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Biochem > Test 2 Chapters 6,7 > Flashcards

Test 2 Chapters 6,7 Flashcards

1
Q

What does “salting in” do?

A

Increase in solubility of a protein with increasingly low salt concentration.

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2
Q

What is salting out

A

The decrease in solubility of a protein with increasing high salt concentrations.

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3
Q

When salting out what does adjusting pH to match pI result in?

A

Makes the protein less soluble b/c no net charge.

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4
Q

What is gel electrophoresis? And what is it used for?

A 
Migration of ions in electric field.
Uses: 
Identification: if band matches a known band.
Purify
Quantity: use a molecular weight loader
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5
Q

Types of gel electrophoresis and their uses:

A
  1. PAGE: Separates by size, shape, and charge.
  2. SDS-PAGE: Separates by charge alone.
  3. 2D gel electrophoresis: protein settles at pH corresponding to pI
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6
Q

What do the proteins travel toward in PAGE? And what is the pH?

A

Toward the anode (+); pH=9

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7
Q

What is PAGE electrophoresis also known as? And why

A

Native gel, the protein is left in native conformation and is not denatured.

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8
Q

What does the SDS in SDS-PAGE stand for?

And what is it and what does it do?

A

Sodium dodecosulfate: [CH3-(CH2)10-CH2-SO3] Na

It is an amphophilic detergent, that forms micelles with negatively charged molecules on the external surface, and denatures proteins by hydrophobic interaction.

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9
Q

When running SDS-PAGE, what are common chemicals to reduce disulfide bonds aiding to unwind the protein

A

2-mercaptoethanol (BME)

Dithiothriotol (DTT)

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10
Q

How does 2D get electrophoresis work.

A

Through a combination of SDS-PAGE and IEF (Isoelectric focusing). This gives each protein a unique position based on M.W. and pI.
The protein will settle at the pH corresponding to pI

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11
Q

Reasons for protein sequencing

A 
Structure/function relationships
Diagnostic tests: 
>(therapies for mutations) (ID them)
>Evolutionary relationships
>>sequence comparison
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12
Q

Steps of protein sequencing.

A
  1. Separate the sub-unites
  2. Cleave polypeptides
  3. Edman Degredation
  4. ID via chromatography
  5. -repeat-
  6. Determine sequence
  7. Repeat w/o reducing agent to ID cystine
  8. Add to Database
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13
Q

If you want to alkylate a thiol group what is used, and why?

A

Iodoacetate; prevents the reformation of disulfide bonds.

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14
Q

What type of molecule is used to cleave peptide bonds.

A

Endopeptidase (protease)

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15
Q

When cleaving peptide bonds what does trypsin?

A

Cleaves peptide bond between arginine and lysine, as long as proline does not follow.

Cleaves scissile bond.

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16
Q

Example of chemical cleavage?

A

Cyanogen bromide

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17
Q

What does cyanogen bromide do?

A

C-side of Met

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18
Q

What is the process for removing the N-terminus amino acid one at a time.

A
  1. Phenylisothiocyanate (PITC); AKA “Edmund’s reagent”
    »reacts to form (PTC) adduct on N-terminus
  2. Trifluoroacetic acid: cleaves the N-terminus.
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19
Q

What are the three common databases?

A

ExPASy
PIR
Uniprot

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20
Q

What does mass spectrometry measure?

A

Mass spectrometry accurately measures the mass-to-charge (m/z) ratio for ions in the gas phase.
M= ion’s mass
Z = its charge

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21
Q

What problem did electrospray ionization fix?

A

Allows for the protein to remain intact.

Intact charged macromolecules: (lysine, histamine, arginine, N-terminus (per subunit).

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22
Q

What conditions are ESI normally run under?
And
How are the samples separated?

A

Acidic and by weight and charge

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23
Q

What is a tandem mass spectrometer and what does it do?

A

It is two mass spectrometers in a coupled series.
First the polypeptide is fragmented into short chains.
Fast process and can measure post-translational modifications, but is unable to distinguish:
Ile from Leu and Gln from Cys (0.036 Da difference)

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24
Q

Where is cytochrome C (Cyt C) found and what does it do?

A

In mitochondria, and carries electrons from complex 3 to 4.

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25
Q

If a specific residue is found in the same spot in all samples what can be infered?
Example: 73 lysine

A

This means it is vital for function since if it was changed it may no longer function.

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26
Q

What does it mean if there is a lot of variability at a certain place in the code:
Example
At 89 there are constant changes.

A

This indicates it is not as vital since the variability would other ways cease it from functioning.

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27
Q

How does protein evolution occur, and what are the types?

A

A change in genes which can change the primary structure of a protein.
Types:
>Beneficial or Neutral: rare and due to natural selection or genetic drift.
>Deletions: by far the most common.

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28
Q

How do proteins reveal evolutionary relationship?

