Lecture 3 (combined)

Western Blot technique (steps)

Used to answer: is a protein there? how much of the protein is there?

1. Lyse (burst open) cells, spilling out all their proteins

2. Sort proteins by size (SDS-PAGE): load proteins into a gel and run electricity through it. proteins are coated with SDS, a detergent that gives them all a negative charge, so they all get pulled down towards the positive electrode at a rate determined ONLY by size.

3. Transfer (blot) proteins into a membrane (like pressing a wet newspaper onto a paper towel to make a copy) → this preserves the proteins’s exact positions

4. Bathe the membrane in a blocking solution so that your antibodies stick ONLY to the target protein

5. Antibody tagging (primary antibody: add an antibody that binds ONLY to your target protein) (secondary antibody: add a second antibody that ONLY binds to antibodies, tagged with something that produces light)

6. Add a substrate that reacts with the tag on the secondary antibody → produces light → expose it to a camera → wherever your protein sat on that gel, you see a dark band

Immunoprecipitation (IP) technique (steps)

Used to investigate quaternary structure and answers the question: “what proteins does my protein interact with?”

1. Lyse cells (burst cells open) to release proteins into a solution

2. Add an antibody “bait” into the solution that specifically binds to your protein of interest

3. Fish antibody out of solution (precipitation), this is due to the antibody being attached to resin beads/magnets

4. Wash the beads to rinse off everything so that you ONLY see your protein of interest and whatever other protein is bound to it

5. Run a western blot for the protein bound to your protein of interest → If you see a band for the pound protein, that means it interacts with your protein of interest

BU-MS technique

Used to answer: “what IS this protein, down to its exact sequence/identity?”

1. Chop the protein into peptide pieces with trypsin

2. separate and weigh those pieces with mass spec

3. shatter them again to read their sequence

4. match the pattern to a database to identify the protein.

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Aquaporin

• protein that facilitates the travel of water through the cell membrane

• homotetramer → 4 of the same subunits (hydrophilic residues inside, hydrophobic residues outside)

• water passes though the hydrophilic residues

Globins

Family of proteins that sense and carry oxygen

Myoglobin

• Single molecule of heme bonded to a globin

  • delivers oxygen to muscles

  • single polypeptide (153 AAs)

  • 8 alpha helices connected by bends, together they bind the heme group

Heme

• molecule that helps carry oxygen. because oxygen is poorly soluble and can’t be carried throughout long distances, evolution drove heme to carry oxygen

  • Its protoporphyrin ring binds Fe²⁺, and Fe²⁺ binds oxygen

  • nitrogens stabilizes iron

  • Fe²⁺ is not stable in free heme, so proteins that contain heme stabilize oxygen by tucking the heme into their 3D structure

Histidine residues and oxygen binding to heme

• Histidines on the heme facilitate oxygen binding

• proximal His: Stabilizes the Fe²⁺ center

• distal His: stabilizes O2 binding

• (stabilizing is done via ionic bonding)

• Proximal and distal His are highly conserved

Hemoglobin

• oxygen-carrying protein in red blood cells

  • 4 subunits of myoglobin-like proteins

  • 4 hemes

  • 2 conformations: R state (relaxed) and T state (tense)

  • allosteric

Explain what this means: oxygen binding to hemoglobin is cooperative

• hemoglobin becomes better at grabbing oxygen as it grabs more oxygen → this is because it has 4 oxygen binding spots (cooperative binding)

• this matters because if it’s in the lungs, with a high affinity for oxygen → it quickly loads up oxygen. however, in the tissues, with low oxygen affinity, hemoglobin releases O2 quickly as it releases O2.

