databases.

• Scientists determine the DNA sequence of a protein-coding gene.

• The genetic code is used to predict the corresponding amino acid sequence.

• This approach is relatively cheap and easy.

• Key advantage: It does not require direct protein sequencing experiments.

How does tandem mass spectrometry (MS/MS) determine protein sequences?

• 1. Digestion: A protease breaks the protein into smaller peptides (hydrolysis!).

• 2. MS-1: Separates the peptides and selects one peptide type to
  go into the collision cell

• 3. Collision cell: Fragment each peptide molecule once (usually
  the peptide bond) in a random fashion.

• 4. MS-2: Measures the masses of the resulting fragments.

• Differences between fragment masses help identify the amino acid sequence.

• Key advantage: Only tiny amounts of protein are required.

Why is trypsin the preferred protease for tandem mass spectrometry?

• Trypsin first cleaves proteins after Lys (K) or Arg (R), producing peptides that generally end with these residues.

• These peptides are then fragmented further inside the collision cell.

• b-type fragments: Retain the N-terminus of the trypsin-generated peptide.

• y-type fragments: Retain the C-terminus, including the terminal Lys or Arg.

• Lys and Arg can carry positive charges, helping y-type fragments produce the highest mass spectrometry signals.

• Key concept: Trypsin facilitates the detection and analysis of y-type fragments.

How can amino acid sequences be determined from y-type fragment masses in tandem mass spectrometry?

• MS-2 measures the masses of y-type fragments containing the peptide's C-terminus.

• Chromatogram - Each peak corresponds to a y-type fragment formed by
  breakage of a peptide bond at a different point in the peptide chain

• Going from the largest to the smallest, each successive peak has one less amino acid than the peak before

• Subtract consecutive fragment masses to determine the mass of the missing amino acid.

• Match each mass difference to the corresponding amino acid residue mass.

• Moving from largest to smallest fragments reveals the sequence from N-terminus to C-terminus.

• Example: 989 − 932 = 57 Da, corresponding to Gly.

Useful Slide

Important: Because we're moving from the largest y-type fragment to the smallest, we're identifying amino acids from the N-terminus toward the C-terminus.

Imagine the original peptide is:

Gly–Ser–Ala–Arg

The y-type fragments would be:

• Gly–Ser–Ala–Arg = 371 Da

• Ser–Ala–Arg = 314 Da

• Ala–Arg = 227 Da

• Arg = 156 Da

Notice how each smaller fragment loses one amino acid from the N-terminal end?

What are NCBI and BLAST, and how are BLAST results interpreted?

• NCBI (National Center for Biotechnology Information): Provides biological databases containing DNA, RNA, protein sequences, genomes, health-related mutations, and access to PubMed.

• BLAST (Basic Local Alignment Search Tool): Compares an input sequence against known sequences in biological databases.

• BLAST produces a list of the best matches, called hits.

  • 100% identity: Positive match with a known sequence.

  • Good identity: Suggests a homolog (evolutionarily related protein).

  • No identity: May indicate a new protein; biochemical tests are needed to investigate its identity.

Review: What are the four levels of protein structure?

• Primary: Linear amino acid sequence.

• Secondary: Local backbone folding patterns (α-helices and β-sheets).

• Tertiary: Overall 3D structure of a single polypeptide.

• Quaternary: Arrangement of multiple polypeptide subunits.

How can rotation around covalent bonds change a protein's three-dimensional structure?

• Rotation around certain single covalent bonds changes the spatial arrangement of atoms.

• Groups connected by single bonds can rotate about bond axis

• Chain flexibility arises from bond rotation, not bond bending

• Normal 109* tetrahedral or 120* trigonal planar bond angles are present

• Bond rotation allows the peptide chain to adopt a variety of shapes

What is the difference between conformation and configuration in proteins?

• Conformation: Spatial arrangement of atoms that can change through rotation around single bonds.

• Conformational changes do not require breaking covalent bonds.

  • For macromolecules such as proteins, we are usually concerned
    with conformations

• Configuration: Spatial arrangement that cannot change through simple single-bond rotation.

• Changing configurations generally requires breaking and reforming covalent bonds.

• Examples of configuration: Cis/trans double-bond isomers and D/L stereoisomers.

How is X-ray diffraction used to study protein structure?

