L 7. Protein Structure & Function II Flashcards
explain protein families
- within each family, amino acid sequences and the 3D shape closely resemble each other
- proteins within families may have different roles bc they have different enzymatic activity
define ligand
any substance that is bound to a protein
how to enzymes regulate metabolic pathways
one enzyme can catalyze a molecule and another enzyme will catalyze the same molecule and so on
how do enzymes catalyze reactions
- it lowers the activation energy
- so it requires less energy for a reactant to turn into a product
how do ligands bond to proteins
- they have to physically interact
- the ligand binds through noncovalent bonds
- the enzyme will fold and create a pocket for substrate to bind
ligand-protein binding - what happens at the pocket of the enzyme
- amino acids within particular areas in the protein will cause noncovalent bonds to interact with the ligand
- this will cause a conformational change in the protein resulting in the protein completing its task
structure of a protein - binding sites
bind and orient substrate/ligand
structure of a protein - catalytic site
reduces chemical activation energy
what are the ways a protein and ligand can bind
- Orientation/proximity
- Rearrangement of e-
- Strain
protein-ligand binding - orientation/proximity
- enzyme binds to two substrate molecules
- orientates them precisely to encourage a reaction
protein-ligand binding - rearrangement of e-
- binding of substrate to enzyme rearranges electrons in the substrate
- creates partial negative charge and positive charges that favor a reaction
protein-ligand binding - strain
- enzyme strains the bound substrate molecule
- forcing it towards a transition state to favor a reaction
explain enzyme substrate interaction
- substrate + enzyme are separate
- enzyme-substrate binds
- enzyme-product (substrate becomes product)
- enzyme + product separate
explain feedback inhibition (negative regulation)
- Prescence of end product stops the pathway
- Prevents further reactions down that pathway
negative feedback - What would happen if there were no feedback regulation?
the cell would keep making enzymes it does not need and wastes energy
negative feedback - how exactly does the end product stop the pathway
- End product binds on regulatory sites
- enzyme undergoes conformational change to stop pathway
how does protein phosphorylation change the function of the protein
- phosphorylation causes a conformational change
- turns it on/off depending on protein
protein phosphorylation - explain how it works
- Kinase – adds a phosphate
- Phosphatase – removes a phosphate
explain other types of covalent protein modifications
different post-translational modifications can alter proteins
explain motor protein myosin
- myosin is an ATPase
- can walk along actin filaments during muscle contraction by hydrolyzing ATP
explain protein complexes
- they are made of individual proteins that collaborate to perform a specific task
- the function is coordinated by the hydrolysis of ATP
explain how you can gather proteins to study them
- Take out of source tissue, if it is abundant
- Producing them artificially, If not abundant but you know the gene that expresses the protein
explain homogenization
- breaking cells and tissues
- can do it in 4 ways:
1. break cells with high-frequency sound (ultrasound)
2. use a mild detergent to make holes in the plasma membrane
3. force cells through a small hole using high pressure
4. sheer cells between a close-fitting rotating plunger and the thick walls of a glass vessel
how do you isolate protein complexes
- Immobilize protein and fish out proteins that you know interacts with it
- Can then break the interaction on proteins and break off a singular specific protein
what is column chromatography
- Separate proteins based on a specific criteria
- three kinds of criteria:
1. ion exchange
2. gel-filtration
3. affinity
column chromatography - ion exchange
- Fish things out based on charge
- Beads are a positive charge
- negative charge molecules will bind to beads
- the negative molecules will slowly change pH and collect fractions
- the negative charge molecules takes longer to take out the column
- positive molecules come out first
column chromatography - gel filtration
- a porous substrate takes in proteins
- Smaller molecules get stuck in pores and come out after bigger molecules
- Big molecules move around pores and comes out first
column chromatography - affinity
- Use a bead that has an affinity for a protein to capture the protein
- needed protein binds and you wash off other proteins, allowing only that specific protein to come out
what are antibodies
- they are proteins that tightly bind to their targets (antigens)
- they have specificity
- they are used to separate and identify specific proteins
explain B cell clonal expansion
- a B cell makes an antibody and it stimulates the cell to divide
- this results in a clone of cells
- some then develop into plasma cells that secret the same antibody
- other develop into memory cells that have the antibody but do not secrete it
what are the two ways antibodies can be produced
- polyclonal
- monoclonal
production of antibodies - polyclonal
Multiple cells are activated that recognizes different parts of a protein
production of antibodies - monoclonal
One B cell and it only binds to one protein
how can you use antibodies to purify molecules
Don’t have to activate protein, antigen can activate protein and you can study the protein
Sodium-Dodecyl Sulphate Polyacrylamide gel electrophoresis (SDS-PAGE)
- Denature protein and boil so they are mineralized
- Coat them with something that gives it a negative charge
- All proteins go towards positive side of the gel
- Protein that are lower molecular weight go through faster than ones that are bigger
Western blot
- Add a membrane to a gel and apply current in different direction
- the current pulls protein out of gel and onto membrane
- proteins then come in contact with different antibodies and it amplifies the signal
- the signal indicates how much protein is there and how long the protein sticks around
