Cell Biology and Protein Function

Protein Shape Change

• Proteins constantly change their 3D structure due to:
  • Binding of ions (e.g., calcium).
  • Binding of other proteins.
  • These interactions alter the properties of side chains and, consequently, the protein's conformation.
• Protein-protein interactions can modify a protein's affinity for other proteins. For example:
  • Protein A binds to protein B, changing protein A's 3D structure.
  • This change alters the properties of a specific domain within protein A, affecting its affinity for protein C.

Phosphorylation by Kinases

• Phosphorylation: Addition of a phosphate group to a protein, typically by enzymes called kinases.
  • Kinases transfer a phosphate group from ATP to one of three amino acids (serine, threonine, or tyrosine).
• Phosphate group: Negatively charged.
  • Adding a phosphate group introduces a negatively charged property within a protein domain.
  • This alters the protein's 3D structure.
• Example: Phosphorylation of serine at position 10 in an alpha helix.
  • The alpha helix folds, becoming closer to another alpha helix due to the negative charge introduced by the phosphate group.
  • This conformational change can modify the protein's function:
    • Gain or lose affinity for other proteins.
    • Acquire a new function.

Experimental Strategies to Study Protein Function

• Goal: to study protein functions.
• many different types of protein functions.
• Experimental strategies: protein function.

Gain-of-Function Studies

• Objective: to determine the function of a protein.
• Method: overexpress a protein in cells (ectopic expression).
• Example: studying the function of the RAS protein.
  • RAS is normally expressed in cells at a regulated copy number (e.g., 10 copies).
  • In a gain-of-function study, express a much higher number of copies (e.g., 1000 copies).
  • Observe the effects on the cell. If cells start proliferating rapidly, it suggests RAS is involved in cell division.
• Central dogma relevance: utilizes recombinant DNA technology to overexpress the protein.
  • Make multiple copies of the DNA that codes for RAS.
  • Introduce the DNA into cells, which then transcribe and translate it to produce thousands of copies of the protein.

Ectopic Protein Expression

• Expression Vector Use:
  • Initially done outside of the cells in a test tube.
  • Expression vector is a circular outline.
• Process:
  • Take a coding sequence (e.g., for RAS protein).
  • Insert the coding sequence into a cloning vector.
  • Make millions of copies of this cloning vector containing the RAS coding sequence to have enough material to work with.
  • Insert the cloning vector into the cells so the cells can express the RAS coding sequence.
• Specific Features of Cloning Factor:
  • Site of insertion of the DNA coding sequence: Requires a multiple cloning site.

Components of a Cloning Vector

• Three Important Components:
  • Multiple Cloning Site (MCS): Site within the cloning vector that allows insertion of the coding sequence for the protein of interest.
    • Allows the coding sequence to be expressed.
  • Promoter: DNA sequence that regulates the assembly of RNA polymerase in front of a coding sequence.
    • To increase transcription, use a strong promoter (a DNA sequence on which RNA polymerase frequently assembles).
  • Origin of Replication: Site on which DNA polymerase assembles.
    • Allows the cloning vector to be replicated in millions of copies.

Cloning Vector Amplification

• Use bacteria to replicate the cloning vector because they easily copy circular DNA.
• Selective Growth:
  • Culture may contain bacteria with and without the expression vector.
  • Use a resistance gene (e.g., insulin resistance gene) in the vector.
  • Only bacteria that have taken up the DNA will survive, resulting in a pure DNA sample of the expression vector containing the coding sequence for the RAS protein.

Introducing Expression Vector into Cells

• Goal: Make the cell take up the expression vector which has a RAS coding sequence under a strong promoter so that the cell will transcribe the gene.
• Methods to introduce genetic material into culture cells:
  • Chemical Mediated Transfection:
    • Calcium phosphate mediated transfection: Expose cells to harsh chemicals that transiently open up the membrane, allowing DNA to enter.
  • Lipid Mediated Transfection (Lipofection or Liposome Mediated):
    • Encapsulate the negatively charged DNA into a lipid layer or capsule.
    • The lipid fuses with the cell membrane, allowing the DNA to enter.
    • COVID vaccines use this method.
  • Electroporation: Zap the cell with an electric current to create temporary pores in the membrane.
  • Viral Mediated Transduction: Engineer viruses to carry the DNA of interest by replacing the viral protein with our DNA.
    • The virus infects the cells and delivers our DNA into them.
• Reliability:
  • Virus-mediated transduction is the most efficient but takes time to prepare.
  • Start with a lot of cells for electroporation.
• DNA vs. mRNA:
  • If you do DNA virus, the DNA virus will incorporate that coding sequence permanently into the genome of the host cells.