• Crystals are ordered arrays of molecules

• Atoms and molecules have similar dimensions to the wavelength of X-rays (0.7-1.5 Å)

• When X-rays reflect off a regular repetitive structure, e.g., molecular crystal or fibre, they are deflected by an angle dependent on wavelength of X-ray and spacing of pattern

• X-rays scatter when they pass through an ordered sample, producing a diffraction pattern.

• If X-ray wavelength is known, dimensions of the repeating pattern of molecules can be calculated.

• Some important structural parameters were identified from early studies of diffraction patterns of fibrous proteins such as α-keratin and β-keratin.

  • α-Keratin: Major repeat of 5.4 Å and minor repeat of 1.5 Å.

  • β-Keratin (fibroin): Major repeat of 7.0 Å and minor repeat of 3.5 Å.

Why are peptide bonds rigid and planar?

• The peptide bond has two resonance forms, one with a double bond

• Resonance restricts rotation around the peptide C–N bond.

• The peptide group is therefore rigid and planar.

  Bond type	Length
  Typical C–N single bond	1.49 Å
  Peptide C–N bond	1.32 Å
  Typical C=N double bond	1.27 Å

• Note: C-N bond between a single bond at 1.49 Å and a double bond at 1.27 Å

• Peptide bonds generally adopt a trans configuration (behaves more like a double bond than a single bond).

Which bonds in a polypeptide backbone can rotate, and what are the phi and psi angles?

• Phi (φ): Rotation around the N–Cα bond.

• Psi (ψ): Rotation around the Cα–C bond.

• These rotations allow the polypeptide backbone to adopt different conformations.

• Peptide C–N bonds (α-amino and α-carboxylate): Rotation is restricted because of resonance.

• The backbone consists of relatively rigid peptide planes connected by rotatable bonds (only the two bonds to α-C can rotate freely)

How do phi and psi rotations contribute to protein secondary structure?

• Restricted bond rotation limits freedom of motion, so that only a few regular structures can form

• The peptide backbone changes direction by 109* at each tetrahedral α-C, defining two possible regular repeating patterns:

• In a helical shape, every α-C bond down the peptide chain turns in same direction (e.g. clockwise)

• In an extended shape, the α-C bonds turn in alternate directions down the peptide chain

• If there is no regular, repeating structure, get random coil

  • α-Helix: Coiled backbone arrangement.

  • β-Strand: Extended backbone arrangement.

  • Random coil: Irregular backbone arrangement.

What are the structural characteristics of an α-helix?

• Helix forms when amino acids all have same orientation, and α-C bonds turn in same direction (i.e. all clockwise)

• Pauling’s models showed a very stable structure with 3.6 amino acids per turn of helix

• #1 C=O lines up with H–N #5 to form hydrogen bonds that make α-helix stable

• Distance between each turn of helix is 5.4 Å

• 5.4 ÷ 3.6 = 1.5 Å is distance along the helix per amino acid

• These distances match α-keratin patterns exactly, hence the name α-helix

Hydrogen Bonding Calculation and Helix Length Calculation

• Remember: Hydrogen bonding: C=O of residue n bonds with N–H of residue n+4.

• Length = 1.5 x Number of Residues

What is the difference between parallel and antiparallel β-sheets?

• β-Sheets consist of extended β-strands stabilized by backbone hydrogen bonds.

• Parallel β-sheet: Adjacent strands run in the same N→C direction.

• Antiparallel β-sheet: Adjacent strands run in opposite N→C directions.

• Hydrogen bonds are generally more directly aligned in antiparallel β-sheets.

Which amino acids prefer β-sheet structures, and why?

• β-Sheets provide more space for bulky or awkwardly shaped side chains.

• Trp, Tyr, Phe (WYF): Have bulky aromatic side chains.

• Val, Ile, Thr (VIT): Have branching at the β-carbon.

• Cys (C): Contains a relatively large sulfur atom.

• Memory trick: WYF, VIT, C.

• These amino acids tend to favour β-sheet structures.

Review: What X-ray diffraction patterns are associated with α-keratin and β-keratin?

How does amino acid composition influence secondary structure formation?

• Secondary structure depends on the local majority of amino acid preferences.

• α-Helix formers: Ala, Arg, Gln, Glu, His, Leu, Lys, Met, (Phe).

• β-Sheet formers: Trp, Tyr, (Phe), Val, Ile, Thr, Cys.

• Phe: Can favour either structure.

• The local majority helps predict whether a segment forms an α-helix or β-sheet.

What are secondary structure breakers, and what is their role in protein folding?

