Module 2 - Proteins Flashcards
1
Q
Do saturated fatty acids have double bonds?
A
No
2
Q
Do unsaturated fatty acids have double bonds?
A
Yes
3
Q
What is the role of double bonds in fatty acids?
A
They make unsaturated fats fluid, ensuring the smooth functioning of the membrane
No double bonds -> long + rigid chain blocking chemicals into cell -> floats around body as cholestrol
4
Q
Which can be used by the body for energy: glucose or fructose?
A
glucose
5
Q
How are ring monosaccharides formed?
A
The aldehyde or ketone group of a sugar molecule reacts with a hydroxol (-OH) group closing the molecule into a ring
6
Q
Isomers
A
Molecules with differences in the arrangement of atoms resulting in changes in chemical properties
7
Q
Example of 2 isomers
A
glucose and galactose
8
Q
Hydrolysis
A
the addition of a water molecule
9
Q
Condensation
A
the removal of a water molecule
10
Q
What are the building blocks of proteins?
A
amino acids
11
Q
What are the building blocks of fats?
A
fatty acids
12
Q
What are the building blocks of nucleic acids?
A
nucleotides
13
Q
What are the building blocks of polysaccharides?
A
sugar units
14
Q
Signalling molecules (ligands)
A
molecules carrying a message that bind to specific receptors
15
Q
Ion channels
A
pore-forming membrane proteins allowing ions to pass through
16
Q
Ion pumps
A
modulate ion transport in + out of a cell or organelle
17
Q
Transmembrane carriers
A
involved in the movement of ions + molecules across the membrane
18
Q
What are molecuels accompanied by when passing through a membrane?
A
proteins
19
Q
Chaperones
A
helps hydrophilic molecules to travel over hydrophobic membrane by binding to partially folded polypeptide chains, helping them to progress along the most energetically favourable folding pathway
20
Q
Example of chaperon
A
insulin for glucose
21
Q
Enzymes
A
protein acting as a catalyst in the movement of ions and molecules across the membrane
22
Q
Antibodies
A
protein made by plasma blood cells (a type of white blood cell) in response to an antigen (= substance causing the body to make a specific immune response)
23
Q
What happens if even 1 of the components of a receptor changes?
A
neurotransmitter loses affinity for it
24
Q
What happens when a ligand (e.g. NT) binds to a receptor?
A
ligand-gated ion channel opens allowing ion to flow across the plasma membrane, ultimately changing the configuration of the receptor
25
What determines the 3D shape of a protein?
amino acid sequence
26
What determines the function of a protein?
its shape
27
What is the structure of an amino acid?
1) general core structure
2) side chain making it a unique amino acid
28
What makes up the general core structure of an amino acid?
1) amino group (H2N)
2) carboxyl group (COOH)
3) H atom
29
What happens when an amino acid is ionized?
It becomes neutral - H from the carboxyl group moves to the amino group resulting in COO (-) and H3N (+)
30
What do isomers have in common?
Identical melting + boiling temperatures, densities and solubilities - differ in chemical reactivity
31
What are the 2 types of isomers of amino acids?
D and L amino acids
32
What amino acid isomer is used by the body
L amino acids
33
Are hydrophilic amino acids polar/covalent or non-polar?
polar (unequal sharing of electrons btw atoms)
34
Are hydrophobic amino acids polar or non-polar?
non-polar (electron pair is shared equally between the two bonded atoms)
35
What are the 3 categories of hydrophilic amino acids?
1) basic
2) polar with uncharged R groups
3) acidic
36
Example of basic hydrophilic amino acid
lysine
37
Example of hydrophilic polar amino acid with uncharged R group
asparaginine
38
Example of hydrophilic acidic amino acid
aspartic acid
39
Example of hydrophobic amino acid
trytophan
40
Example of special amino acid
glyceine
41
Covalent bond
two atoms share electrons between themselves
42
Non-covalent bond
either exchange electrons or do not exchange electrons at all
43
Where are hydrophobic amino acids located?
centre of a protein
44
Where are hydrophilic amino acids located?
surface of a protein
45
Hydrophobic amino acids have __ side chains
long
46
Hydrophilic amino acids have __ side chains
short
47
Why is water polar?
Because of its shape - H ends of the molecule are (+) charged and the O end is (-) charged
48
Why do H2O molecules attract each other and other polar molecules?
Because of its shape - H ends of the molecule are (+) charged and the O end is (-) charged
49
Peptide bonds aka
amide bond
50
How is a peptide/amide bond formed?
amine group (H3N) of one amino acid bonds with the carboxyl group (COOH) of another -> condensation reaction -> peptie bond formed
51
What kind of bond links amino acids?
peptide/amine bonds
52
What are proteins (chain of amino acids) made up of?
