BIS 102 Midterm 2

Cheng M2 Spring 2026

Sanger’s Nobel Prize

Sanger’s breakthrough in analyzing insulin protein 1° sequencing (1958)

Pauling’s Nobel Prize

Pauling’s interpretation of chemical bonds in protein 2° structure (1954)

Anfinsen’s Nobel Prize

Anfinsen’s dogma in recognizing protein RNase 3° folding (1972)

Merrifield’s Nobel Prize

Merrifield’s “peptide synthesis” of a full-function 3° RNase (1984)

Levinthal’s Nobel Prize

Levinthal’s resolving the paradox of 3° folding kinetics

Perutz’s Nobel Prize

Discovered first tertiary structure - 3° myoglobin proteins (1962)

Klug’s Nobel Prize

Virus assembly - 4° quaternary structures (1982)

Agents affecting protein structures: Urea

disrupts hydrogen bonds

Agents affecting protein structures: SDS

disrupts hydrophobic cores of proteins

Agents affecting protein structures: Heat/High Temps

disrupts weak forces

Agents affecting protein structures: Beta-Mercaptoethanol (MCE)

disrupts disulfide bonds

Agents affecting protein structures: Distilled Water

disrupts ionic interactions

Agents affecting protein structures: Organic Solvents

interact with hydrophobic amino acid side chains

Primary (1°) Level

the sequence of amino acids in the polypeptide chain dictated by covalent bonds

Secondary (2°) Level

local structures - H-bonds within the main chain (alpha helix and beta sheets)

Tertiary (3°) Level

overall three-dimensional shape involving side chain in all weak-force interactions (globular proteins)

Quaternary (4°) Level

subunit organization based on weak-force interactions between polypeptide chains (Hemoglobin)

Sanger Sequencing: Step 1

Separate Polypeptide chains

• subunits are associated by weak force interactions

• expose to pH extremes, 8M Urea, Guanidinium Hydrochloride or high salt concentrations disrupts the polar interactions (H bonds)

Sanger Sequencing: Step 2

Cleave (reduce) disulfide bridges

• Use performic acid or BME or DTT coupled with iodoacetate (or 3-bromopropylamine)

Sanger Sequencing: Step 3

Determine N- and C-terminal analysis

• N-terminal analysis: Edman’s reagent (PTC), recovered as PTH derivatives

• C-terminal analysis

  • Carboxypeptidase A: any residues except PRO, ASP, GLU, ARG, LYS

  • Carboxypeptidase B: only work on ARG or LYS

  • Carboxypeptidase Y: on any C-terminal residue

Sanger Sequencing: Step 4

Cleave each chain into smaller fragments and determine the composition & sequence of each chain

• Trypsin → Cleaves on the C-terminus of ARG and LYS

• Chymotrypsin → Cleaves at Carboxyl groups of Aromatic residues: PHE, THR, TRP, LEU

Sanger Sequencing: Step 5

Repeat step 4 - using a different cleavage procedure to generate a different & overlapping set of fragments

• CNBR (cyanogen bromide) → MET

Sanger Sequencing: Step 6

Reconstruct the sequence of the protein from the sequences of overlapping fragments (Use sequencing strategy table)

Sequencing Strategy Table

Enzyme	Cleavage Site
Trypsin	R, K (not before P)
Lys-C	K (not before P)
Arg-C	R
Glu-C	E, D
Asp-N	D, E (N-terminal)
Chymotrypsin	F, W, Y, L
CNBr	M (Chemical)

Cleavage Procedure Method (ENZYMES): Trypsin

Susceptible Residue(s): ARG or LYS

Cleavage Procedure Method (ENZYMES): Chymotrypsin

Susceptible Residue(s): PHE, TRP or TYR; LEU

Cleavage Procedure Method (ENZYMES): Clostripain

Susceptible Residue(s): ARG

Cleavage Procedure Method (ENZYMES): Staphylococcal Protease

Susceptible Residue(s): ASP or GLU

Cleavage Procedure Method (ENZYMES): Endopeptidase Lys-C

Susceptible Residue(s): LYS

Cleavage Procedure Method (CHEMICAL METHODS): Cyanogen bromide

Susceptible Residue(s): MET

Basic Structure: α-Helix

• Residues per turn: 3.6 amino acids

• Rise per residue: 1.5 Angstroms

• Rise per turn (pitch): 3.6 × 1.5 Å = 5.4 Angstroms

Hydrogen Bonding in α-Helix

Hydrogen bonds form between every i+4 residue in the helical backbone

• Carbonyl oxygen (H-bond acceptor): bonds with the i+4 amide hydrogen

• Amide hydrogen (H-bond donor): bonds with the i-4 carbonyl oxygen

Rules about α-Helix: Why are proline and glycine not included in α-Helix?

