Biochem

What does a reducing agent do to disulfide bonds?

It breaks disulfide bonds: $R−S−S−R \rightarrow 2R−SH$. Examples: DTT and β-mercaptoethanol.

What conditions promote disulfide-bond formation?

Oxidizing conditions. $2R−SH \rightarrow R−S−S−R$. Oxidize to connect. Reduce to release.

Which amino acid forms disulfide bonds?

Cysteine, because its side chain contains a thiol group: $−SH$.

Where do disulfide bonds commonly form inside a cell?

The endoplasmic reticulum because it has an oxidizing environment. The cytosol is reducing and discourages disulfide bonds.

One band on nonreducing SDS-PAGE but two bands on reducing SDS-PAGE means what?

The protein contains two different subunits joined by a disulfide bond. It is a disulfide-linked heteromultimer.

What does SDS do during SDS-PAGE?

SDS denatures proteins and gives them similar negative charge-to-mass ratios, so separation occurs mainly by size.

Which proteins migrate fastest in SDS-PAGE?

The smallest proteins. Smallest travels fastest.

Which proteins move fastest through size-exclusion chromatography?

The largest proteins. Large proteins cannot enter the bead pores, so they take the shorter path and elute first.

What is primary protein structure?

The amino acid sequence from the N-terminus to the C-terminus, held together by peptide bonds.

What can change a protein’s primary structure?

A mutation that changes the amino acid sequence or cleavage of peptide bonds. Denaturation does not normally change primary structure.

What forms secondary protein structure?

Hydrogen bonding between the peptide backbone groups. Examples: Alpha helices, Beta sheets.

What interactions stabilize tertiary and quaternary structure?

Hydrophobic interactions, Hydrogen bonds, Ionic interactions, Disulfide bonds. Tertiary is one chain. Quaternary involves multiple chains.

Peptide-bond formation is what type of reaction?

A condensation reaction that releases water: $\text{amino acid}+\text{amino acid} \rightarrow \text{peptide}+H_2O$.

What reaction breaks a peptide bond?

Hydrolysis, which consumes water: $\text{peptide}+H_2O \rightarrow \text{smaller products}$.

Between which groups does a peptide bond form?

Between the carboxyl group of one amino acid and the amino group of another. The bond formed is: $−CO−NH−$.

In what direction does solid-phase peptide synthesis build a peptide?

From the C-terminus toward the N-terminus. The C-terminal amino acid is attached to the bead first.

Which amino acids are positively charged near physiological pH?

Lysine (K), Arginine (R), Histidine (H), can also be positively charged depending on pH.

Why can adding several lysine or arginine residues slow peptide synthesis?

Their positively charged side chains repel one another, making it harder for the incoming amino acid to approach the growing peptide.

Which amino acids have negatively charged side chains near physiological pH?

Aspartate (D), Glutamate (E).

What is the Henderson-Hasselbalch equation?

$pH=pK_a+\log\left(\frac{[A^-]}{[HA]}\right)$. $A^-$ is deprotonated and $HA$ is protonated.

What happens when pH=pK_a?

$[A^-]=[HA]$. The group is 50% protonated and 50% deprotonated.

What is the protonation pattern at pK_a±2?

At $pH=pK_a−2$, approximately 99% protonated. At $pH=pK_a+2$, approximately 99% deprotonated.

What does a lower $K_d$ mean?

Higher binding affinity. Lower $K_d = \text{stronger binding}$.

What is the difference between affinity and avidity?

Affinity: strength of one binding interaction. Avidity: total combined strength of multiple binding interactions.

What is cooperativity?

Binding at one site changes the affinity of another binding site. This is different from simply having multiple binding sites.

What does a longer molecule with multiple binding sites often have compared with a short fragment?

Greater apparent affinity due to increased avidity and opportunities for rebinding.