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CHEM 472 Biochem > Exam 2: Chp 5 > Flashcards

Exam 2: Chp 5 Flashcards

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1
Q

Describe the structure and function of fibrous proteins?

A

simple, linear structure, and insoluble in water; structural role

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2
Q

Describe the structure of globular proteins?

A

Spherical, hydrophobic amino acids inside, hydrophilic amino acids outside

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3
Q

Describe the structure and location of membrane proteins?

A

fewer hydrophilic amino acids, hydrophilic amino acids on outside of protein, and insoluble in water; embedded in a cell membrane

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4
Q

What is a protein primary structure?

A

the amino acid sequence

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5
Q

What is a protein secondary structure?

A

3D structures localized on part of the polypeptide chain; typically regular and repeating

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6
Q

What is a protein tertiary structure?

A

the whole 3D structure of one polypeptide chain formed via folding

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7
Q

What is a protein quaternary structure?

A

Arrangement of multiple folded polypeptide chains into one larger protein

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8
Q

What are three types of secondary protein structures?

A

⍺-helix
β-sheet
β-turn

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9
Q

What kind of interactions facilitate secondary, tertiary, and quaternary structure formation?

A

Noncovalent bonds

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10
Q

What kind of interactions facilitate primary structure formation?

A

Covalent bonds

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11
Q

What are three types of crude protein isolation/purification?

A

Centrifugation, salting out, or dialysis

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12
Q

How does centrifugation isolate/purify proteins?

A

Separates them based on density

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13
Q

How does salting out isolate/purify proteins?

A

Increasing salt concentration causes the salt to solvate solvate water ions, decreasing solubility of the protein

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14
Q

What are some limits of protein isolation/purification via salting out?

A

It only works to a certain point and its not efficient for separating a protein from a protein

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15
Q

How does dialysis isolate/purify proteins?

A

Diffusion across a semipermeable membrane separates proteins from small molecules

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16
Q

What is the definition of chromatography?

A

Solution of compounds is passed through a stationary phase

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17
Q

How does ion exchange chromatography purify/isolate proteins?

A

Separates proteins based on charge using a matrix with positively and negatively charged groups

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18
Q

How does size exclusion chromatography isolate/purify proteins?

A

Small proteins are slowed because they must travel through the inside of porous beads in a matrix; the large proteins travel between beads.

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19
Q

How does hydrophobic interaction chromatography isolate/purify proteins?

A

A matrix with hydrophobic groups that interact with non polar proteins, separating them from polar proteins

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20
Q

How does affinity chromatography isolate/purify proteins?

A

Specific ligand is bound to the protein of interest then covalently coupled to a matrix, all other compounds are washed away

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21
Q

What is high-performance liquid chromatography?

A

Any chromatography method with high molecular interactions made small scale, packed tightly, and ran with high pressure to push the protein through

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22
Q

What is a benefit of high-performance liquid chromatography?

A

produces a high resolution (good separation)

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23
Q

What are limits of high-performance liquid chromatography (HPLC)?

A

size and pressure

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24
Q

How does gel electrophoresis purify/isolate proteins?

A

An electric field propels proteins through an agarose or polyacrylamide gel that separates them based on size

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25
Q

How are proteins purified/isolated during SDS-PAGE electrophoresis

A

Based on size

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26
Q

What does SDS-PAGE stand for?

A

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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27
Q

How does isoelectric focusing electrophoresis isolate/purify proteins

A

Proteins stop moving through pH gradient when their net charge reaches zero based on their relative amounts of acidic or basic groups

28
Q

How does 2D electrophoresis isolate/purify proteins?

A

First isoelectric focusing is performed, then the gel matrix is rotated 90° and SDS-PAGE is performed

29
Q

What is a purification scheme?

A

Several protein purification/isolation methods in sequence

30
Q

What is the goal of a purification scheme?

A

to maximize specific activity

31
Q

What is specific activity?

A

The ration of a protein of interest to total protein

32
Q

What is of sacrifice of purification schemes?

A

material is lost during each step

33
Q

What is acid hydrolysis?

A

The process of decomposing a polypeptide into individual amino acids using 6 M HCl and 110°C

34
Q

What is the first step in determining primary structure (amino acid sequence) of a protein? (Step 1)

A

If quaternary structure exists then polypeptide chains should be separated and purified

35
Q

How do you separate and purify polypeptide chains?

