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BIOL 3600 > Chapter 3- Proteins Part 2 > Flashcards

Chapter 3- Proteins Part 2 Flashcards

1
Q

What is difficult but important in understanding proteins?

A

Protein purification

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2
Q

Why is protein purification difficult?

A
  1. there are tens of thousands of proteins
  2. and all are similar but different (made from 20 AA)
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3
Q

What is necessary in order to purify a protein?

A

an assay (test)

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4
Q

What is an enzyme assay?

A

measures enzyme activity in a sample

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5
Q

List an example of an enzyme assay.

A

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

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6
Q

What are the two type of separation techniques?

A

chromatography and electrophoresis

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7
Q

Why are separation techniques important?

A

they are important tools for protein purification

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8
Q

How does chromatography separate the individual proteins?

A

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.

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9
Q

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

A

We learn different characteristics of the protein as well.

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10
Q

What is electrophoresis most commonly used for?

A

characterization

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11
Q

How does electrophoresis separate molecules?

A

charge, size (due to the matrix)

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12
Q

How does SDS page separate proteins?

A

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

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13
Q

What does PAGE gel do?

A

slows down migration

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14
Q

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

A

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.

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15
Q

What is Isoelectric focusing (IEF)?

A

charge is a function of pH and is separated with this

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16
Q

How does IEF separate proteins?

A

separates based on the pI of a protein.

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17
Q

What is within the matrix of a isoelectric focusing?

A

pH gradient

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18
Q

pI stands for what? And what does that men?

A

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

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19
Q

What facilitates protein purification and characterization?

A

Genetic engineering

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20
Q

What does genetic engineering do?

A

It manipulates genetic information

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21
Q

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

A

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

22
Q

What is a good example of tagged proteins?

A

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.

23
Q

What is important in characterizing proteins?

A

Amino acid sequencing

24
Q

How are proteins sequenced now a days?

A

theoretically

25
Q

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

A

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.

26
Q

What are the important tools for studying proteins?

A

Limited proteolysis, immuno-techniques, mass spectrometry

27
Q

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

A

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.

28
Q

Proteases are what….?

A

proteins

29
Q

What is an example of immuno -techniques?

A

antibodies

29
Q

What is the general structure of an antibody?

A
  1. antigen binding site
  2. 3 domains
  3. 4 chains (2 heavy chains and 2 light chains)
  4. the chains linked by disulfide bonds
30
Q

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

A

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)

31
Q

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

A

want diversity at the ends of the molecules

32
Q

What are antibodies made from?

A

made in response to an antigen

33
Q

What are important characteristics about immuno-techniques?

A

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.

34
Q

What is a good example of immunotechniques?

A

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.

35
Q

how can you use immuno-techniques in protein purification?

A

you can make an antibody for that specific protein

36
Q

Explain mass spectrometry.

A

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

37
Q

Why do we use mass spectrometry?

A

to observe and characterize a protein in a mixture

38
Q

How is mass spectrometry different than theoretical sequencing?

A

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

39
Q

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

A

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

40
Q

What is proteomics?

A

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

41
Q

What does proteomics use?

A

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

42
Q

Peptides can be synthesized using what?

A

automated or solid state methods

43
Q

Why would you want to synthesize peptides?

A

to raise antibodies (to do immunotechniques)

44
Q

How can the tertiary structure of small proteins be determined?

A

using MultiD-NMR

45
Q

What are some limitations of NMR? And why?

A

the size of the molecule for protein

the limitation happens because a signal depends on the rate at which a protein tumbles

46
Q

How does NMR work?

A

It distinguish one nucleus from another

47
Q

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

A

140 AA small

It means that a majority of proteins can’t do NMR because the average is around 450

48
Q

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

A

X-ray crystallographic methods

you have to learn how to crystallize the protein

49
Q

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

A

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

50
Q

How does x-ray crystallography work?

A

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

51
Q

What do they use to get x-ray crystallography?

A

The Patterson function

BIOL 3600 (12 decks)

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