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BIOCH 320 > 2 - primary structure + purification > Flashcards

2 - primary structure + purification Flashcards

1
Q

whats an oligopeptide

A

synthetic, series of aa joined by peptide bonds

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2
Q

whats a polypeptide

A

long chain of aa (natural)

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3
Q

whats a conjugated protein

A

protein + prosthetic group(something that isnt protein)

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4
Q

how long are most proteins

A

100-1000

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5
Q

what ultimately determines the structure and function of polypeptides

A

primary sequence

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6
Q

what is Mr

A

relative molecular mass/molecular weight, same as Da but no units

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7
Q

what is 1 Da

A

1/12 of Carbon 12

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8
Q

what is Da of free aa

A

128

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9
Q

what is Da of residue

A

110

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10
Q

does primary sequence information include the disulfide bonding pattern

A

Yes

but they are still i think 3 structure

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11
Q

which terminus does methylation occur

A

C

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12
Q

which terminus does amidation occur

A

C

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13
Q

which terminus does acetylation occur

A

N

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14
Q

which terminus does acylation occur

A

N

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15
Q

what does methylation do to charge

A

makes it no charge

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16
Q

what does amidation do to charge

A

makes it no charge

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17
Q

what does acetylation do to charge

A

makes it no charge

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18
Q

what does acylation do to charge

A

makes it no charge

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19
Q

what is diff with acylation and acetylation

A

acylation is general (COR group added) but acetylation adds COCH3 (R=CH3)

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20
Q

what does formylation do to charge

A

makes it no charge

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21
Q

do proteins with different functions have the same sequence

A

no

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22
Q

do proteins with same functions have the same sequence

A

yes or very similar

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23
Q

what happens when you change aa sequence (to structure and function)

A

changes them

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24
Q

what are 2 things that a sequence can predict

A

structural information and cell localization

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25
Q

what is the most useful way to analyze primary sequence

A

compare with other sequences do generate similarities in structure and/or function

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26
Q

what is homology modelling

A

you can build a model if a sequence if it is close to another amino acid sequence

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27
Q

what are invariant residues

A

amino acids which do not vary at all between a set of sequences

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28
Q

what is the significance of invariant residues

A

critical in protein function

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29
Q

what are conservative substitutions

A

when one amino acid is substituted with another similar one

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30
Q

what are non-conservative substitutions

A

when one amino acid is substituted for another with dissimilar characteristics

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31
Q

what is the significance of non-conservative substitutions

A

may reflect differences in functions between two proteins or less important/critical locations in structure

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32
Q

what does it take for an amino acid replacement to be considered conservative

A

if there are at least 2 similarities (polarity, size, functional groups…)

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33
Q

what is sequence alignments

A

2 or more aligned to make the best or closest match

-largest number of invariant or conservative substitutions

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34
Q

what is % identity

A

identical positions/total positions

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35
Q

what is % similarity

A

conservative positions/total positions

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36
Q

is % similarity or % identity bigger

A

% similarity

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37
Q

what does a -# in matrices for scoring alignments mean

A

low likelihood that you would find that aa swapped out in another sequence

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38
Q

what does a +# in matrices for scoring alignments mean

A

high likelihood to be swapped out for in another sequence

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39
Q

what is a “gap” in protein structure

A

2 sections of aa sequence that may align well but have different lenghts

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40
Q

what causes gaps in protein structure

A

insertions or deletions at the genetic level

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41
Q

what do gaps correlate to in the actual protein structure

A

loops on the surface, or the ends that change (C or N terminus)

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42
Q

what happens with a mutation in a loop vs middle of helix

A

loop- likely ok, keep function

helix-bad

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43
Q

what happens if the amino acid mutation is non-functional

A

it may be detrimental and not retained

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44
Q

what happens if the amino acid mutation is conservative

A

neutral so the change may be retained

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45
Q

are all aa positions equally sensitive to change

A

no

some more than others

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46
Q

what does the degree in sequence between polypeptide correspond to

A

length of time since divergence

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47
Q

what are homologs

A

two proteins which share a common ancestor

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48
Q

what are “any two proteins which arose out of a single gene”

A

homologs

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49
Q

what are 2 ways that homologs can arise

A

duplication or speciation events

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50
Q

what are orthologs

A

homologs (two proteins which share a common ancestor) which arise out of speciation events

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51
Q

what are paralogs

A

homologs (two proteins which share a common ancestor) which arise out of gene duplication events

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52
Q

how do orthologs compare in different organisms (function wise)

A

carry out the SAME function (speciation)

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53
Q

how do paralogs compare in different organisms (function wise)

A

may have the same or different functions (gene duplication)

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54
Q

are beta-globin and alpha-globin para or ortho logs

A

paralogs (gene duplication)

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55
Q

is human beta-globin vs mouse beta-globin para or ortho logs

A

orthologs (speciation)