Detecting Protein Expression

• Verify that the protein is being expressed after introducing the protein sequence into a cell.
• In this case, green fluorescent protein sequence into a cell.
• Need to have a pathway to detect the presence of protein.

Antibodies for Protein Detection

• Antibodies: Proteins with high specificity to specific target proteins.
  • Antibody molecule consists of a heavy chain and a light chain.
  • Variable regions: Regions with high specificity to specific proteins.

Immunostaining

• Method: detecting RAS proteins.
• Done on fixed cells (cells treated with paraformaldehyde to preserve architecture).
• Process:
  • Incubate cells with primary antibody, which has affinity for the protein of interest (e.g., RAS protein).
  • The antibody binds to the protein, and a secondary antibody with a fluorescent tag is added.
• Information Gained:
  • Presence and location of the protein within the cell.
  • The intensity of fluorescence indicates the level of protein expression.
• Benefits:
  • Preserves cell architecture, allowing visualization within the nucleus.
  • Can be quantitative to compare the level of protein expression.
• The reason that you use secondary antibodies because to expand the signal.
  • If you have one protein and one antibody, it's hard to see.

Western Blot

• Immunostaining is done on intact cell, western blot is cell lysate.
• Blood is done on cell lysate (contents within the cells after breaking them open).
  • The contents of a cell after breaking them open
• SDS PAGE (separation of proteins by size). SDS-page separates proteins by size.
• Process:
  • Separate the proteins using SDS-PAGE.
  • Transfer the separated proteins to a membrane.
  • Incubate the membrane with a primary antibody.
  • Add a secondary antibody conjugated to a chemical or fluorescent protein for detection.
• Information Gained:
  • Presence of the protein, indicated by a sharp band.
  • The thickness and intensity of the band reflect the amount of protein present.
• Phosphorylation State Detection:
  • Use antibodies specific to the phosphorylation state of a protein (e.g., ERK).
  • Compare total ERK to phospho-ERK to determine the percentage of active ERK in the sample.
• Active ERK will be bound, while inactive will not.
• Example: Comparing total ATM to phospho-ATM in irradiated vs. non-irradiated samples.

Protein Transport

• Cytoplasmic Proteins: Easy, they stay in cytoplasm.
• Mitochondria Proteins: transported to the mitochondria after translation.
  • Translation is complete, then they get delivered to their final destination
• Endomembrane System: Organelles constantly exchanging membranes through vesicular transport.
  • Because they share membranes
• Co-translational Import starts co translational import.
  • Translation is complete, you end up in ER. From there, go to Golgi, and then transported to final destination.
• This is why you see ribosome attached to the ER surface.

Nuclear Pores

• The gates into the nucleus are the nuclear pores.
• Lining the pore for the proteins to go in on.
• Those protein complex is called MPC, nuclear pore complex.
• Molecules and proteins go in and out of the nucleus.
• Proteins and RNAs Transported:
  • Proteins are made in the cytoplasm and imported into the nucleus.
  • Transcription occurs in the nucleus, producing mRNA, which is then transported out for translation.
• Size Cutoff:
  • Small proteins (10nm or smaller) freely diffuse into the nucleus without regulation.
  • Larger proteins require an active transport mechanism.
• This domain of the protein is recognized by UPS delivery then.

Nuclear Localization Signal (NLS)

• Address embedded is the amino acid signal.
• Called nuclear localization signal, in short, NLS.
• Protein Domain:
  • NLS domain of the protein is what's recognized by the UPS delivery.
• Experiment:
  • Take one sample out of cytoplasmic protein or nuclear protein?
  • Take nuclear protein.
• Then expectation?
  • Will stay in the cytoplasm.
• Gain of function study tells you whether something is sufficient
• NLS-Coated Gold Particles:
  • Old particles being coded with NLS are coated with gold.
  • NLS is sufficient to bring anything inside the nucleus.