A

Sequence alignment, and in Cyt C: conserved residues.

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29
Q

What are homologous proteins?

A

Evolutionarily related proteins

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30
Q

vs % sequence identity

A

How similar two proteins are without being related.

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31
Q

Invariant residue definition?

A

Conserved, usually essential for function

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32
Q

Conservatively substituted residue definition.

A

Change residue to one with similar properties.
Asp—->Glu
Ser—–>Thr

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33
Q

Hypervariable residues definition

A

A.a. not specific to function

|&raquo_space;may be a glorified “space holder”

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34
Q

How does most protein evolution occur?

A

Gene duplication and gene segment duplication.

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35
Q

What are the differences between adult hemoglobin and fetal hemoglobin?

A

Adult: a2B2
Fetal: a2y2
Fetal has higher affinity for oxygen.

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36
Q

What does paralogous mean?

A

Two genes with different functions that evolved during gene duplication.

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37
Q

Pseudogene?

A

Gene with no new function and doesn’t retain the original function.

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38
Q

What does Othologous mean?

A

Homologous proteins generated by speciation, that often have the same function.

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39
Q

Rate of sequence divergence

A

Rate of evolution varies among proteins.
>b/c rate of successful mutations vary
>relatively lower for a particular protein.

Rate is dependent on a.a. relevance to function.
>structural stability.

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40
Q

What is a domain?

A

Evolutionarily conserved structures (40-200 a.a.)

Usually have structural similarities.

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41
Q

What is domain shuffling and related concepts?

A

Gene segments that encode for protein domains are copied and inserted into other positions in a genome to generate new genes that encode proteins.
Many proteins made up of multiple domains,
Evolution is faster with domain shuffling.

42
Q

Defining structures of primary structure?

A

A.a. sequence and disulfide bonds.

43
Q

Defining structures of secondary structure?

A

Spatial arrangement of back bone with minor considerations of side chains.
> a helix
> B sheets

44
Q

Defining structures of tertiary structure?

A

Spatial arrangement considering side chains; determines where hydrophobic and hydrophilic subunits are.

45
Q

Defining structures of quartenary structure?

A

Includes subunits of proteins that multiple.

46
Q

What is the nature of the peptide bonds.

A

Rigid planar structure.

47
Q

About 10% of Proline are?

A

CIS

48
Q

Backbone C main chain can be described by torsional angles.

A 
Cα----N  = Φ 
Cα----C = Ψ
49
Q

What do the different colors indicate on the Ramachandran Diagram.

A

Blue = sterically allowed Φ and ψ angles for all residues except Gly and Pro.

Green = indicate the more crowded (outer limit) angles.

Yellow = represent conformational angles of several secondary structures.

50
Q

Two exceptions to the Ramachandran diagram?

A

Proline: Φ angles limited to around -60

Glycine not sterically hindered.

51
Q

What are the two common secondary structure, structures?

A

α - helix

Β - sheets

52
Q

Features of an α-helix?

A

Right handed turn
3.6 residues per turn
5.4 A pitch
Hydrogen bonding between C=O (nth residue) and N-H (n + 4 residue)

53
Q

What are the defining features of B-sheets?

A 
H-binding with neighboring strand
Pleated sheets
2 main types:
>parallel (has looping effect)
>anti-parallel
R-groups within a strand alternate sides
Slight right hand twist, for each strand.
54
Q

What are the features of a reverse turn?

A 
180 degree flip back bone
Usually found on protein surface
> H-bonding
4 a.a.
> Two types
>> Type 1
>> Type 2 - contains proline
55
Q

Keratin is an example of what type of protein?

A

Fibrous

56
Q

What are the two types of keratin and what are they found in?

A

a - keratin: Hair, nails, horns, hooves, skin

B - keratin: scales, feathers, beaks

57
Q

What are the features of a - keratin?

A 
2 a-helices in a left handed coil (coiled coil) (super helix)
7 residue pseudo-repeat
1 and 4 are usually non-polar
>>roughly same as a turn
>>results in a hydrophobic strip.
58
Q

From keratin to hair.

A
  1. Two keratin polypeptides form a dimeric coild coil.
  2. Two staggered rows of head to tail associated coiled coils form a protofilament.
  3. The protofilaments dimerize to form a protofibril, four of which form a microfibril.
59
Q

Keratin has many _____ residues.

The number of which dictates if it is _____ or _____.

A

Cysteine; soft and hard.

60
Q

What is the most abundant vertebrate protein?

A

Collagen

61
Q

Where is collage found?

A

Skin, bone, teeth, blood vessels, cartilage, and tendons.

62
Q

What is the structure of collage?

A 
Triple helix, composed of 3 L-handed helices wrapped in a right handed twist.
>>Composed of:
>>> ~33% Gly
>>> 15-30% hydroxylated proline
>>> Some hydroxylated lysine
63
Q

How do collagen fibers crosslink?

