Lecture 2 (combined)

Memorize triad (on iPad)

done

n/a

n/a

n/a

v

n/a

n/a

Primary structure + draw an example

An amino acid chain connected by a covalent peptide bond (C-N).

• Nitrogen (from amino group) of the first amino acid connects with the carbon (carboxyl group) of the second amino acid

  • N-terminus: beginning of primary structure, refers to the nitrogen

  • C-terminus: end of primary structure, refers to the carbon

Residue

Amino acid (more specifically, each amino acid in the polypeptide chain)

Peptide bond in relation to the ribosome and tRNAs + explain the process of translation

The ribosome is the “machine” that builds peptide bonds using tRNAs to bring in the right amino acids in the right order (aka translation)

1. The ribosome reads one mRNA codon at a time → each codon specifies which amino acid to bring

2. tRNA carries one specific amino acid and has an anticodon that matches the mRNA codon

3. Ribosome lines up the growing chain

4. Peptide bond forms — the ribosome catalyzes the reaction between the growing chain and the new amino acid

5. Ribosome shifts forward to the next codon, the used tRNA leaves, and a new tRNA comes in

6. Repeat until polypeptide chain is fully built

start codon

Met (AUG)

stop codons

UGA, UAG, UAA

Why is AUG special, and how does the ribosome know when it means "start" vs. just "insert methionine"?

• AUG codes for methionine AND is the universal start codon

• The ribosome can't tell which one from the codon alone → Context decides:

  • surrounding sequences and other factors at the genome dictate which one it is

What are the two ways to study peptides (broadly)?

1. separate them based on mass

2. Separate them based on the unique chemistry of their side chains

Class example: Insulin and peptide sequencing

• alpha and beta chains come from two different genes, but they come together to form insulin

• insulin is a conserved sequence (meaning that compared to a lot of other animals, our insulin sequence is very similar to there), meaning it is important/not very changed throughout evolution

Which enzymes chop up peptides? What do they chop up? Why?

Trypsin, Chymotrypsin → break peptide bonds

• Trypsin: cuts C-terminal after Lysine or Arginine → This is because Trypsin’s active site has a negatively charged pocket (Asp residue at its bottom) → because Lysine and Arginine are positively charged and basic residues their side chains get pulled into that pocket

  • *only if there is not a Proline as the C terminus residue → this is because Proline forms a rigid ring, making it difficult for Trypsin to access the peptide bond

• Chymotrypsin: cuts after large hydrophobic residues (The, Trp, Tyr)

Secondary structure

Polypeptide beginning to twist into four patterns, defined by weak and strong interactions

• peptide bonds, H bonds between NH and C=O groups

Disulfide bridge

2 cysteines in proximity form a covalent bond (S-S) → either between regions on the same chain OR between chains

In secondary structure, what is proline, hydrophobic/hydrophillic interactions, H bonding, and cysteine used for?

“Tools” that help peptides start making structures

• Proline: kinks

• Hydrophobic/hydrophillic side chains: to attract like, to repel unlike

• H bond: for weak interactions

• Cysteine: for strong bonding

Alpha helix

polypeptide chain that twists into a rod-like structure

• secondary structure, rod-like shape

• inside: backbone

• outside: R-chain groups (exposed for maximum reactivity)

• some AAs are more likely to be in an alpha helix formation than others

• Hydrogen bonds between carbonyl oxygens (C=O) and amine hydrogen 4 amino acids downstream

Beta pleated sheets

Polymers of amino acids are lined up either parallel or antiparallel to each other

• secondary structure

• antiparallel: line up perfectly

• parallel: same direction, so they can’t line up perfectly

Tertiary structure

3D shape that the polypeptide chain forms

• Secondary structures combine to form motifs (more complex structures)

Quaternary structure

2 or more polypeptide chains that bind together to form superstructures

Denaturation and renaturation

Denaturation: loss of protein structure and function

renaturation: When bonds that are broken are remade

Proteostasis

The life cycle of a protein

1. Transcription (DNA→RNA) → Translation (RNA→amino acid chain, made by ribosome)

2. Chaperones help the new chain fold

3. Protein can exist in 3 interconvertible states:

• Native State = correctly folded, functional

• Folding Intermediates = partially folded, in progress

• Aggregates = misfolded clumps (bad — disease-associated)

• These states can convert back and forth (folding ↔ unfolding, aggregation ↔ disaggregation

• If a protein can't be fixed, it's degraded

How do chaperones help protein folding, and what happens without them?

