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Proteins > Proteins Exam 1 > Flashcards

Proteins Exam 1 Flashcards

1
Q

silent mutation

A

no change in AA sequence

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2
Q

non-sense mutation

A

causes truncated protein by creation of stop codon

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3
Q

missense mutation

A

causes a change in AA sequence

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4
Q

frameshift mutation

A

causes all AA after frameshift to be mutated
insertion
deletion

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5
Q

Fred Sanger

A

1918-2013
inventor of sanger protein sequencing method

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6
Q

N - alpha C

A

phi bond 111degrees

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7
Q

alpha C - C bond

A

psi bond 120 degrees

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8
Q

Edmans Sequencing

A

Slow with multiple steps
Need pmol minimum of protein
Huge problem with background noise from reactants incorrectly bound
Only modification that can be detected is disulfide bonds
Cannot determine blocked (on N-terminus) or post-translationally modified amino acids

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9
Q

Properties of Mass Spectrometry

A

Fast
fmol of protein
Amenable to High-Throughput
Blocked/Modified peptides

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10
Q

Linus Pauling

A

1901-1994
Proposed alpha helix and beta sheet structures

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11
Q

alpha helix

A

3.6 AA per turn
1 – 10 loops
Side chains point out
Stabilized primarily by
Hydrophobic Interactions
N of an amino acid forming H-bonds with C of carboxyl group 4 AA ahead of the first AA
Electromagnetic interactions
Polarity of a-helix

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12
Q

what bonds stabilize alpha helices

A

Hydrophobic Interactions
Entropy because phi and psi angles fixed ~60°
H-Bonds
Vander Waal forces
Salt bridges
Helix dipole

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13
Q

what is the secondary structure loop

A

Interconnecting and non-structured elements connecting structured secondary elements

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14
Q

what do beta turns usually contain

A

proline and glycine

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15
Q

who developed X-ray crystallography techniques for polypeptide structure determination

A

max perutz and john kendrew

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16
Q

who developed NMR based techniques for polypeptide structure

A

richard ernst, Kurt Wuthrich and Albert Overhauser

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17
Q

what is a domain

A

Subsection of a polypeptide
Usually linked by a unstructured region of the polypeptide
Often contain separate functional abilities

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18
Q

what is tertiary structure controlled by

A

Constraints by type and positioning of secondary structure
Interactions between amino acid residues

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19
Q

what are the stabilizing factors in tertiary structure

A

Hydrophobic Interactions
Electrostatic Interactions
Covalent Linkages
Disulfide bonds

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20
Q

tertiary structure motif

A

A tertiary structural and/or functional sequence element found in multiple polypeptides of diverse origin that consist of similar secondary structures arranged in similar spatial organization that may play similar functional roles.

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21
Q

tertiary structure motif examples

A

helix-loop-helix
four helix bundle
zinc finger
greek key topology
hairpin structure
beta sandwich
beta barrel

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22
Q

what do beta barrels form

A

pores and channels

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23
Q

family definition

A

Polypeptides that have high levels of Sequence, Structural, and/or Functional similarities. Traditionally, evolutionarily related polypeptides. Now may refer to polypeptides whose structure and/or function only are similar but are not evolutionarily related.

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24
Q

superfamily definition

A

Polypeptide families that have Sequence, Structural, and/or Functional similarities (less than individual polypeptides at the family level). Traditionally, evolutionarily related polypeptide families. Now may refer to those whose structure and/or function are similar but are not evolutionarily related.