Both are helix breakers!

• Proline has no backbone NH/amid to form an H-bond

• Glycine only has a hydrogen as a side chain so the rotation around φ and ψ is so unconstrained -- too flexible 

Rules about α-Helix: Role of Serine in α-Helix Stability

Serine’s hydroxyl group can form hydrogen bonds with water or other polar groups

• If a polar or H-bond-forming residue is 3-4 positions away, transient H-bonding can destabilize the helix, leading to breaks

Rules about α-Helix: Steric Hindrance and Electrostatic Repulsions in α-Helix

• Bulky residues = W-W, F-F, Y-Y

  • cause steric hindrance

• Electrostatic repulsions: Like charges = +/+ or -/- repel each other, destabilizing the helix

Basic Structure: β strands

• Rise per residue (pleated sheets): 3.25 å for parallel sheets and 3.47 å for antiparallel sheets

  • Parallel sheets composed of fewer than 5 strands, Antiparallel sheets composted of at least 2 strands

Beta (Reverse) turns

• Reverse turns involve 4 amino acids (sharp turn) and allows the peptide chain to reverse direction for forming the antiparallel β-sheet

• Carbonyl Oxygen (acceptor) of one residue is H-bonded to the amide Hydrogen (donor) of the other residue -- in sequence, three residues away from each other (i+3, the 4^th residue)

Anfinsen’s Tertiary (3°) structure discoveries: Uniqueness

Uniqueness of protein folding: the sequence has no other configuration with comparable free energy → free energy minimum must be unchallenged

Anfinsen’s Tertiary (3°) structure discoveries: Kinetic Accessibility

Kinetic accessibility: the path from unfolded to folded state must be smooth → avoid highly complex changes like knots or high-order conformations

Anfinsen’s Tertiary (3°) structure discoveries: Stability

Stability: small changes in the surrounding environment cannot change the minimum energy configuration → protein remains stable in its folded state despite minor environmental fluctuations

Free Energy Surface - Funnel Model

• The free energy surface resembles a funnel.

• The native state is at the bottom of the funnel, representing the most stable conformation.

• This is not like a soup plate, which would imply multiple low-energy states.

• Steep free energy surface ensures stability by making it difficult for the protein to unfold or adopt alternative conformations (protects native state)

Weak Force Interactions Types

Weak chemical forces restrict organisms to a small window of environmental conditions and fitness (pH, temp, salt concentration) which includes:

• van der Waals interactions

• Hydrogen bonding

• Ionic interactions

• Hydrophobic interactions

Van der Waals interactions

• Induced electric interactions occur when atoms are close together

• Significant when many contacts form complementary surfaces

Hydrogen bonding

• Donor and acceptor pair: Direction dependent

• The donor is hydrogen covalently bonded to an electronegative O or N

• The acceptor is a lone pair on O or N

Ionic interactions

• Stronger than H bonds/Not directional

• Strength can be influenced by solvent properties

• Ionization of amino acids

Hydrophobic interactions

• Occur when nonpolar groups are added to water

• Water molecules H-bonding

• Nonpolar groups interfere with water H bonding and aggregate/need to minimize exposure

Cryo-Electron Microscopy 2017: Structural Preservation

Structural preservation is achieved by rapid freezing methods that generate vitrified water for Cryo-EM imaging

Cryo-Electron Microscopy 2017: Low-Dose Imaging

Low-dose imaging addresses challenges and breakthroughs associated with imaging biological structures using EM techniques

Cryo-Electron Microscopy 2017: Recording with Direct Electron Detectors

Direct Electron Detectors improve upon CCD cameras by enhancing signals through reduced noise in vitrified water

Cryo-Electron Microscopy 2017: Data Processing - Signal Enhancement

Data processing involves mathematical functions that determine cryoEM structures, where superimposing 2D images enhances signal clarity for higher resolution

Chaperons

Protein complexes that adjust hydrophobic interactions by providing a 2nd chance at protein folding → subvert kinetic trapping

Protein Life Cycle: Synthesis

Step 1 - The life cycle begins with the synthesis of a ribosome of a polypeptide chain, whose primary structure is dictated by an mRNA.