• Breakers: Gly, Pro, Asn, Asp, Ser (GPNDS).

• Can interfere with regular secondary structure formation.

• Professor's rule: Two breakers within four amino acids can interrupt secondary structure.

• Breaker clusters promote turns and flexible loops.

• These regions allow the polypeptide backbone to change direction, sometimes by approximately 180°.

How can you predict secondary structure from an amino acid sequence?

• Identify secondary structure breakers (GPNDS).

• Look for clusters containing at least two breakers within four amino acids.

• Use these clusters to divide the sequence into segments.

• Examine the amino acid preferences within each segment.

• Majority α-helix formers: Predict an α-helix.

• Majority β-sheet formers: Predict a β-sheet.

What is the difference between native and denatured proteins?

• Native state: Protein is folded into its functional 3D tertiary structure.

• Denaturation: Loss of normal protein folding.

• Denatured proteins may become unstructured or aggregated.

• Protein function is typically lost upon denaturation.

• Causes: Heat, disruptive solvents, and harsh detergents (e.g., SDS).

• Denaturation is often irreversible.

• Denaturation is generally avoided during protein purification.

What is the simplest possible tertiary structure and three examples?

• Tertiary structure is the overall pattern of folding of the whole polypeptide chain

• The simplest possible tertiary structure is continuous secondary structure

• α-keratin is totally α-helix

• Fibroin (a β-keratin) is totally anti-parallel β-sheet

• Collagen (tendon, bone and connective tissue) has a unique triple-helix structure

  • Collagen structure requires sequence with repeating units of three amino acids -Gly-Pro-X-

• Secondary structure is rigid, so these are fibrous proteins

How do secondary structure breakers contribute to globular protein formation?

• Globular proteins require the polypeptide chain to fold back on itself.

• Folding requires “breaks” in secondary structure, which is rigid

• Breakers: Gly, Pro, Asn, Asp, Ser (GPNDS).

• Clusters of 2–3 breakers within four amino acids can interrupt secondary structure.

• These interruptions form flexible loops and turns.

• Flexible regions allow the polypeptide to change direction and form compact tertiary structures.

How does the hydrophobic effect drive protein folding?

• The hydrophobic effect is a major force driving protein folding.

• Nonpolar amino acids: Cluster in the protein's interior to minimize contact with water.

• Polar amino acids: Tend to remain on the exterior, interacting with water or ions.

• Polar side chains can form hydrogen bonds with surrounding water.

• This arrangement helps stabilize the folded structure of soluble globular proteins.

How do amino acid packing and van der Waals forces stabilize protein tertiary structure?

• Amino acid side chains pack together like jigsaw puzzle pieces.

• Good packing maximizes close atom-to-atom contacts.

• Close contacts produce weak van der Waals (London dispersion) forces.

• Each interaction contributes approximately 0.1–1 kJ/mol.

• Hundreds of close contacts collectively help stabilize the protein.

• Poor packing produces fewer favourable interactions.

Summary: How does the amino acid sequence determine a protein's tertiary structure?

• Amino acid preferences influence α-helix and β-sheet formation.

• Secondary structure breakers (GPNDS): Introduce flexible loops and turns.

• Nonpolar amino acids: Tend to fold inward due to the hydrophobic effect.

• Polar amino acids: Tend to remain exposed to water.

• Side-chain packing: Pattern of large and small side chains is arranged so that secondary structure components (e.g. helices) pair up with best possible fit (Maximize Van der Wall Forces)

• These factors collectively determine the protein's stable native structure.

Review: What are the three major tertiary structure patterns described in Lecture 8?

• Mostly α-helical: Proteins containing primarily α-helical segments.

• Mostly β-strand: Proteins containing primarily β-strands.

• Mixed α/β: Proteins containing alternating α-helical and β-strand segments.

• Amino acid sequence influences which tertiary structure pattern forms.

How do α-helix bundles form, and what is an example?

• Proteins rich in α-helix-forming amino acids can form α-helix bundles.

• Secondary structure breakers separate individual helices (set the limits of each helix)

• Nonpolar amino acids every 3–4 residues create a hydrophobic stripe along each helix.

• Nonpolar stripes face inward when helices pack together.

• Example: Myoglobin is a bundle of 8 α-helix sections

How can antiparallel β-sheets have one polar face and one nonpolar face?

• Antiparallel β-strands run in opposite N→C directions.

• Their hydrogen bonds are relatively straight, making them more stable.

• β-strand side chains alternate between opposite sides of the sheet.

