Bio 40 Lec 4: Primary Protein Structure & Protein Purification Methods

What defines a protein's primary structure?

Amino acid sequence and the location of disulfide bonds. Primary structure is defined purely by covalent bonds.

Why are Cys14 and Cys17 invariant residues in cytochrome c across species?

They are involved in binding the heme cofactor -- essential for function.

What single amino acid change causes sickle cell anemia, and who predicted its molecular basis?

A single Glu to Val substitution in beta-hemoglobin. Linus Pauling predicted its molecular basis.

In a phylogenetic tree built from protein sequence data, how are branch distances typically measured?

Number of amino acid changes per 100 residues; branch points represent common ancestors.

Distinguish peptides, polypeptides, and proteins by size.

Peptides: 1-40 aa; Polypeptides: >40 aa (long enough to fold stably); Proteins: typically 10k-100k Da mass, though much larger examples exist.

What are the largest known polypeptide and largest known protein, and what structural feature do they share?

Titin (muscle, ~27,000 aa) is the largest polypeptide; glutenin (wheat, Mr >10 million) is the largest protein -- both have coiled structures giving elasticity.

How does the lactate dehydrogenase (LDH) enzymatic assay work, and what wavelength is monitored?

In standard cytotoxicity assays, lactate dehydrogenase converts lactate + NAD+ to pyruvate + NADH; NADH absorbs at 340 nm, so activity is followed by monitoring 340 nm absorbance.

When would you use an ELISA assay instead of an enzymatic assay to detect a protein?

When the protein has no enzymatic activity to assay directly -- use a specific antibody instead.

Why is a protein least soluble at its isoelectric point (pI)?

At the pI, net charge = 0, so there's minimal charge-based repulsion between protein molecules, allowing aggregation.

Explain the two opposite effects of ion concentration on protein solubility in 'salting out.'

Low ion concentration: ions shield protein charge, increasing solubility. High ion concentration: ions compete with protein for water, decreasing solubility.

In gel filtration chromatography, why do large proteins elute before small proteins?

Large proteins are excluded from the porous beads and pass through faster; small proteins enter the pores and are retarded, eluting later.

In ion-exchange chromatography, distinguish anion exchange from cation exchange, and name two factors that affect binding.

Anion exchange (+ support) binds (-) solutes tightest, eluting last. Cation exchange (- support) binds (+) solutes tightest, eluting last. Binding also depends on other charged solutes and pH.

To calculate the net charge on a polypeptide, what pK values do you use for the N- and C-termini, and why don't internal peptide bonds contribute charge?

N-terminal amino group pK ≈ 8-8.5, C-terminal carboxyl pK ≈ 3.1; only the termini and ionizable side chains contribute charge, since the peptide bond itself eliminates charge from the groups that formed it.

Which purification method exploits a protein's specific 3-D structure/function (e.g. an active site), and how do you elute the bound protein?

Affinity chromatography, using an immobilized ligand that the protein specifically binds; elute with excess free ligand or by changing pH/ionic strength.

Why does SDS-PAGE separate proteins by mass rather than by their native charge or shape?

SDS coats the protein with a large uniform negative charge, masking intrinsic charge, and denatures it into a rod -- so mobility depends only on mass.

What are the two main criteria used to assess protein purity during a purification?

Visualization as a single band on SDS-PAGE, and increasing specific activity (even as total activity decreases due to protein loss).

During a successful protein purification, how do specific activity and total activity typically change?

Specific activity should increase (less contaminating protein diluting it); total activity typically decreases (protein is lost at each step).

What is the ninhydrin reaction used for?

Gives a purple/bluish color with free amino acids, used to detect/quantify them after hydrolysis.

Why is alkylation performed after reduction when preparing a protein for sequencing?

Reduction (DTT/beta-mercaptoethanol) breaks disulfides to free -SH, but they can re-form; alkylation (iodoacetate) permanently blocks -SH so disulfides can't reform.

Describe the Edman degradation cycle and what makes it useful for sequencing (vs. older methods).

PITC reacts with the N-terminal residue to form a PTC derivative; mild acid cleaves it off as a PTH derivative, identifying that one amino acid, while leaving the rest of the polypeptide intact.

What is the difference between the PTC derivative and the PTH derivative in Edman degradation?

PTC is the intermediate derivative formed on the intact peptide; PTH is the final cleaved derivative of the single removed amino acid, used for identification.

How do carboxypeptidases A, B, C, and Y differ in specificity for C-terminal sequencing?

C and Y cleave any amino acid; A does not cleave Pro/Arg/Lys; B cleaves only Arg and Lys.

State the cleavage specificity of trypsin, chymotrypsin, and cyanogen bromide (CNBr).

Trypsin: carboxy-side of Lys/Arg. Chymotrypsin: carboxy-side of Phe/Tyr/Trp. CNBr: carboxy-side of Met.

Give the formulas for predicting the number of peptide fragments from CNBr cleavage and from trypsin cleavage.

CNBr: # peptides = # Met + 1. Trypsin: # peptides = # Lys + # Arg + 1.

Why should any peptide fragment produced by CNBr cleavage end in methionine (except possibly the last fragment)?

Because CNBr cleaves on the carboxy-side of Met, so every fragment (except the final C-terminal one) ends with the Met that was cleaved after.

How do you use two different cleavage reagents to reconstruct a protein's complete primary sequence?

Use two different cleavage reagents to generate two different, overlapping sets of peptide fragments, then align the overlaps.

What are the 3 titratable groups of glutamate, and their pKa values?

Alpha-carboxyl (pKa 2.1), side-chain carboxyl (pKa 4.1), alpha-amino (pKa 9.5).

On a titration curve, where do you mark a group's pKa -- at the half-equivalence point or the full equivalence point?

The half-equivalence point (e.g. 0.5, 1.5, 2.5 equivalents), because pKa = pH at 50% dissociation of that group.

Walk through glutamate's net charge as it goes from fully protonated to fully deprotonated (adding 3 equivalents of base).

+1 (start) → 0 (after pKa 2.1) → -1 (after pKa 4.1) → -2 (after pKa 9.5).

Calculate glutamate's isoelectric point (pI) from its pKa values (2.1, 4.1, 9.5).

pI = (2.1 + 4.1) / 2 = 3.1.

For any triprotic amino acid (not just glutamate), what is the general method for finding which two pKa's to average to get the pI?

Find the net-charge-zero species on the titration curve, then average the two pKa's that flank that specific species.