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25
Q

Van’t Hoff Analysis

A

Melting Curve (transition curve) and Van’t Hoff’s analysis allow determination of the thermodynamics of protein stability
Things that can be determined
Fraction of protein natured vs. denatured.
Keq of protein(s) at a given temperature
ΔH
ΔS
ΔG
E= the sum of (E NOE + E bond + E VWF + E angle)

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26
Q

Van’t Hoff Analysis Assumptions

A

Assumptions:
DH and DS are not temperature dependent
Limitation because Keqcan be measured only over a narrow range of temperatures

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27
Q

Post-Translational modifications

A

phosphorylation and sumoylation
glycosylation
methylation
acetylation
acylation
amidation
sulfation
proteolytic Cleavage

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28
Q

glycosylation

A

attachment of sugars

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29
Q

where does glycosylation happen

A

extracellularly

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30
Q

two types of glycosylation

A

n-linked and o-linked (S,T)

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31
Q

functions of glycosylation

A

Guide protein folding/assembly
Targeting and Trafficking of Proteins
Aid Ligand binding to receptors and elucicating the biological response
Stabilize Protein
Regulate ½-life of proteins (sialic cap on N-Terminus)

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32
Q

methylation

A

addition of a methyl group, mono, di, or tri

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33
Q

on which amino acids does methylation occur

A

R or K

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34
Q

function of methylation

A

Linked with protein function, but exact action is often not predictable.
Effects must be empirically determined.
Mono-, di-, or tri-methylation can have opposite, combined or opposing effects.

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35
Q

acetylation

A

N-acetyltransferases transfer acetyl group from acetyl CoA

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36
Q

where does Acetylation occur

A

on the N terminus of some proteins, can occur co-translationally or post-translationally

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37
Q

What structure is added in acylation

A

addition of lipids to proteins

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38
Q

where is acylation found?

A

Found in all eurkaryotes and in viruses
Found on a variety of proteins including structural, cytoplasmic and membrane proteins

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39
Q

most common lipids attached in acylation

A

Palmitic acid: 16-C saturated fatty acid
Ester or thioeser linkage to S, T or C
Post-translationally added
Complex Regulation
Myristic acid: 14-C saturated fatty acid
Linked to N-terminus of G
Co-translational Modification by myristoyl CoA:N-myristoyl transferase

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40
Q

function of acylation

A

Function is not well-defined:
Not likely interaction with membranes
Maybe protein complex stabilization or protein:protein interactions

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41
Q

amidation

A

amide group attached to C-terminus

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42
Q

where is amidation found

A

on short peptides

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43
Q

amidation function

A

Function not well understood
May help binding of regulatory peptides to certain receptors
May stabilize the structure and functions of proteins

44
Q

sulfation

A

added sulfate groups mediated by sulfotransferases

45
Q

where does sulfation occur

A

higher eukaryotes
YST

46
Q

function of sulfation

A

protein:protein interaction

47
Q

proteolytic Cleavage

A

cleavage of protein after translation

48
Q

function of proteolytic cleavage

A

Create Functional mature protein from “pre”-protein
EPO
Target protein to organelle or extracellularly

49
Q

what to consider for protein purification

A

level of protein expression
source of protein
location of protein
fusion tag
physiochemical properties
purpose of purification

50
Q

physiochemical properties

A

size
solubility
charge
hydrophobicity

51
Q

Function of immunoassays

A

Usually just measure protein presence, but not activity

52
Q

why are immunoassays good

A

High Affinity molecules
High Specificity
High sensitivity
Short assay times
Easily automated
Recombinant proteins with Fusion Tags

53
Q

bioassays

A

know protein is present and active

54
Q

pros and cons of bio assays

A

pros: examine function of protein
cons: costly, imprecise

55
Q

which is preferred bioassays or immunoassays

A

bioassays

56
Q

plant and fungus cell disruption

A

Waring Blender
Glass Beads with freeze/thaw

57
Q

Animal cell disruption

A

Potter homogenizer
Dounce homogenizer
Osmotic Shock
Freeze/Thaw
Waring Blender (for tough tissues)

58
Q

microbial cell disruption

A

Chemical methods
Lysozyme
Detergents, ionic
Organic Solvents
Antibiotics
Alkaline Conditions
Chaotropic Agents
Physical Methods
Sonication
Glass beads
Microbial Homogenization

59
Q

cheapest way to get rid of whole cells and cell debris

A

filtration
0.2 micrometer pore sizes removes all bacteria

60
Q

materials of filters

A

cellulose acetate or cellulose citrate
nylon
PTFE

61
Q

PEG

A

polyethylene glycol

62
Q

affinity partitioning

A

uses a ligand of high specificity to protein

63
Q

diafiltration

A

Same as ultrafiltration except reservoir of filtrate kept constant
Often pellet continuously resuspended and recycled through filter.