• Without a chaperone: unfolded protein folds on its own, but slowly → during this slow window it's exposed and can stick to other unfolded proteins, forming a nonfunctional aggregate.

• With a chaperone: the unfolded protein is captured inside the chaperone's cavity, which shields/isolates it from other proteins. Protected from unwanted interactions, it folds correctly into its native shape, then is released ("removed for use") as a functional folded protein.

What is GC-MS and how does it work

used anytime you need to figure out what specific small molecules are in a sample (better for small stuff, not big stuff like DNA)

1. Inject liquid into port → turns into gas

2. Sample gas is pushed through a column using a different gas → its molecules stick differently.

3. Ones that stick better move through the column faster, the ones that don’t are delayed

4. MS blasts molecules with electrons → turns into fragments → fragments separated by mass → fragmentation pattern generated

What is SDS PAGE and what is it used for?

used anytime you need to know what proteins are in a sample, how big they are, or how much of them there is

1. Get proteins and treat them with heat and SDS (a detergent that makes them all - charged)

2. Put proteins into wells on the top of the gel (- electrode on top, + electrode on bottom) → they all travel to the + electrode

3. small proteins → closer to bottom, large proteins → closer to top

Isoelectric point of a protein

pH at which the net charge is 0

• average of the pKa of the carboxyl and amine group of the protein

What is 2D electrophoresis, and what is it used for?

Good if there are a lot of proteins you need to identify

• Dimension 1: isoelectric focusing

  • lay proteins on gel strip with built in pH gradient

  • run electricity through it → each protein moves until its isoelectric point

• Dimension 2: SDS page (as usual)

What is column chromatography, and what is it used for?

Separates proteins from a mixture

1. Fill tube with beads

2. pour protein mixture on top of the column

3. Elute (wash through) using solvent

4. Separate based on:

   1. Size: big molecules come out first, small come out last

   2. charge: + charge proteins stick to beads and don’t move, - charge proteins flow through and don’t stick (cus beads are - charge) and come out first, neutral proteins also flow through

   3. Affinity: target protein sticks, everything else washes through

5. Collect fractions (liquid that pours through the bottom)

6. Check fractions for protein of interest

Peptide tags

A peptide sequence you attach onto a protein of interest, so you don’t have to use an antibody

Fluorescence

A glowing protein you can use to attach onto other proteins, so you don’t have to use an antibody (peptide tag)

6xHis tag

Peptide tag that gives your protein a metal binding property

• 6 His in a row, His is basically a metal magnet

Immunochemistry and antibodies in rabbits

• antibodies are generated to target a protein of interest

• ex) generating antibodies in rabbits: inject antigen in rabbit → antigen activates B-cells → plasma B cells provide antibodies → collect antibodies from rabbits

Problems with antibody generation

1. limited to what is immunogenic → some proteins don’t provoke a good immune response

2. generates antibodies specific for many different cell motifs → grabs lookalike proteins with similar motifs, not JUST your protein of interest

3. native vs denature protein antibodies → The immune system "sees" different things depending on the protein's shape at injection — a folded protein produces an antibody that recognizes 3D shape, while a denatured/unfolded protein produces an antibody that recognizes the linear amino acid sequence

What is immunohistochemistry? + immunofluorescence

Used to study in-situ observations

1. cut cells thin and attach to a microscope slide

2. primary antibody finds target protein in sample

3. secondary antibody finds primary antibody based on its constant (Fc) region

4. makes a brown color where antibody is bound (due to color changing enzyme)

immunofluorescence → secondary antibody is a fluorescent protein, not an enzyme