Chapter 3- Proteins Part 2

Question 1

What is difficult but important in understanding proteins?

Answer 1

Protein purification

Question 2

Why is protein purification difficult?

Answer 2

1. there are tens of thousands of proteins
2. and all are similar but different (made from 20 AA)

Question 3

What is necessary in order to purify a protein?

Answer 3

an assay (test)

Question 4

What is an enzyme assay?

Answer 4

measures enzyme activity in a sample

Question 5

List an example of an enzyme assay.

Answer 5

When an experiment turns yellow we know a specific enzyme is present

Question 6

What are the two type of separation techniques?

Answer 6

chromatography and electrophoresis

Question 7

Why are separation techniques important?

Answer 7

they are important tools for protein purification

Question 8

How does chromatography separate the individual proteins?

Answer 8

We can make the stationary phase of anything that will interact with proteins differentially. We can make the stationary phase out of a + charged moiety. And then we can separate based on how negatively charged something is. - charged moiety or we can do a hydrophobic column.

Question 9

During separation techniques what additionally do we learn about the protein?

Answer 9

We learn different characteristics of the protein as well.

Question 10

What is electrophoresis most commonly used for?

Answer 10

characterization

Question 11

How does electrophoresis separate molecules?

Answer 11

charge, size (due to the matrix)

Question 12

How does SDS page separate proteins?

Answer 12

takes detergent that is negatively charged and separates by their size or molecular mass.

Question 13

What does PAGE gel do?

Answer 13

slows down migration

Question 14

How does SDS page separate via size if the field is charged?

Answer 14

because it is a uniform negative charge it binds to proteins uniformly so it is exclusively separating by molecular weight or size. The smaller molecules run through the gel faster and the larger ones are slower.

Question 15

What is Isoelectric focusing (IEF)?

Answer 15

charge is a function of pH and is separated with this

Question 16

How does IEF separate proteins?

Answer 16

separates based on the pI of a protein.

Question 17

What is within the matrix of a isoelectric focusing?

Answer 17

pH gradient

Question 18

pI stands for what? And what does that men?

Answer 18

isoelectric point, it is the pH which the net charge is “0”

Question 19

What facilitates protein purification and characterization?

Answer 19

Genetic engineering

Question 20

What does genetic engineering do?

Answer 20

It manipulates genetic information

Question 21

What do tagged proteins do? What does the tag have?

Answer 21

they allow us to take a region of DNA and add a fluorescent green tag so that we can characterize the protein (ex. where is it going what is it doing)

the tag has high affinity

Question 22

What is a good example of tagged proteins?

Answer 22

6 Histidine proteins and a Nickle attached to it. Can purify from everything else because this specific example is one of the only ways that Nickle can attach to.

Question 23

What is important in characterizing proteins?

Answer 23

Amino acid sequencing

Question 24

How are proteins sequenced now a days?

Answer 24

theoretically

Question 25

What is theoretical sequencing? Why is it easier to do? And where do you translate DNA?

Answer 25

When DNA is encoded first and then translated to an AA sequence. Because DNA is easier to sequence than protein. DNA is translate in silico.

Question 26

What are the important tools for studying proteins?

Answer 26

Limited proteolysis, immuno-techniques, mass spectrometry

Question 27

What is proteolysis? What does the limited part in proteolysis mean?

Answer 27

Breaks peptides bonds or cutting proteins of molecules. The high specificity proteases only cut certain peptide bonds making it limited. If it wasn't so specific we would not be able to find out what sequence it is.

Question 28

Proteases are what....?

Answer 28

proteins

Question 29

What is an example of immuno -techniques?

Answer 29

antibodies

Question 29

What is the general structure of an antibody?

Answer 29

1. antigen binding site 2. 3 domains 3. 4 chains (2 heavy chains and 2 light chains) 4. the chains linked by disulfide bonds

Question 30

What are some general characteristics of an antibody regarding the chains?

Answer 30

The heavy and light chains come together to form Fab domains, which have the antigen-binding sites at the ends. The two heavy chains form the Fc domain. (constant) They have a Y or T shape because they are flexible. has quaternary structure (because it has 4 tertiary structures)

Question 31

What is unique about the antigen binding sites located where they are?

Answer 31

want diversity at the ends of the molecules

Question 32

What are antibodies made from?

Answer 32

made in response to an antigen

Question 33

What are important characteristics about immuno-techniques?

Answer 33

The structure is specific so that the binding can be specific to certain antigens. have a tag that sticks very specifically they have perfect molecular recognition or specific molecular matching.

Question 34

What is a good example of immunotechniques?

Answer 34

To obtain antibodies that recognize a particular protein, a biochemist injects the protein into a rabbit twice, 3 weeks apart. The injected protein acts as an antigen, stimulating the reproduction of cells producing antibodies that recognize it. Blood is drawn from the immunized rabbit several weeks later and centrifuged to separate blood cells from the supernatant, or serum. The serum, called an antiserum, contains antibodies to all antigens to which the rabbit has been exposed. Only some of them will be antibodies to the injected protein.

Question 35

how can you use immuno-techniques in protein purification?

Answer 35

you can make an antibody for that specific protein

Question 36

Explain mass spectrometry.

Answer 36

it measures the inertia of the ions from lightest ion to heaviest. by moving the ions with a laser.

Question 37

Why do we use mass spectrometry?

Answer 37

to observe and characterize a protein in a mixture

Question 38

How is mass spectrometry different than theoretical sequencing?

Answer 38

detects ions on the basis of their mass-to-charge ratio

Question 39

Why does the mass for a protein reflected on mass spectrometry different than its theoretical sequencing mass?

Answer 39

Due to Post Translational Modification. If something is phosphorylated its going to weigh 80 Daltons more than its supposed mass.

Question 40

What is proteomics?

Answer 40

the discovery based study of proteins (the totality of proteins)

Question 41

What does proteomics use?

Answer 41

sequence databases (DNA) separation techniques (make mixtures simpler) mass spectrometry bioinformatics software

Question 42

Peptides can be synthesized using what?

Answer 42

automated or solid state methods

Question 43

Why would you want to synthesize peptides?

Answer 43

to raise antibodies (to do immunotechniques)

Question 44

How can the tertiary structure of small proteins be determined?

Answer 44

using MultiD-NMR

Question 45

What are some limitations of NMR? And why?

Answer 45

the size of the molecule for protein the limitation happens because a signal depends on the rate at which a protein tumbles

Question 46

How does NMR work?

Answer 46

It distinguish one nucleus from another

Question 47

What is the maximum amount of AA that NMR is useful for? And what does this mean?

Answer 47

140 AA small It means that a majority of proteins can't do NMR because the average is around 450

Question 48

Protein tertiary and quaternary structure can be determining using what? What are the limitations?

Answer 48

X-ray crystallographic methods you have to learn how to crystallize the protein

Question 49

What is to be said regarding the ease of crystallizing a protein?

Answer 49

the more flexible they are the less able we are to crystalize the protein

Question 50

How does x-ray crystallography work?

Answer 50

you have to focus it to defract a pattern to find the electron density. At the end the crystal of a molecule is lined up in regular patterns

Question 51

What do they use to get x-ray crystallography?

Answer 51

The Patterson function