64
Q

chromatography

A

separation of proteins based on differential partitioning between a stationary phase and a mobile phase

65
Q

chromatography steps

A

Equilibrate column to correct conditions

Load sample onto column

Irrigate column

Elute proteins by changing properties of mobile phase

Monitor Proteins in each fraction by absorbance at 280 nm

Assay fractions with protein for protein of interest

66
Q

chromatography size exclusion chromatography

A

Also called “gel permeation” chromatography
Matrix is porous, not adsorptive
Large proteins run fast, small proteins slow
As do irregular shaped proteins
Difference is that small proteins can enter more matrix pores

67
Q

chromatography size exclusion column composition

A

Usually made of cross-linked polymer fibers of molecules like dextran, agarose, acrylamide, and vinyl polymers

68
Q

con of size exclusion chromatography

A

dilutes protein

69
Q

pro of ion exchange chromatography

A

Most popular and most employed in at least one step of protein purification
Easy to use
Very high resolution of proteins
Concentrates proteins
Scalable for industry

70
Q

ion exchange chromatography

A

Based on reversible charge of protein at different pHs of mobile phase
pH values above the pI are more negative, pH values below the pI are more positive

anionic and cationic

71
Q

anionic ion exchange chromatography

A

Anionic
Bind negatively charged proteins
Groups attached to matrix are positively charged
Aminoethyl
Diethylaminoethyl

72
Q

cationic ion exchange chromatography

A

Cationic
Bind positively charged proteins
Groups attached to matrix are negatively charged
Sulfo
Carboxymethyl

73
Q

reverse phase chromatography

A

organic solvent to elute

74
Q

hydrophobic interaction chromatography

A

Patches of hydrophobic residues on protein surface
Can exploit these patches of hydrophobicity to purify protein
Uses hydrophobic groups

75
Q

what resins and groups are used in hydrophobic interaction chromatography

A

Resins
Agarose
Sepharose
Groups
Octyl-
Phenyl-

76
Q

two ways to elute via hydrophobic interaction chromatography

A

Eluted by increasing ionic strength of solution
NaCl or ammonium sulfate
Attracts water away from protein
Eluted by Lowering polarity of buffer
EtOH
Ethylene glycol

77
Q

hydroxyapetite chromatography

A

Natural mineral in rock and bones
Mechanism of binding not well understood
Elution by irrigation with potassium phosphate buffer
Used as last resort

78
Q

most powerful kind of chromatography

A

affinity

79
Q

affinity chromatography

A

Most powerful because of its specificity and selectivity
Elution buffer changes disrupt bonds between ligand and protein
pH
Ionic strength
Agents reducing solution polarity
Detergents
Ethylene glycol
Competing ligands
Combination of the above

80
Q

limitations of affinity chromatography

A

Reagents expensive
Reagents often unstable
Techniques to couple ligand are complex, hazardous and time-consuming
Ligands leaching from column
Reduces capacity of system
Noxious products end up in the eluant

81
Q

immunoaffinity chromatography

A

Antibodies covalently attached to matrix
Very high specificity
Sometime difficult to desorb
Often changes buffer pH to desorb
Glycine-HCl at pH 2.2 – 2.8

82
Q

lectin affinity chromatography

A

Lectins from plants and some invertebrates
Binds glycoproteins
Examples of lectins
Concanavalin A
Soybean lectin (SBL)
Wheat germ agglutinin (WGA)
Bind at close to neutral pHs
Desorption occurs by changes in buffer or adding free sugar molecules

