🧬 Chapter 8: Determination of Protein Structure

This chapter focuses on experimental methods used to determine protein structure, especially molecular mass, oligomeric state, shape, and primary sequence. These methods are essential because structure determines function, and many proteins are too large or flexible for direct atomic-resolution methods alone.

🔹 8.1 Introduction: Scope of the Chapter

Big picture 🧠

• Protein structure determination is stepwise:
  1. Primary structure (amino-acid sequence)
  2. Molecular mass
  3. Subunit composition
  4. Overall shape
• These properties constrain and guide higher-resolution structural techniques (e.g. X-ray crystallography, NMR, cryo-EM).

Why multiple methods are needed

• No single method gives everything
• Some methods work only under denaturing conditions, others under native conditions
• Comparing results across techniques helps identify:
  • Oligomerization
  • Conformational changes
  • Post-translational modifications (PTMs)

🔹 8.2 Molecular Mass Determination

Key concept

Molecular mass is not trivial to determine because proteins differ in shape, charge, and interactions with solvents.

🔸 8.2.1 SDS-PAGE (Denaturing electrophoresis)

Principle ⚡

• SDS (sodium dodecyl sulfate):
  • Denatures proteins
  • Binds uniformly (~1.4 g SDS / g protein)
  • Masks native charge
• Proteins migrate based primarily on molecular mass

What SDS-PAGE tells you

• Approximate subunit molecular mass
• Presence of multiple polypeptides
• Changes due to reduction of disulfide bonds

Key assumptions (important for exams!)

• Proteins are:
  • Fully denatured
  • Fully coated with SDS
• Migration ∝ log(molecular mass)

Strategy used (shown in figures)

1. Run molecular mass standards
2. Plot mobility vs log(Mr)
3. Interpolate unknown protein

📌 Coomassie Blue staining

• Detects ~0.1–1 µg protein
• Limited sensitivity for very small proteins

🔸 SDS-PAGE with reducing vs non-reducing conditions

Reducing agents

• β-mercaptoethanol or DTT
• Break disulfide bonds

Interpretation

• If band shifts upon reduction → disulfide-linked subunits
• If no shift → subunits not covalently linked

🔸 Cross-linking + SDS-PAGE 🔗

Purpose

• Determine oligomeric state (monomer, dimer, trimer…)

How it works

• Chemical cross-linkers covalently join nearby subunits
• SDS-PAGE then reveals:
  • Monomers
  • Cross-linked dimers, trimers, etc.

⚠️ Must optimize:

• Cross-linker concentration
• Protein concentration

🔸 8.2.2 Ferguson plots

Why Ferguson plots exist

• SDS-PAGE mobility also depends on gel concentration
• Ferguson analysis separates:
  • Molecular mass
  • Shape effects

Method 📈

1. Run SDS-PAGE at multiple acrylamide %
2. Plot log(mobility) vs gel %
3. Extrapolate to zero gel concentration

What you learn

• True molecular mass
• Whether protein deviates from globular shape

🔸 8.2.3 Gel filtration (Size-exclusion chromatography)

Principle 🧫

• Separation based on hydrodynamic radius
• Larger molecules elute earlier

Important parameters

• Void volume (V₀)
• Elution volume (Ve)
• Partition coefficient (Kav)

K_ = rac{V_e - V_0}{V_t - V_0}

What gel filtration tells you

• Native molecular mass
• Oligomeric state
• Shape (globular vs elongated)

📌 Compare:

• SDS-PAGE mass (denatured)
• Gel filtration mass (native)

🔸 8.2.4 Ultracentrifugation

Two major approaches:

(A) Sedimentation velocity 🚀

• Measures rate of sedimentation
• Gives:
  • Sedimentation coefficient (s, in Svedbergs)
  • Shape and mass information

s = rac{v}{omega^2 r}

(B) Sedimentation equilibrium ⚖️

• Measures mass distribution at equilibrium
• Independent of shape
• Highly accurate molecular mass determination

📌 Considered a gold standard for molecular mass

🔸 8.2.5 Mass spectrometry (MS)

Why MS is powerful 💥

• Extremely accurate
• Detects:
  • Exact molecular mass
  • PTMs
  • Heterogeneity

(A) Electrospray ionization (ESI)

• Produces multiply charged ions
• Allows analysis of large proteins
• Requires deconvolution of charge states

m/z = rac{M + nH}{n}

(B) MALDI-TOF

• Mostly singly charged ions
• Easier spectra
• Slightly less accurate for very large proteins

MS and modifications

• Detect:
  • Phosphorylation
  • Acetylation
  • Glycosylation
• Mass shifts directly reveal modification type

🔹 8.3 Primary Structure Determination

Key concept

The amino-acid sequence defines everything downstream in structure and function.

🔸 Approaches to sequencing

1️⃣ Edman degradation ✂️

• Sequential removal of N-terminal amino acids
• Limited to ~30–50 residues
• Requires:
  • Free N-terminus
  • Purified protein or peptide

📌 Often used on peptide fragments

2️⃣ Enzymatic cleavage strategies

Common proteases:

• Trypsin (after Lys/Arg)
• Chymotrypsin (after aromatic residues)
• Endoproteinase Glu-C

➡️ Overlapping peptides are essential!

🔸 8.3.2 MS/MS sequencing

Tandem mass spectrometry

1. Peptide ion selected
2. Fragmented
3. Fragment masses reveal sequence

Fragment types

• b-ions
• y-ions

📌 Extremely powerful for:

• Rapid sequencing
• PTM localization
• Complex mixtures

🔸 8.3.3 DNA-derived sequences 🧬

When protein sequencing is hard

• Use gene sequence instead

Steps:

1. Clone gene
2. Determine DNA sequence
3. Translate to amino-acid sequence

⚠️ Caveat:

• DNA sequence does not reveal PTMs

🔹 Final take-home messages 🎯

• Molecular mass ≠ one value → depends on method and conditions
• Combine:
  • SDS-PAGE
  • Gel filtration
  • Ultracentrifugation
  • Mass spectrometry
• Primary structure is best determined by:
  • MS/MS
  • DNA sequencing
• Always compare native vs denatured data