1) polypeptide backbone
2) amino terminus (N-terminus)
3) carboxyl termines (C-terminus)
4) amino acids linked by peptide bonds
53
What are the 2 ends of a chain of amino acids?
1) amino/N terminus
2) carboxyl/C terminus
54
What is the backbone of a chain of amino acids
polypeptide backbone
55
What kind of bonds are involved in the folding of a protein (tertiary structure)?
non-covalent (e.g. hydrogen) between side chains
56
Primary level of protein structure
linear sequence of amino acids
57
Secondary level of protein structure
the localized organisation of parts of a polupeptide chain (e.g. the alpha helix or beta sheet)
58
Tertiary level of protein structure
3D arrangement of polpeptide chain
59
Quaternary level of protein structure
association of 2 or more polypeptides into a multi-subunit complex
60
How are alpha-helices formed?
Hydrogen bonds: the N-H of every peptide bond is hydrogen bonded to the C = O of the neighbour peptide bond, forming a spiral helix
61
Where are alpha-helices found?
cell membrane proteins e.g. receptors
62
What determines whether a protein's secondary structure is an alpha helix or beta sheet?
ribosomes
63
What determines where proteins are used
ribosomes
free ribosomes -> protein used in the cell
endoplasmic reticulum ribosomes -> proteins out of the cell are packaged to be used in the cell membrane
64
How are beta sheets formed?
Hydrogen bonds (H + O) formed in a polypeptide chain in a way that forms either a chain running in the direction opposite to that of its neighbours (antiparallel chains - so it folds on itself) OR neighbouring segments o the polypeptide backbone running in the same orientation (parallel chains)
65
What is a coiled-coil motif?
2 or more helices wound around one another
66
How are coiled-coil motifs formed?
form when 2 (or more) helices have most of their non-polar (hydrophobic) side chains on one side, so they can twist around eachother with these side chains facing inwards
67
What kind of bonds help proteins maintain folding?
covalent bonds (e.g. disulfide)
68
How are proteins secreted from a cell?
through the extracellular matrix
69
What are protein domains?
functional protein domains - complex structure + unique function
70
Example of a quaternary level structure protein
Hemoglobin = 2 alpha-globins and 2 beta-globins
71
Protein families
a similar structure (amino acid sequence) but differ in function
72
Native state of protein
molecules of any protein adopt a single conformation known as the native state, which is the most stably form of the molecule
72
Native state of protein
molecules of any protein adopt a single conformation known as the native state, which is the most stably form of the molecule
72
Native state of protein
molecules of any protein adopt a single conformation known as the native state, which is the most stably form of the molecule
73
Proteasome
proteins that degrade unneeded or damaged proteins through the ubiquitination process
74
3 steps of an enzymatic reaction
1) substrate binding (-> enzyme-substrate complex)
2) product formation (-> enzyme-product complex)
3) product release (-> product released from enzyme)
75
Hydrolases
catalyze hydrolytic cleavage reaction
76
Nucleases
break down nucleic acids by hydrolizing bonds between nucleotides
77
Proteases
break down proteins by hydrolyzing bonds between amino acids
78
Isomerases
catalyze the rearrangement of bonds with a single molecule
79
Polymerases
catalyze polymerization reactions e.g. synthesis of RNA
80
Kinases
catalyze the addition of a phosphate group to molecules
81
Phosphatases
catalyze hydrolytic removal of a phosphate group from a protein i.e. dephosphyrylation
82
Oxido-reductase
one molecule is oxidized while the other is reduced
83
ATPases
hydrolyze ATP
84
As concentration increases, the rate of an enzymatic reaction
increases and then plateaus when substrate-binding sites on the enzyme molecules are occupied
85
What is the relevane of enzyme kinetics?
can we inhibit or slow them down
86
Thorium
a rate-limiting enzyme in SE synthesis that determines how much SE will be produced
87
Competitive inhibitor
competes with substrate at the active site of an enzyme - it linearly increases the rate of the reaction in contrast to if there was no inhibitor present (which eventually plateaus)
88
What are the 2 ways in which proteins are regulated?
1) reversible binding of other molecules
2) protein phosphorylation: covalent addition of a phosphate group to 1 or more of its amino acid side chains
89
Non-competitive inhibitor (allosteric modulator)
does not bind to active site but another site -> works similar to if there was no inhibitor present but it slows down the reaction and plateus at a lower point
90
What 4 ways can enzyme activity be regulated?
