L 7. Protein Structure & Function II

Question 1

explain protein families

Answer 1

• 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

Question 2

define ligand

Answer 2

any substance that is bound to a protein

Question 3

how to enzymes regulate metabolic pathways

Answer 3

one enzyme can catalyze a molecule and another enzyme will catalyze the same molecule and so on

Question 4

how do enzymes catalyze reactions

Answer 4

• it lowers the activation energy
• so it requires less energy for a reactant to turn into a product

Question 5

how do ligands bond to proteins

Answer 5

• they have to physically interact
• the ligand binds through noncovalent bonds
• the enzyme will fold and create a pocket for substrate to bind

Question 6

ligand-protein binding - what happens at the pocket of the enzyme

Answer 6

• 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

Question 7

structure of a protein - binding sites

Answer 7

bind and orient substrate/ligand

Question 8

structure of a protein - catalytic site

Answer 8

reduces chemical activation energy

Question 9

what are the ways a protein and ligand can bind

Answer 9

• Orientation/proximity
• Rearrangement of e-
• Strain

Question 10

protein-ligand binding - orientation/proximity

Answer 10

• enzyme binds to two substrate molecules
• orientates them precisely to encourage a reaction

Question 11

protein-ligand binding - rearrangement of e-

Answer 11

• binding of substrate to enzyme rearranges electrons in the substrate
• creates partial negative charge and positive charges that favor a reaction

Question 12

protein-ligand binding - strain

Answer 12

• enzyme strains the bound substrate molecule
• forcing it towards a transition state to favor a reaction

Question 13

explain enzyme substrate interaction

Answer 13

1. substrate + enzyme are separate
2. enzyme-substrate binds
3. enzyme-product (substrate becomes product)
4. enzyme + product separate

Question 14

explain feedback inhibition (negative regulation)

Answer 14

• Prescence of end product stops the pathway
• Prevents further reactions down that pathway

Question 15

negative feedback - What would happen if there were no feedback regulation?

Answer 15

the cell would keep making enzymes it does not need and wastes energy

Question 16

negative feedback - how exactly does the end product stop the pathway

Answer 16

• End product binds on regulatory sites
• enzyme undergoes conformational change to stop pathway

Question 17

how does protein phosphorylation change the function of the protein

Answer 17

• phosphorylation causes a conformational change
• turns it on/off depending on protein

Question 18

protein phosphorylation - explain how it works

Answer 18

• Kinase – adds a phosphate
• Phosphatase – removes a phosphate

Question 19

explain other types of covalent protein modifications

Answer 19

different post-translational modifications can alter proteins

Question 20

explain motor protein myosin

Answer 20

• myosin is an ATPase
• can walk along actin filaments during muscle contraction by hydrolyzing ATP

Question 21

explain protein complexes

Answer 21

• they are made of individual proteins that collaborate to perform a specific task
• the function is coordinated by the hydrolysis of ATP

Question 22

explain how you can gather proteins to study them

Answer 22

1. Take out of source tissue, if it is abundant
2. Producing them artificially, If not abundant but you know the gene that expresses the protein

Question 23

explain homogenization

Answer 23

• 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

Question 24

how do you isolate protein complexes

Answer 24

• 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

Question 25

what is column chromatography

Answer 25

- Separate proteins based on a specific criteria - three kinds of criteria: 1. ion exchange 2. gel-filtration 3. affinity

Question 26

column chromatography - ion exchange

Answer 26

- 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

Question 27

column chromatography - gel filtration

Answer 27

- 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

Question 28

column chromatography - affinity

Answer 28

- 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

Question 29

what are antibodies

Answer 29

- they are proteins that tightly bind to their targets (antigens) - they have specificity - they are used to separate and identify specific proteins

Question 30

explain B cell clonal expansion

Answer 30

- 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

Question 31

what are the two ways antibodies can be produced

Answer 31

1. polyclonal 2. monoclonal

Question 32

production of antibodies - polyclonal

Answer 32

Multiple cells are activated that recognizes different parts of a protein

Question 33

production of antibodies - monoclonal

Answer 33

One B cell and it only binds to one protein

Question 34

how can you use antibodies to purify molecules

Answer 34

Don’t have to activate protein, antigen can activate protein and you can study the protein

Question 35

*Sodium-Dodecyl Sulphate* Polyacrylamide gel electrophoresis (SDS-PAGE)

Answer 35

- 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

Question 36

Western blot

Answer 36

- 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