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56
Q

do you use orthologs or paralogs to make evolutionary trees

A

orthologs

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57
Q

how can you purify proteins based on solubility

A

salting out

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58
Q

how can you purify proteins based on ionic charge

A
  • ion exchange chromatography
  • electrophoresis
  • isoelectric focusing
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59
Q

how can you purify proteins based on polarity

A

hydrophobic interaction chromatography

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60
Q

how can you purify proteins based on size

A
  • gel filtration/ size exclusion chromatography
  • SDS PAGE
  • dialysis
61
Q

how can you purify proteins based on binding specificity

A

affinity chromatography

62
Q

what do you want to minimize during purification

A

denaturation and degradation

63
Q

what are the 3 most important + 2 other things to control in protein purification

A

pH (use buffers)
temperatures (slow cold)
enzymes (inhibit protease maybe)

then adsorption (how protein sticks to glass, plastics)
long term stability (oxidation, denaturation)
64
Q

how do you directly monitor concentrations of protein of interest vs. other compounds

A

enzyme activity! and spectrophotometry (if protein is associated with specific profile, like color)

65
Q

how do you indirectly monitor concentrations of protein of interest vs. other compounds

A

antibody-linked/immunoassays (use if protein is antigen for specific antibody)

66
Q

definition of activity

A

ability to convert substrate into produce, usually indicated in units

67
Q

what does 1 unit=

A

1 μmol substrate converted per min

68
Q

what is specific activity definition

A

activity as a fraction of total protein

69
Q

what is specific activity formula

A

units/mg total protein

70
Q

what happens to specific activity as time goes on in purification

A

RISE (less contaminants)

71
Q

what happens to activity as time goes on in purification

A

LOW (less % recovery)

72
Q

when is spectroscopy good

A

specific absorption in some proteins (prosthetic groups, hemoglobin, cytochrome)

73
Q

when are immunoassays good

A

non-enzymatic proteins or ones with no appropriate enzyme assay

74
Q

when can you use immunoassays

A

if there are appropriate antibodies available

75
Q

how does centrifugation work / what it do

A

fractionation of compounds based on mass/sedimentation rate

76
Q

what is differential centrifugation / how does it separate

A

separates compounds by sedimentation rate

77
Q

what kind of compounds sediment fastest in differential centrifugation

A

large proteins sediment faster than smaller ones (experience less friction and SA or something)

78
Q

what is density/isopycnic centrifugation

A

separates compounds by density

79
Q

what kind of process is density/isopycnic centrifugation

A

equilibrium process - density gradient in sample tube determines final location/position

80
Q

how does density/isopycnic centrifugation separate/work

A

density gradient in sample tube determines final location/position

81
Q

does time matter is differential centrifugation

A

ya think so

you can spin too much i think

82
Q

does time matter is density/isopycnic centrifugation

A

no, once it hits equilibrium then it will always stay there

83
Q

what is solubility/ precipitation in protein purification

A

some proteins solubilize/precipitate under different salt concentrations and pH

84
Q

what causes salting in/salting out

A

some proteins solubilize/precipitate under different salt concentrations and pH

85
Q

what is a common salt used in solubility/ precipitation in protein purification

A

ammonium sulphate (NH4)2SO4

86
Q

what is often combined with solubility/ precipitation in protein purification

A

centrifugation

87
Q

what kind of technique is dialysis

A

size based

88
Q

how do you use dialysis / why

A

to remove small molecules from preparations (anything smaller than the pores in the membrane will go through the membrane and leave the bag)

89
Q

what does column chromatography allow for

A

differential separation of compounds based on physical characteristics

90
Q

what does rate of travel through a column depend on

A

relative affinity for the solid/stationary or liquid/mobile phase

91
Q

what are some types of stationary phase

A

gel, beads, resin, polymer

92
Q

what happens to migration rate with affinity to the solid phase

A

slows migration rate

93
Q

what happens to migration rate with affinity to the liquid phase

A

increases migration rate

94
Q

what is the mobile/liquid phase

A

the fluid that constantly flows from one end of column to the other

95
Q

when do you use cation exchange

A

when you have a positively charged protein that you want to purify

96
Q

when do you use anion exchange

A

when you have a negatively charged protein that you want to purify

97
Q

what is the setup in cation exchange

A

stationary phase is negative, more positive are attracted to beads (elute slowly)

98
Q

what is the setup in anion exchange

A

stationary phase is positive, more negative are attracted to beads (elute slowly)

99
Q

how do you increase more release from the ion exchange column

A

increase salt concentration to elute things that bind weak, the more salt to elute medium binding, etc

100
Q

what does the charge in the ion-exchange chromatography depend on

A

protein pI and pH of buffer

101
Q

what pH do you use to elute in cation exchanger

A

0.5-1.5pH below the pI of interest (want it to be positive)

102
Q

what pH do you use to elute in anion exchanger

A

0.5-1.5pH above the pI of interest (want it to be negative)

103
Q

why dont you want a big gap in pH and pI in cation or anion exchange

A

too big gap = denature (too charged)