Protein Life Cycle: Folding

Step 2 - As synthesis proceeds, the polypeptide begins to fold into its native conformation

Protein Life Cycle: Processing

Step 3 - Folding may be accompanied by processing events such as 

• proteolytic cleavage of an N-terminal leader sequence (the most common form of post-translational processing)

• formation of disulfide bonds (S—S). 

Protein Life Cycle: PTM

Step 4 - Post-Translational Modifications found in proteins that may, e.g. attach a fatty acid molecule

Protein Life Cycle: Translocation

Step 5 -  the modified peptide to a membrane.

Protein Life Cycle: Activation

Step 6 - If needed, binding an allosteric effector may trigger the adoption of a catalytically active conformation.

Protein Life Cycle: Aging

Step 7 - Proteins become damaged by chemical attacks, deamidation, or denaturation over time.  

Protein Life Cycle: Ubiquitination

Step 8 - Damaged proteins may be “marked" by the covalent attachment of several ubiquitin molecules (Ub).

Protein Life Cycle: Recycling

Step 9 - The ubiquitinated protein is subsequently degraded to its component amino acids, which become available for the synthesis of new proteins

Human diseases linked to abnormalities of protein folding

• Alzheimer’s disease

• Familial amyloidotic polyneuropathy

• Cancer

• Creutzfeldt-Jakob Disease

• Hereditary emphysema

• Cystic fibrosis

Current theory of protein folding (in resolving Levinthal’s paradox)

Protein folding is a cooperative and sequential process which may involve intermediate states, including transition states, and is often guided by the protein’s primary structure. Specific proteins may require helpers like enzymes or chaperones to fold properly.

How long does most protein folding take, and what does this imply about the process?

Most folding is completed in about a second, implies that the process is highly efficient and not random. This rapid folding supports the idea of a guided, cooperative mechanism rather than a trial-and-error search through all possible conformations.

What is a “hydrophobic collapse,” and how does it contribute to protein folding?

A hydrophobic collapse is a process where nonpolar residues aggregate or coalesce. This collapse helps drive the folding process by reducing the exposure of nonpolar regions to water, leading to a more stable and compact structure.

What are the subsequent steps in protein folding after secondary structures form?

After secondary structures form, the next steps involve:

• The cooperative aggregation of folding nuclei to form domains.

• Long-range interactions between secondary structures or other hydrophobic regions.

• The formation of a “molten globule” state, where assembled domains take on a more defined structure

Thermodynamics of protein folding

Protein folding is driven by enthalpy (H) being predominant over entropy (S) in the Gibbs free energy equation. This means that heat is released as the polypeptide folds into a more ordered state. The energy landscape of folding can be visualized as a 3D approach involving:

• Conformational space (all possible shapes the protein can take).

• Reaction coordinate (the pathway of folding).

• Free-energy minimum (the most stable, lowest-energy state).

How to increase protein stability

• Increased H-bonds

• Fewer hydrophobic residues are exposed to the solvent

• Charged residues on the surface

• Ion-pair networks on the surface

• Electrostatic optimization

• Shorter loops

• Clustering of aromatic residues

• Improved packing of the protein core

Characteristics of the core of a globular protein structure

The helices and sheets in the core of a globular protein are typically constant and conserved in sequence and structure 

Characteristics of the surface of a globular protein structure

• Surface is composted of loops/coils and tight turns that connect helices and sheets of the core

• Surface elements can interact with small molecules or other proteins

• Basis for enzyme-substrate interactions, cell signaling, and immune responses

X-ray Crystallography

Intense X-rays are directed at a pure protein crystal, causing diffraction patterns that are processed to create electron density maps.

Best for high-resolution atomic structures of stable crystallizable proteins

X-ray Crystallography Pros and Cons

Pros: most productive method, representing the majority of. the PDB entries

Cons: requires large amounts of pure protein, crystallization is slow and difficult, flexible proteins are challenging