A

Via covalently bonded side chains, and joint stiffness.

64
Q

What was the third type of secondary structure?

A

Non-repeating “coils”, still order but irregular.

>not a random coil since that would denote no secondary structure.

65
Q

What are the features of a tertiary structure, and how can they be visualized?

A

Folded secondary structure, each atom has an address.

Molecular graphics program can be used to visualize it.

66
Q

Equation for fractional saturation of oxygen of Mb

A

Yo2 = (pO2)/(KD)+(pO2)

When pO2 = KD, Yo2 =0.5

67
Q

What is the p50 for Hb?

A

Hb p50 = 26 torr

68
Q

What is the p50 for Mb

A

Mb p50 = 2.8

69
Q

What is the Bohr effect?

A

As you go down in pH the Hb has less affinity for oxygen.
Effects:
Enhances oxygen transfer
T–>R causes some pKs to decrease (His & Lys)

70
Q

Explain the cross link structure of collagen.

A

Gly-X-Y
>X is often proline
»this prevents alpha helix formation
>Y is often Hyp, and sometimes Hyl.
> Glycine fits easily in the center because of its little steric hinderence.
>Staggard so that cross section results in all 3

71
Q

What is x-ray crystallography?

A

fun x-ray beam and diffraction pattern.

72
Q

What are the 4 structural motifs?

A
  1. BaB
  2. B hairpin
  3. Greek key
  4. alpha-alpha
  5. Omega loop
  6. Zinc Finger
73
Q

Side chain locations:

Often found in the internal hydrophobic region of the protein

A

non-polar a.a.

74
Q

Side chain locations:

Mostly found on the polar surface of the protein

A

Charged groups, except the backbone

Turns and loops

75
Q

Side chain locations:

Where are the uncharged polar side chains?

A

Both internal and external, this decreases overall polarity of the group this is the backbone.

76
Q

Side chain locations:

What might you find at a catalytic site?

A

Histidine

77
Q

Side chain locations:

Structure of trans-membrane proteins?

A

Hydrophobic patch that allows two hydrophobic areas to interest.

78
Q

a, b, a/b protein classification:
Cyt b ?
and motif?

A

a

motif: aa

79
Q

a, b, a/b protein classification:
Immunoglobulin fragment?
and motif?

A

B

motif: Greek key and B hairpin

80
Q

a, b, a/b protein classification:

Lactate dehydrogenase

A

a/B

motif: BaB

81
Q

Protein topology:

What is a B-barrel composed of?

A

B hairpins

82
Q

Protein topology:

What is a jelly role barrel composed of?

A

Greek keys

83
Q

Protein topology:

What is a aB-barrel composed of?

A

BaB

84
Q

Quaternary structure:

Homodimer?

A

same subunits

85
Q

Quaternary structure:

Heterodimer?

A

different subunits

86
Q

Quaternary structure:

Protomer

A

Identical units of oligomers (few to 20 a.a.)

aB is a protomer

87
Q

Quaternary structure:

What is a dimer of two protomers?

A

Diprotomer

88
Q

Quaternary structure:

What interactions occur between subunits?

A

Hydrophobic, hydrogen bonds, interchain disulfide bonds.

89
Q

What is a chaotropic agent?Example of a chaotropic agent?

A

Increase solubility of non-polar groups.

Guandidium, urea

90
Q

Mechanism of protein folding?

A
  1. Begins folding as translated.
  2. Secondary structure forms due to hydrophobic collapse, and forming molten globule (beginning tertiary structure).
  3. Domains fold individually
  4. Then sub units associate.
91
Q

How are wrong disulfide bonds corrected?

A

Using the protein: Disulfide isomerase

This corrects non-native disulfide bonds by breaking and reforming the correct bonds.

92
Q

Concept of molecular chaperones?

A

Isolate folding proteins. (in vivo high protein concentration)

93
Q

Example of chaperones?

A

Heat shock proteins

94
Q

What heat shock proteins were discussed in class?

A 
HSP 70 (DNA-K)
>Cotranslational or immediately after translation
Gro EL/ES chaperonin
>induce conformational changes
HSP 90 cell signaling
95
Q

Inherited protein misfolding?

A

Amyloidoses

96
Q

Four prion diseases?

A

Mad cow–>BSE
Kuru
CJD
Scrappie

97
Q

Mb structure

A 
8 a helices
globular
Heme group
Porphyrin derivative (4 pyroles)
Fe (II) coordinated by 5 nitrogens and held in place by Val and Phe
98
Q

What role does the E7 histidine play?

A

Prevents heme oxidation Fe(II) to Fe(III)

99
Q

color of deoxy Mb

A

Purple

100
Q

Color of oxy Mb

A

Scarlet

Biochem (7 decks)

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