Lecture 2 Amino Acid Chemistry, Peptides, and Protein Methods

What does a buffer do? How is it related to amino acids?

It resists changes in pH when acid or base is added.

Amino acids can also buffer because their ionizable groups can gain or lose hydrogen ions.

What is the ideal condition for an ionizable group buffer? Why?

When the pH is approximately equal to the pKa as the group is about 50% protonated and 50% deprotonated. So the solution has both forms available to absorb added acid or base.

Primary Structure

Amino acid sequence of the protein held together by peptide bonds. From N-terminus (Amino group) to C-terminus (Carboxyl group).

Secondary Structure

Local folding patterns in protein backbone, stabilized by hydrogen bonds. The two most common are alpha-helix and beta-sheet.

Tertiary Structure

The overall 3D shape of one polypeptide chain, stabilized by non-covalent interactions between amino acid side chains. These include:
- Hydrogen bonds.
- Ionic interactions.
- Hydrophobic interactions.
- Van der Waals interactions.
- Disulfide bonds.

Quaternary Structure

How multiple polypeptide chains assemble together. Not all proteins have this structure, only applying when a protein has more than one subunit.

Why are covalent bonds stronger than non-covalent interactions?

Covalent bonds are very stable because the energies required to break them are much greater than the thermal energy available at room temperature or body temperature.

What are the types of peptides?

- Dipeptide: 2 amino acids, 1 peptide bond.
- Tripeptide: 3 amino acids, 2 peptide bonds.
- Oligopeptide: A few amino acids.
- Polypeptide: Many amino acids, molecular weight < 10 kDa.
- Protein: Thousands of amino acids, molecular weight > 10kDa.

How is peptide bonds formed and broken?

Formed through condensation.
Broken through hydrolysis.

How do you estimate the number of residues?

Protein molecular weight / Average molar weight of a single free amino acid.

What is the average molecular weight of amino acid? What about water?

128 daltons. 18 daltons.

What is a dalton equal to?

g/mol

What is the amino acid and groups that form disulfide bonds? How is the disulfide bond formed?

Two cysteine side chains can have sulfydryl/thiol groups.

When oxidized, a disulfide bond is formed, where the paired form is called cystine.

What are the outcomes of forming disulfide bonds? What are examples?

1. Stabilizes 3D protein folding and connects protein chains together. Insulin has disulfide bonds connecting two chains.

2. Increases protein stability outside the cell. Common in proteins secreted or found outside the cells, like antibodies, insulin, digestive enzymes, extracellular matrix proteins.

Where do disulfide bonds form? Where do they go?

In the ER for proteins that enter the secretory pathway. These proteins travel through the Golgi and may be secreted outside the cell, inserted into the plasma membrane, sent to lysosomes, or retained in the ER.

Which proteins do not usually form stable disulfide bonds? Why?

Cytosolic and nuclear proteins because they are in reducing environments.

How do ionic bonds form? Which side chains/groups?

From the attraction of fully positively charged ion (cation) for a fully negatively charged ion (anion).

Basic and acidic amino acids.

What are the outcomes of ionic bonds?

1. Help enzymes bind substrates.
2. Help proteins bind DNA, RNA, or membranes.
3. Contribute to protein-protein interactions.

How do hydrogen bonds form? Which side chains/groups?

From interaction of partially positively charged hydrogen atom in a molecular dipole with unpaired electrons from another atom (usually oxygen or nitrogen).

What are the outcomes of hydrogen bonds?

1. Stabilizes protein secondary structure.
2. Form alpha-helices and beta-sheets.
3. Stabilizes DNA base pairing (A-T, G-C bonds).
4. Helps enzymes recognize substrates.
5. Helps water interact with other molecules such as glucose.

What do hydrophobic interactions refer to?

Tendency of nonpolar molecules to aggregate in aqueous solutions, minimizing their contact with water.

In context of hydrophobic interactions. What is an unfavorable interaction? What is a favorable interaction?

Unfavorable is when nonpolar groups are exposed to water, so water molecules become ordered around them.

Favorable is when nonpolar groups cluster around, leaving more water free to interact with itself.

How do hydrophobic amino acids help protein folding? What is the process and what does it create?

They help decide which parts of the protein should face water and which parts should be buried inside.

Hydrophobic side chains cluster together, move away from water, and become buried inside the protein. This creates a hydrophobic core.

Why does protein purification work? What are its steps?

Different proteins have different physical and chemical properties.

1. Starting material.
2. Separation principle.
3. Outcome.

What is the process for obtaining starting material for protein purification?

Cells or tissue is lysed and its protein lysate is obtained. This is centrifuged and the soluble protein extract is obtained in the supernatant. The sediment includes nonsolubles.

What are the different types of lysis for protein purification?

1. Detergent-based lysis: Detergents disrupt membranes.
2. Mechanical/physical disruption: Homogenization, grinding, beat beating, sonication.
3. Freeze-thaw: Repeated freezing and thawing ruptures cells.
4. Enzymatic lysis: Enzymes digest cell walls or extracellular barriers.

What does sequential purification do to the proteins?

It decreases total protein, but increases concentration of protein of interest.

What are the different protein purification procedures?

- Ion-exchange chromatography. (Charge)

- Size-exclusion chromatography. (Size/shape)

- Affinity chromatography. (Specific binding)

How can enzyme purification be monitored?

Specific activity: Number of enzyme units per mg of total protein.

What is the principle behind ion-exchange chromatography?

Proteins have different net charges depending on their amino acid composition and pH of buffer.

- Cation exchanger: Positively charged proteins bind to negatively charged resin.

- Anion exchanger: Negatively charged proteins bind to positively charged resin.

Proteins that bind weakly come out earlier, while proteins that bind strongly come out later and increase salt concentration.

What is the principle behind size-exclusion chromatography?

Also called gel filtration chromatography.

- Large proteins cannot enter pores in beads very well, taking shorter path and coming out first.

- Small proteins enter the pores, take longer path, and come out later.

Large proteins elute first and small proteins elute later.

What is the principle behind affinity chromatography?

Column contains ligand that specifically binds the protein of interest, which is always tagged. (6xHis-tagged protein binding to nickel resin)

- Unwanted proteins flow through first.

- Bound protein of interest is finally eluted by outcompeting the His-tagged protein with high conc. of imidazole. It will flow through in this second round.

Why does protein purification work?

Because different proteins have different physical and chemical properties.

What can a purified protein be used for?

Structure studies, activity assays, and functional analysis.

What are the procedures for protein analysis?

- Polyacrylamide Gel Electrophoresis.

- Western blotting.

- Activity assay.

What is the usage of polyacrylamide gel electrophoresis?

Visualize and characterize purified proteins to estimate degree of purity and approximate molecular weight.

What is the principle behind polyacrylamide gel electrophoresis?

Uses cross-linked polymer polyacrylamide gels that gives all proteins a similar charge-to-mass ratio electrophoresis in presence of sodium dodecyl sulfate (SDS). It will separate proteins by molecular weight, where smaller proteins migrate more rapidly. This will be visualized through coomassie blue dye binding to the proteins.

What are the advantages of Western blotting?

It detects a specific protein, not all proteins. It can also detect protein modifications such as phosphorylation, cleavage, ubiquitination, or acetylation.