A

Disrupt their non-covalent interactions using pH, high salt, urea, or gaunidium chloride and isolate via chromatography

36
Q

When determining protein primary structure; what is done after polypeptide chains are purified and separated? (Step 2)

A

Disulfide bonds are cleaved

37
Q

What are two ways to cleave disulfide bonds?

A

1) Oxidation using performic acid
2) Reduction using 2-mercaptoethanol or dithrothrietol

38
Q

When determining protein primary structure; what is done after disulfide bonds are cleaved? (Step 3)

A

Determine the N-terminal amino acid and the C-terminal amino acid

39
Q

How is the N-terminal of a polypeptide chain determined?

A

Cleavage using an Edman reagent and identification of the PTH derivative

40
Q

How is the C-terminal of a polypeptide chain determined?

A

Cleavage using carboxypeptidase

41
Q

Why can carboxypeptidase or the Edman reagent not be used to determine long amino acid sequences?

A

Limited to 20-50 Ads because lost product builds up and muddles the reaction

42
Q

When determining protein primary structure; what is done after the N-term and C-term AAs are determined? (Step 3)

A

The polypeptide is cleaved into smaller fragments and their sequences are determined using Edman or carboxypeptidase reactions

43
Q

What are two ways polypeptides are cleaved into smaller fragments when determines AA sequences?

A

1) Enzymatically using a protease
2) Chemically

44
Q

What proteases are used to enzymatically cleave a polypeptide during determination of primary structure?

A

Trypsin and Chymotrypsin

45
Q

What end of the peptide bond is susceptible to cleavage by chymotrypsin? What amino acids?

A

C; Phe, Trp, and Tyr (rarely Leu)

46
Q

What end of the peptide bond is susceptible to cleavage by trypsin? What amino acids?

A

C; Arg and Lys

47
Q

What end of the peptide bond is susceptible to cleavage by staphylococcal protease? What amino acids?

A

C; Asp and Glu

48
Q

What end of the peptide bond is susceptible to cleavage by cyanogen bromide? What amino acids?

A

C; Met

49
Q

What is used to chemically cleave a polypeptide chain during primary structure determination? What does it do?

A

cyanobromide; cleaves the C-terminal side of Met and turns it into homoserine lactone

50
Q

During primary structure determination; what is done after the initial cleavage and sequencing of a primary structure?

A

The polypeptide sequence is once again cleaved with a different method and the sequences are determined

51
Q

During primary structure determination; what is done once the polypeptide sequence is cleaved and sequenced in too different ways?

A

The full sequence is constructed using overlapping fragments of the sequence fragments

52
Q

What are homologous proteins?

A

Proteins with similar sequences found in different species that typically indicate evolutionary relatedness

53
Q

What are post translational modifications? an example?

A

Covalent modifications after proteins synthesis that typically modify AA side chains; disulfide bonds

54
Q

Which terminal does the peptide bond form on the amino acid containing the insoluble resin?

A

The N-terminal on the nucleophilic amine group

55
Q

Which terminal does the peptide bond form on the amino acid containing the FMOC protecting group?

A

The activated C-terminal

56
Q

What is used to activate the carboxyl of an amino acid that is adding to the N-terminal of another amino acid?

A

DIPCDI (diisopropylcarbodimide)

57
Q

What is used to protect the amine group of an amino acid that is adding to the N-terminal of another amino acid?

A

FMOC-Cl (flourenylmethyloxylcarbonyl-Cl)

58
Q

How is FMOC cleaved once a polypeptide is made?

A

Using weak organic base because it is base labile - piperidine

59
Q

How is the insoluble residue removed once a polypeptide is made?

A

An acid, such as HF, because it is acid labile

60
Q

How is a polypeptide purified from byproducts once it is made?

A

Filtered out because the byproducts are soluble and the insoluble resin keeps the polypeptide seperate

61
Q

What is fp in terms of ligand and protein kinetics?

A

the fraction of protein that exists bound to ligand

62
Q

What is a saturation curve?

A

ligand concentration on x-axis and fp on y axis

63
Q

What is Kd in terms of ligand and protein kinetics?

A

A measure of binding affinity recording the concentration of ligand where 1/2 of protein has ligand bound

64
Q

What does a smaller Kd mean?

A

the protein and ligand have a higher binding affinity

65
Q

Function of 2-mercaptoethanol?

A

To cleave disulfide bonds

CHEM 472 Biochem (12 decks)

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