1) allosteric transitions
2) phosphorylation/dephosphorylation
3) proteolytic activation
4) compartmentalization
91
Purpose of phosphorylation
vital for cellular storage + transfer of free energy using energy carrier molecules
92
Proteolytic activation
activating an enzyme by peptide cleavage i.e. breaking peptide bonds
93
Compartmentalization
storing enzymes in specific compartments to keep them from doing damage or provide the right conditions for activity
94
How do antibodies function?
bind tightly with high specificity to a target molecule, inactivating it directly or making it for destruction
95
Antigen
target of antibody
96
What is the purpose of cell lysis?
to free the content of the cell so proteins are in the solution
97
What do you need for cell lysis?
lysis buffer
98
What components of the lysis buffer enable the solubilizing of the cell membrane?
Igepal + triton
99
What component of the lysis buffer stabilizes the protein?
glycerol
100
What components of the lysis buffer enable enzyme inhibition?
EDTA + EGTA
101
What components of the lysis buffer inhibits the removal of a phosphate group i.e. acts as a phophatase inhibitor?
Trisodium vanadate + sodium fluoride
102
Denatured proteins are
looser + more random in structure
102
Denaturation
modifying protein structure by breaking weak hydrogen links
103
What is the purpose of bicinchoninic acid (BCA) sodium salt?)
interacts with copper in a complex non-covalent interaction in a basic environment (BCA-Copper reaction) to give a blue colour
104
Blueness is proportional to
amount of peptide bonds present in proteins
105
Steps of BCA-copper reaction
1) Peptide bonds of a protein in a basic environment complex a bivalent Cu2+ ion in a way that it become monovalent, Cu1+, and stabilizes the bond
2) BCA molecules take over Cu1+ ion to make a complex with 2 BCA molecules which turn blue by absorbing a lot of light
106
What is sodium dodecylsulphate (SDS)?
denaturing agent that is (-) charged enabling proteins to migrate into electrical field
107
Polyacrylamide Gel Electrophoresis (PAGE)
induction of movement by an electric field
108
What is the goal of SDS-PAGE?
to separate protein based on molecular weight
109
Upper part (stacking) of gel is _ charged
negatively
110
Lower part (running) of gel is _ charged
positively
111
What 2 gels are used in SDS-PAGE
1) running
2) stacking
112
What is the pH of the running gel and what does that contribute?
The pH of the running gel is 8.8 (more acidic)
113
What is the purpose of the stacking gel being negatively charged?
causes them proteins to move slower
114
What is the purpose of the running part of the gel being positively charged?
causes them to more faster through the gel leaving proteins behind
115
What causes proteins to move through the gel?
negative charge
116
What is the function of TEMED in SDS-PAGE
acts as a catalyst of the chemical reaction, reduces need for energy in the reaction
117
What is the function of ammonium persulfate (APS) in SDS-PAGE?
initiate reaction
118
What is the function of Polymerized acrylamide (polyacrylamide) in SDS-PAGE?
forms a mesh-like matrix suitable for the separation of proteins of typical size
119
What are the components of the running/stacking gel in SDS-PAGE?
1) milliQ water
2) acrylamide
3) tris hydrochloride (8.8 + 6.8)
4) SDS
5) APS
6) TEMED
120
Large proteins move __ through the gel
slower
121
Small proteins move __ through the gel
faster
122
What determines how quickly proteins move through the gel in SDS-PAGE?
size
123
Purpose of glycine in SDS-page?
pushes proteins into running gel
124
What is Coomassie staining? What is its purpose
It is a blue dye that stains proteins - the more faint the less protein bonds present, the more blue the more present
125
What molecular weight marker do we look at in Coomassie staining?
kDa (1kDa = 100 Da)
126
How do you calculate dilution factor of a concentration of lysate?
substitute average absorption of the 3 wells for each dilution as y into the line of best fit
127
How do you calculate the total protein concentration?
add dilution factors together
128
How do you prepare lysate?
1) aspirate medium (move to another tube)
2) wash cells left with PBS twice gently to remove external proteins
3) add lysis buffer to flask and incubate on use for 2 minutes
4) scrape external proteins using cell scraper on bottom of flask)
5) triturate the lysate and transfer to eppendorf tube
6) incubate for 30 mins on ice vortexing every 5 minutes
129
How do you prepare dilutions of lysate?
combine with lysis buffer in eppendorf tubes depending on desired concentration e.g. 50% would be 25 ul of lystae + 25 ul of lysis buffer
130
What protein standard was used in protein determination of SDS-PAGE?
Bovine Serum Albumin (BSA)
131
How do you make dilutions of your protein?
Mix 50 ul BSA with 450 ul lysis buffer to get a concentration of 4mg/ml, then mix 250 ul BSA with 250 ul lysis buffer to get a concentration of 2mg/ml and continue A-H dilutions
132
How do you measure the absorptions of your protein assay?
