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CHEM 472 Biochem > Exam 2: Chp 5 > Flashcards

Exam 2: Chp 5 Flashcards

(65 cards)

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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
How are proteins purified/isolated during SDS-PAGE electrophoresis
Based on size
26
What does SDS-PAGE stand for?
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
27
How does isoelectric focusing electrophoresis isolate/purify proteins
Proteins stop moving through pH gradient when their net charge reaches zero based on their relative amounts of acidic or basic groups
28
How does 2D electrophoresis isolate/purify proteins?
First isoelectric focusing is performed, then the gel matrix is rotated 90° and SDS-PAGE is performed
29
What is a purification scheme?
Several protein purification/isolation methods in sequence
30
What is the goal of a purification scheme?
to maximize specific activity
31
What is specific activity?
The ration of a protein of interest to total protein
32
What is of sacrifice of purification schemes?
material is lost during each step
33
What is acid hydrolysis?
The process of decomposing a polypeptide into individual amino acids using 6 M HCl and 110°C
34
What is the first step in determining primary structure (amino acid sequence) of a protein? (Step 1)
If quaternary structure exists then polypeptide chains should be separated and purified
35
How do you separate and purify polypeptide chains?
Disrupt their non-covalent interactions using pH, high salt, urea, or gaunidium chloride and isolate via chromatography
36
When determining protein primary structure; what is done after polypeptide chains are purified and separated? (Step 2)
Disulfide bonds are cleaved
37
What are two ways to cleave disulfide bonds?
1) Oxidation using performic acid 2) Reduction using 2-mercaptoethanol or dithrothrietol
38
When determining protein primary structure; what is done after disulfide bonds are cleaved? (Step 3)
Determine the N-terminal amino acid and the C-terminal amino acid
39
How is the N-terminal of a polypeptide chain determined?
Cleavage using an Edman reagent and identification of the PTH derivative
40
How is the C-terminal of a polypeptide chain determined?
Cleavage using carboxypeptidase
41
Why can carboxypeptidase or the Edman reagent not be used to determine long amino acid sequences?
Limited to 20-50 Ads because lost product builds up and muddles the reaction
42
When determining protein primary structure; what is done after the N-term and C-term AAs are determined? (Step 3)
The polypeptide is cleaved into smaller fragments and their sequences are determined using Edman or carboxypeptidase reactions
43
What are two ways polypeptides are cleaved into smaller fragments when determines AA sequences?
1) Enzymatically using a protease 2) Chemically
44
What proteases are used to enzymatically cleave a polypeptide during determination of primary structure?
Trypsin and Chymotrypsin
45
What end of the peptide bond is susceptible to cleavage by chymotrypsin? What amino acids?
C; Phe, Trp, and Tyr (rarely Leu)
46
What end of the peptide bond is susceptible to cleavage by trypsin? What amino acids?
C; Arg and Lys
47
What end of the peptide bond is susceptible to cleavage by staphylococcal protease? What amino acids?
C; Asp and Glu
48
What end of the peptide bond is susceptible to cleavage by cyanogen bromide? What amino acids?
C; Met
49
What is used to chemically cleave a polypeptide chain during primary structure determination? What does it do?
cyanobromide; cleaves the C-terminal side of Met and turns it into homoserine lactone
50
During primary structure determination; what is done after the initial cleavage and sequencing of a primary structure?
The polypeptide sequence is once again cleaved with a different method and the sequences are determined
51
During primary structure determination; what is done once the polypeptide sequence is cleaved and sequenced in too different ways?
The full sequence is constructed using overlapping fragments of the sequence fragments
52
What are homologous proteins?
Proteins with similar sequences found in different species that typically indicate evolutionary relatedness
53
What are post translational modifications? an example?
Covalent modifications after proteins synthesis that typically modify AA side chains; disulfide bonds
54
Which terminal does the peptide bond form on the amino acid containing the insoluble resin?
The N-terminal on the nucleophilic amine group
55
Which terminal does the peptide bond form on the amino acid containing the FMOC protecting group?
The activated C-terminal
56
What is used to activate the carboxyl of an amino acid that is adding to the N-terminal of another amino acid?
DIPCDI (diisopropylcarbodimide)
57
What is used to protect the amine group of an amino acid that is adding to the N-terminal of another amino acid?
FMOC-Cl (flourenylmethyloxylcarbonyl-Cl)
58
How is FMOC cleaved once a polypeptide is made?
Using weak organic base because it is base labile - piperidine
59
How is the insoluble residue removed once a polypeptide is made?
An acid, such as HF, because it is acid labile
60
How is a polypeptide purified from byproducts once it is made?
Filtered out because the byproducts are soluble and the insoluble resin keeps the polypeptide seperate
61
What is fp in terms of ligand and protein kinetics?
the fraction of protein that exists bound to ligand
62
What is a saturation curve?
ligand concentration on x-axis and fp on y axis
63
What is Kd in terms of ligand and protein kinetics?
A measure of binding affinity recording the concentration of ligand where 1/2 of protein has ligand bound
64
What does a smaller Kd mean?
the protein and ligand have a higher binding affinity
65
Function of 2-mercaptoethanol?
To cleave disulfide bonds

CHEM 472 Biochem (12 decks)

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