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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
Van't Hoff Analysis
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)
26
Van't Hoff Analysis Assumptions
Assumptions: DH and DS are not temperature dependent Limitation because Keqcan be measured only over a narrow range of temperatures
27
Post-Translational modifications
phosphorylation and sumoylation glycosylation methylation acetylation acylation amidation sulfation proteolytic Cleavage
28
glycosylation
attachment of sugars
29
where does glycosylation happen
extracellularly
30
two types of glycosylation
n-linked and o-linked (S,T)
31
functions of glycosylation
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)
32
methylation
addition of a methyl group, mono, di, or tri
33
on which amino acids does methylation occur
R or K
34
function of methylation
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.
35
acetylation
N-acetyltransferases transfer acetyl group from acetyl CoA
36
where does Acetylation occur
on the N terminus of some proteins, can occur co-translationally or post-translationally
37
What structure is added in acylation
addition of lipids to proteins
38
where is acylation found?
Found in all eurkaryotes and in viruses Found on a variety of proteins including structural, cytoplasmic and membrane proteins
39
most common lipids attached in acylation
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
40
function of acylation
Function is not well-defined: Not likely interaction with membranes Maybe protein complex stabilization or protein:protein interactions
41
amidation
amide group attached to C-terminus
42
where is amidation found
on short peptides
43
amidation function
Function not well understood May help binding of regulatory peptides to certain receptors May stabilize the structure and functions of proteins
44
sulfation
added sulfate groups mediated by sulfotransferases
45
where does sulfation occur
higher eukaryotes YST
46
function of sulfation
protein:protein interaction
47
proteolytic Cleavage
cleavage of protein after translation
48
function of proteolytic cleavage
Create Functional mature protein from “pre”-protein EPO Target protein to organelle or extracellularly
49
what to consider for protein purification
level of protein expression source of protein location of protein fusion tag physiochemical properties purpose of purification
50
physiochemical properties
size solubility charge hydrophobicity
51
Function of immunoassays
Usually just measure protein presence, but not activity
52
why are immunoassays good
High Affinity molecules High Specificity High sensitivity Short assay times Easily automated Recombinant proteins with Fusion Tags
53
bioassays
know protein is present and active
54
pros and cons of bio assays
pros: examine function of protein cons: costly, imprecise
55
which is preferred bioassays or immunoassays
bioassays
56
plant and fungus cell disruption
Waring Blender Glass Beads with freeze/thaw
57
Animal cell disruption
Potter homogenizer Dounce homogenizer Osmotic Shock Freeze/Thaw Waring Blender (for tough tissues)
58
microbial cell disruption
Chemical methods Lysozyme Detergents, ionic Organic Solvents Antibiotics Alkaline Conditions Chaotropic Agents Physical Methods Sonication Glass beads Microbial Homogenization
59
cheapest way to get rid of whole cells and cell debris
filtration 0.2 micrometer pore sizes removes all bacteria
60
materials of filters
cellulose acetate or cellulose citrate nylon PTFE
61
PEG
polyethylene glycol
62
affinity partitioning
uses a ligand of high specificity to protein
63
diafiltration
Same as ultrafiltration except reservoir of filtrate kept constant Often pellet continuously resuspended and recycled through filter.
64
chromatography
separation of proteins based on differential partitioning between a stationary phase and a mobile phase
65
chromatography steps
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
chromatography size exclusion chromatography
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
chromatography size exclusion column composition
Usually made of cross-linked polymer fibers of molecules like dextran, agarose, acrylamide, and vinyl polymers
68
con of size exclusion chromatography
dilutes protein
69
pro of ion exchange chromatography
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
ion exchange chromatography
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
anionic ion exchange chromatography
Anionic Bind negatively charged proteins Groups attached to matrix are positively charged Aminoethyl Diethylaminoethyl
72
cationic ion exchange chromatography
Cationic Bind positively charged proteins Groups attached to matrix are negatively charged Sulfo Carboxymethyl
73
reverse phase chromatography
organic solvent to elute
74
hydrophobic interaction chromatography
Patches of hydrophobic residues on protein surface Can exploit these patches of hydrophobicity to purify protein Uses hydrophobic groups
75
what resins and groups are used in hydrophobic interaction chromatography
Resins Agarose Sepharose Groups Octyl- Phenyl-
76
two ways to elute via hydrophobic interaction chromatography
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
hydroxyapetite chromatography
Natural mineral in rock and bones Mechanism of binding not well understood Elution by irrigation with potassium phosphate buffer Used as last resort
78
most powerful kind of chromatography
affinity
79
affinity chromatography
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
limitations of affinity chromatography
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
immunoaffinity chromatography
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
lectin affinity chromatography
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
dye affinity chromatography
Blue Dextran Dye in gel filtration columns (agarose) Triazine Dye (Cibacron Blue F3G-A) Dye has affinity for proteins
84
advantages of dye affinity chromatography
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
disadvantages of dye affinity chromatography
Mechanism not well understood Has aromatic groups Has sulfonate groups (negative charge) Makes hydrogen bonds
86
immobilized metal ion affinity chromatography
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
chromatofocusing
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
HPLC
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
types of columns in HPLC
made of high-grade steel Ion exchange Gel filtration Hydrophobic interactions
90
what kind of proteins is HPLC good for
small and extracellular proteins
91
advantages of HPLC
Superior resolution Decreased fractionation times
92
binding tags on proteins
His tags and IMAC Glutathione-S-transferase and Glutathione N-terminus or C-terminus Can also be used as antigenic epitope
93
steps to purify recombinant proteins
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
why is it necessary to remove a tag
Potentially affect structure, function or is immunogenic Typically done enzymatically but may be done chemically
95
proteases for tag removal
TEV Protease Enterokinase Thrombin Self-cleaving tags
96
problems with removing a fusion tag
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
general protein stabilizers
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
specific protein stabilizers
Substrates Ligands Antibodies
99
lyophilization
drying of proteins
100
Common AA in alpha helix
AKREQ
101
AA not found in alpha helix
TIP
102
Kinases
Put on phosphate
103
De phosphorylates/phosphotases
Take off phosphate
104
What proteins are phosphorylated
Intercellular
105
Physical ways to degrade proteins
High temps Freeze thaw Agitation Extreme pH
106
Biological ways to degrade proteins
Carbohydrases Phosphotases Proteases
107
Maintain protein stability
Keep in buffer Minimize processing times High purity reagents Store frozen Stable pH

Proteins (3 decks)

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