• Odd-numbered residues project toward one side; even-numbered residues project toward the other.

• Alternating polar and nonpolar amino acids (NPNPNP) can create one polar face and one nonpolar face.

How do antiparallel β-barrels form?

• β-Sheets with one polar face and one nonpolar face can curve to bury their nonpolar surface.

• 3–5 β-strands (Smaller sheet): Form an open fold.

• 6–8 β-strands (Larger sheet): Can wrap around to form an antiparallel β-barrel.

• The nonpolar face points inward, while the polar face remains exposed to water.

• Example: Green fluorescent protein (GFP).

How do parallel β-sheets form, and what are their characteristics?

• Alternating β–α–β–α sequences can form parallel β-sheets.

• α-Helices connect β-strands running in the same N→C direction.

• Connecting helices lie above or below the β-sheet.

• Parallel β-sheets have angled hydrogen bonds and are less stable than antiparallel β-sheets.

• They are generally buried in the protein's interior, away from water.

• They typically contain mostly nonpolar amino acids.

How does a parallel αβ-barrel form?

• Forms from alternating α-helical and parallel β-strand segments.

• Connecting α-helices lie on the same side of the β-sheet.

• The β-sheet wraps around to form a central barrel.

• α-Helices surround the outside of the barrel.

• Example: Triose phosphate isomerase.

How does a parallel αβ-sandwich form?

• Forms from alternating α-helical and parallel β-strand segments.

• Connecting α-helices lie on both sides of the β-sheet.

• The nonpolar β-sheet forms the central layer between two layers of α-helices.

• The β-sheet is often twisted to improve packing.

• Example: Part of lactate dehydrogenase.

What are protein domains, and why are larger proteins considered modular?

• Domains are sections of a polypeptide chain that fold into distinct 3D structures.

• Each of the structures outlined above forms from a chain of 10-20 kDa

• Larger proteins fold up in 10-20 kDa sections; each section is called a domain

  • Ex. A protein of 50 kDa may have 3 or 4 domains

• Larger proteins may contain multiple domains with different folding patterns.

• This makes larger proteins modular.

• Example: Lactate dehydrogenase contains two αβ-sandwich domains.

Review: The main forces that contribute to protein stability and function (2 really important ones!)

• Covalent bonding links amino acids in a chain in a specific sequence

• Non-covalent interactions dictate folding pattern and stability

• Hydrophobic effect and van der Waals effect are the most important non-covalent interactions

• Hydrophobic effect locates non-polar amino acids in core of folded protein, avoiding unfavourable interaction with H2O

  • Contributes ~50% of total energy stabilizing native state

  • –5 kJ/mol per CH, CH2 or CH3 moved out of contact with H2O

• Van der Waals interaction – dipole-dipole interactions between atoms that are close, but not covalently bonded to each other – aka London dispersion forces

  • Contributes significantly if many atoms are in close contact

How do hydrogen bonds and ion pairs contribute to protein stability?

• Hydrogen bonds form between appropriately aligned donors and acceptors.

• Ion pairs (salt bridges) form through electrostatic attraction between oppositely charged side chains.

• Both help stabilize the folded protein.

• They generally contribute less than hydrophobic and van der Waals interactions because most polar groups face the protein's exterior.

  • Charged AAs pair with ions in external solution

  • H-bonding AAs bond to H2O in surroundings

What are disulfide bonds, and how do they stabilize proteins?

• Disulfide bonds form when pairs of Cys –SH groups react with O2, releasing H2O

• Strong covalent S–S bonds formed by oxidation of two cysteine –SH groups.

• Help hold different regions of a folded protein together.

• Few proteins have disulfide bonds; mostly proteins designed to function outside cells since O2 needed

• Example: Insulin.

What did Anfinsen's experiment demonstrate about protein folding?

• The primary amino acid sequence contains the information required for a protein's secondary and tertiary structures.

• Anfinsen denatured ribonuclease using urea and 2-mercaptoethanol.

• When these chemicals were removed, ribonuclease regained its native structure and catalytic activity.

• This process is called renaturation.

What are the roles of urea and 2-mercaptoethanol in protein denaturation?

• Urea: Weakens the hydrophobic effect, allowing proteins to unfold.

• 2-Mercaptoethanol: Reduces disulfide bonds to cysteine –SH groups (converts disulfides back to the original unlinked Cys-SH groups).

• Together, they disrupt interactions that stabilize the native protein structure.