Biorad plate reader
133
Ideal wavelength for SDS-PAGE?
655nm
134
Why is TEMED added last minute to gels?
Will start polymerizing the protein if added before
135
What do the chemical properties of the protein depend on?
sequence of side-chain group
136
How are disulfide bridges made?
oxidation - adding an oxidant
137
How are disulfide bridges broken?
reduction - adding a reductant
138
Ubiquitin
small molecule placed on the protein that marks misfolded proteins to be broken down
139
Michaelis constant (Km)
amount of substrate we must add to have half of the enzyme's activity
140
Substrate inhibition
too much substrate -> enzyme loses activity, slows down
141
Product inhibition
the product of the enzyme interferes with the binding site of the substrate
142
What property of proteins do we use to measure the total amount of proteins?
peptide bonds
143
Purpose of antibodies
used by the immune system to identify and neutralize foreign objects
144
What are the 2 regions of an antibody
1) constant
2) variable
145
Purpose of constant region of an antibody
contains antigen binding sites
146
Purpose of variable region of an antibody
binds to immune cell receptor
147
Constant region has __ chain
heavy
148
Variable region has __ chain
light
149
What are antibodies made up of?
multiple proteins joined by disulfide bridges
150
Hypervariable loop of the variable region
light chain; sequence of amino acids different between antibodies (each antibody is specific to one kind of antigen); present at the end of both arms
151
Specificity of binding of a monoclonal antibody
Binds to a specific epitope on an antigen
152
Specificity of polyclonal antibodies
aspecific binding: binds to all epitopes because they can recognize other molecules that are only a little bit different
153
Specificity of monoclonal anitbodies is __ and of polycloncal is __
high, low
154
Sensitivity of monoclonal antibodies is __ and of polyclonal is __
low, high
155
Monoclonal antibodies are used in
smaller animals e.g. mice
156
Polyclonal antibodies are used in
larger animals e.g. rabbits
157
What are antibodies produced from
B/immune cells
158
How are monoclonal antibodies formed?
1) mouse injected with a specific antigen coupled to an immunogenic carrier protein -> production of antibodies induced
2) mouse sacrificed to enable isolation of immune cells (each with one kind of antibody)
3) immune cells are coupled to tumorous cells to form hybridomas (can live forever)
4) Hybridomas are screened for the production of desired antibody
5) anti-body producing hybridomas are cloned -> monoclonal antibodies
159
How are polyclonal antibodies formed?
1) animal injected with antigen coupled to immunogenic carrier protein
2) after 12 weeks, blood taken, blood purified to get different antibodies, they recognize different parts and sides of 1 antigen
160
Primary antibodies
bind directly to epitope of antigen they are built against
161
Secondary antibodies
bind to heavy chain of primary antibody they are built against
162
Direct detection of antibody
primary antibody is directly conjugated to a label
163
Indirect detection of antibody
primary antibody is bound by a labelled secondary antibody that has been against the host species of the primary antibodies
164
Pro + con of direct antibody detection
high specificity, low sensitivity
165
Pro + con of indirect antibody detection
high sensitivity, low specificity
166
Chromogenic detection (enzyme labelling)
work through chromogenic reactions i.e. soluble + colourless substrate (e.g. DAB) is converted into an insoluble chromogenic (colorful) product
167
Example of chromogenic detection
Horseradish peroxidase (HRP) - hydrogen peroxide H2O2 (which is produced in the mitochondria and is highly reactive) is transformed into water by peroxidase
1. Secondary antibody is conjugated with HRP
2. A solution with DAB and hydrogen peroxide is added
3. A side chain group of one amino acid from HRP binds to the hydrogen peroxide, converting the hydrogen peroxide into water resulting in a cascade of reactions
168
Fluorescence detection
fluorescent molecules capture energy from a light source and release it in the form of a photon - when receiving energy, their electrons will move to a higher orbit and will then fall back, releasing the energy they got in the form of light
169
Which one is more durable? chromogenic or fluorescence detection
chromogenic since fluorescent molecules decay over time
170
Which one only shows one target? chromogenic or fluorescence
chromogenic
171
What happens if more targets are done in chromogenic?
risk of overlap
172
Why can multiple targets be used in fluorescence detection?
shows different colours
173
Which can be analysed quicker? chromogenic or fluorscence
chromogenic (other is more complicated but more accurate)
174
Which is cheaper? chromogenic or fluorescence?
chromogenic
175
How do we decide between chromogenic and fluorescence detection?
depends on which the primary antibody we are looking at works best with