• this is an example of allosteric activity

R state vs T state

T state: tense state

• oxygen has LOW affinity for binding to heme, heme is tucked

R state: relaxed state

• oxygen has HIGH affinity for binding to heme, heme is outward facing

Allosteric protein

A protein where the binding of one site effects the binding properties of another site on the same protein

Ligand

a molecule or ion that binds to another molecule (usually a protein) at a specific binding site

ex) Oxygen binds to hemoglobin, so oxygen is the ligand

Homotropic vs heterotrophic

Homotropic: When the ligand that binds is the SAME molecule as the one whose binding is being affected

• ex) O2 affects the binding of O2 (same molecule)

Heterotrophic: When the ligand that binds is a DIFFERENT molecule than the one whose binding is being affected

• ex) H+ affects O2 binding (different molecules)

Class example: actin/myosin muscle contraction

actin/myosin muscle contraction is an example of protein/ligand interaction

• Muscles contain actin and myosin filaments

Process:

1. A nerve impulse triggers the release of calcium ions

2. Calcium ions bind onto actin filaments → this causes the actin filament to turn, exposing the myosin binding sites

3. The head of each myosin is bound to an ADP and a phosphate

4. Myosin head releases the phosphate and binds to actin filaments through the myosin binding sites

5. The filaments then glide past each other → as myosin and actin move, they release the ADP

6. ATP then binds to myosin heads, severing the bond between actin and myosin

7. ADP and phosphate is then restored on the myosin heads → repeat process

Phosphorylation

• Adds a phosphoryl group to reactive hydroxyls in proteins (Ser, Thr, Tyr)

Process:

1. Signal comes in

2. Protein kinase grabs a phosphoryl group off of ATP

3. Kinase transfers phosphorly group onto a reactive hydroxyl from Ser, Thr, or Tyr → enzyme is now phosphorylated → turns enzyme from inactive to active

4. Enzyme does its job → protein phosphatase removes phosphate group from enzyme

5. Phosphate is released as Pi (inorganic phosphate)

6. Enzyme goes from active → active (switch off)

Kinase: phosphorylates proteins

Phosphatase: removes phosphate groups

Kinases

enzymes that phosphorylate proteins

Phosphatases

enzymes that remove phosphate groups

What is signal transduction via phosphorylation cascade?

• Signal from outside the cell gets “relayed” into the cell by a chain of kinases turning each other on (like dominoes) until it triggers a final response

1. extracellular ligand binds a receptor (a kinase)

2. Triggers kinase cascade, where each active kinase phosphorylates (activates) the next

3. ends in a biological response

Ubiquitination

• Protein waste program

  • Ubiquitin - peptide

1. Ubiquitin’s terminal Glycine (COOH group) and the substrate’s Lysine (NH2 group) come together

2. Bond forms between the carboxyl carbon of Glycine and amino group of Lysine → releases water → result is an isopeptide bond linking Ubiquitin to the substrate

The more ubiquitous attached to a protein, the more it signals for the protein to get trashed

Ubiquitin

Peptide that’s added to a Lysine residue through an isopeptide bond with Ubiquitin’s C-terminus

Ubiquitin and proteasome “shredder”

1. Ubiquitin tags protein

2. protein is fed into the proteasome “shredder”

Part 1: E1-E2-E3 cascade

1. E1(enzyme) : activates ubiquitin

2. E2 (enzyme): takes from E1

3. E3 (ligase): takes from E2, catalyzes attaching ubiquitin onto protein

Part 2: proteasome

1. Ubiquitin tagged protein is recognized by the receptor on the proteasome’s 19S cap

2. DUBs (enzymes that remove ubiquitin tags) pop off, recycling ubiquitin molecules so the cell can reuse them

3. The protein is fed into the 20S core → Beta subunits inside the 20S core cut the protein → protein gets chopped into oligopeptides, which are released

Silent mutation

No change in amino acid

Nonsense mutation

introduces STOP codon

Missense mutation (conservative)

Change in amino acid, but the new amino acid has similar biophysical properties to the original amino acid

Missense mutation (non-conservative)

Change in amino acid, but the new amino acid has different biophysical properties to the original amino acid

Loss of function mutation

protein loses its normal activity

Gain of function mutation

protein does more than normal, does something new, or is active when it shouldn't be

Which amino acid is best for mutation, and why?

Alanine

• simple/unreactive side chain (CH3)

Mutation nomenclature

Format: [Original amino acid][position number of that original amino acid][new amino acid]

ex) G12V = Glycine-12 mutated to Valine (or Gly12Val)

Mimetic mutations

Replacing AAs with AAs that have biological reactivity to mimic a function on that protein

ex) Phosphomimetics

• ex) Aspartate looks and acts like phospho-serine → by replacing Serine with Aspartate, you fake an amino acid that is already phosphorylated