83
Q

dye affinity chromatography

A

Blue Dextran Dye in gel filtration columns (agarose)
Triazine Dye (Cibacron Blue F3G-A)
Dye has affinity for proteins

84
Q

advantages of dye affinity chromatography

A

Dye available in bulk
Coupling is easy and non-toxic
Dye resistant to chemical, physical and enzymatic degradation
Protein binding capacity high
Easy to elute proteins

85
Q

disadvantages of dye affinity chromatography

A

Mechanism not well understood
Has aromatic groups
Has sulfonate groups (negative charge)
Makes hydrogen bonds

86
Q

immobilized metal ion affinity chromatography

A

Pseudo-affinity purification
Exploits weak bonds between metals and basic residues on protein surface
Fe2+
Ni2+
Cu2+
Co2+
Charged with metal
Binds basic groups – usually H
Elution solution uses
lower pH buffer to deprotonate the ion
Chelating agent – eg, EDTA
Used prominently for purification of recombinant proteins

87
Q

chromatofocusing

A

Using a column and mobile phase of two different pHs
Forms a continuous pH gradient along column
Both column matrix and mobile phase buffer need a good buffering capacity
Sample applied in running buffer (mobile phase) whose pH is below that of the column
Negatively charged proteins adsorb into column
Positively charged protein go to part of column where column pH = their pI
But pH of column is constant getting more basic
Thus proteins move sequentially down the column based on the pI from most acidic to most basic
Elute in this order as well
High degree of protein resolution

88
Q

HPLC

A

Other Chromatography is low-pressure
Higher pressures give
Quicker runs
Sharper peaks
Used at analytical or preparative scale
higher elution rates and better elution peaks

89
Q

types of columns in HPLC

A

made of high-grade steel
Ion exchange
Gel filtration
Hydrophobic interactions

90
Q

what kind of proteins is HPLC good for

A

small and extracellular proteins

91
Q

advantages of HPLC

A

Superior resolution
Decreased fractionation times

92
Q

binding tags on proteins

A

His tags and IMAC
Glutathione-S-transferase and Glutathione
N-terminus or C-terminus
Can also be used as antigenic epitope

93
Q

steps to purify recombinant proteins

A

Engineer cells
Equilibrate IMAC column
Grow cells
Add IPTG (Isopropyl b-D-1-thiogalactopyranoside)
Lyse Cells
Apply lysate to column
Incubate to bind protein
Wash column at least 2 times
Eluted with Imidazole
or by lowering pH of elution buffer

94
Q

why is it necessary to remove a tag

A

Potentially affect structure, function or is immunogenic
Typically done enzymatically but may be done chemically

95
Q

proteases for tag removal

A

TEV Protease
Enterokinase
Thrombin
Self-cleaving tags

96
Q

problems with removing a fusion tag

A

Protein of interest may also be cleaved
Harsh chemical conditions denature proteins
Tag removal less than 100% efficient
Poly-his tag triggers aggregation
May require additional separation steps

97
Q

general protein stabilizers

A

Reducing Agents
Protease Inhibitors
Bulk proteins (e.g., BSA)
Amino acids (G, T, A, K)
Carbohydrates – reduce waters of hydration
Glycerol
Polymers (e.g., PEG)

98
Q

specific protein stabilizers

A

Substrates
Ligands
Antibodies

99
Q

lyophilization

A

drying of proteins

100
Q

Common AA in alpha helix

A

AKREQ

101
Q

AA not found in alpha helix

A

TIP

102
Q

Kinases

A

Put on phosphate

103
Q

De phosphorylates/phosphotases

A

Take off phosphate

104
Q

What proteins are phosphorylated

A

Intercellular

105
Q

Physical ways to degrade proteins

A

High temps
Freeze thaw
Agitation
Extreme pH

106
Q

Biological ways to degrade proteins

A

Carbohydrases
Phosphotases
Proteases

107
Q

Maintain protein stability

A

Keep in buffer
Minimize processing times
High purity reagents
Store frozen
Stable pH

Proteins (3 decks)

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