104
Q

what is another name for size-exclusion chromatography

A

gel filtration

105
Q

what is another name for gel filtration chromatography

A

size-exclusion

106
Q

what is the stationary phase in size-exclusion

A

porous beads with small openings

107
Q

what moves fastest gel filtration chromatography (size exclusion)

A

mobile phase moves faster around the beads than through them

108
Q

does size-exclusion chromatography/gel filtration denature

A

no, doesn’t wreck 4 structure

109
Q

when do you use affinity chromatography

A

when you have a specific protein of interesting binding a specific ligand

110
Q

what will bind to the stationary phase in affinity chromatography

A

only proteins that bind to a specific ligand (ligand is covalently bound to stationary phase)

111
Q

what happens once you add the ligand in solution in affinity chromatography

A

it will allows the protein to elute from the column (bound to free ligand instead of column)

112
Q

how can you engineer a protein to help it stick in affinity chromatography

A

things like His tags or GST fusions

113
Q

are engineered proteins for affinity chromatography supposed to change original structure and function

A

ideally not

114
Q

what are his tags

A

6-14 His inserted into polypeptides usually a N or C terminus

115
Q

why do you use His tags

A

to increase protein solubility or help to bind to columns (nickel)

116
Q

what kind of column is good for his tagged proteins

A

nickel

117
Q

how does His bind to Nickel

A

coordination bond from imidazole ring to metal ion

118
Q

how can you elute things from column with his tags

A

changing pH or adding free imidazole

weakens protein/column activity

119
Q

how do you wanna change pH when using his tags and nickel

A

lower pH so it will be protonated and cant coordinate

120
Q

why do you add free imidazole when using his tags and nickel

A

outcompetes with his in protein so it leaves the column

121
Q

desalting columns remove salt and other small molecules from protein solutions. what kind of chromatography is it

A

size exclusion bc small

122
Q

what does electrophoresis do

A

separates proteins based on charge/mass ratio and size

123
Q

what do you do to proteins before electrophoresis

A

add SDS DTT and then heat

124
Q

what is and does SDS do

A

anionic detergent which denatures proteins and makes a consistent charge to mass ratio

125
Q

what is and does DTT do

A

reducing agent to disulphides, allows for more complete denaturation

126
Q

how long do you hear proteins before electrophoresis and how hot

why

A

95 C for 5 mins

for complete denaturation

127
Q

how does SDS bind to aa and how often

A

1SDS binds for every 2 residues

128
Q

why is SDS good to measure molecular weight of proteins

A

since it gives them all a consistent charge to mass ratio and denatures, it separates them based on mass (small migrate quickest)

129
Q

what is added after electrophoresis

A

coomassie blue

130
Q

where does coomassie blue bind

A

to proteins in the gel so that each polypeptide can be seen as a descrete band

131
Q

do what point are proteins denatured in SDS PAGE (primary secondary tri tert which one)

A

primary and some secondary

132
Q

how do you graph the molecular weight estimation via SDS PAGE (what are axes)

A

Log Mr vs migration distance - it will be linear

133
Q

using SDS PAGE, calculated Mr (based on migration) is 10% lower than actual Mr why

A

smaller # means it moves faster

so the protein may be more negative than expected

134
Q

what is western blotting

A

visualization of proteins based on interactions with antibodies

135
Q

is western blotting sensitive or specific

A

very

136
Q

when is western blotting performed and why

A

after SDS PAGE to locate proteins of interest amongst other present proteins

137
Q

what is the western block TRANSFER process

A

run current perpendicular with - cathode above gel and positive anode undergel and membrane under gel (top to bottom:-cathode , filter paper, gel, membrane, filter paper, +anode)

138
Q

what attaches to the protein in western blot

A

a primary antibody to specific antigen on protein

139
Q

what attaches to the primary antibody in western blot

A

secondary antibody with a detectible portion (like enzyme to catalyze a visible reaction-color or luminescence)

140
Q

what is added to the surface of the membrane in western blotting

A

blocking agent to block unoccupied sites on membrane (so like skim milk powder)

141
Q

what is the intensity of the western blot band used for

A

quantifying concentration/amount of protein present

142
Q

how does isoelectric focusing work

A

gel has pH gradient, samples migrate in a direction determined by pI/charge

143
Q

what end is the positive and negative cathod anode put in isoelectric focusing

A

positive cathode at top because anything more negative will run up (low pH on top)

144
Q

is low pH on top or bottom in isoelectric focusing

A

top

145
Q

is isoelectric focusing based on time

A

no, its impossible to run for too long (equilibrium based technique)

146
Q

what are 2 equilibrium based technique seen in protein purification

A

isoelectric focusing

density/isopycnic centrifugation

147
Q

what happens in 2D electrophoresis (axis)

A

isoelectrical focusing (pI) vs SDS PAGE (molecular weight)

148
Q

what should happen to the amount of bands as you purify

A

there should be less and less

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