chapter 4 Flashcards
(136 cards)
1
Q
The shape of a protein is specified by its
A
amino acid
sequence
2
Q
Amino acids are connected together by
A
covalent peptide
bonds
3
Q
Proteins fold into
A
3D shapes
4
Q
Proteins fold into a conformation of
A
lowest energy
5
Q
An amino acid contains an
A
amino group (NH2), a carboxyl group (COOH),
an a-carbon atom and a side chain
6
Q
covalent link between carboxyl group of one amino acid and amino group of next amino acid
A
Peptide bond
7
Q
Linking amino acids together through peptide bonds forms the
A
polypeptide backbone
8
Q
polypeptide backbone
A
a repeating sequence of the core atoms (N-C-C) in
every amino acid
9
Q
Denature proteins with solvents that
A
disrupt non-covalent bonds (urea, detergent)
10
Q
When you remove the solvents from proteins, the proteins will
A
refold
11
Q
Protein folding depends on
A
non-covalent bonds and
hydrophobic interactions
12
Q
Protein folding occurs in the
A
aqueous environment of the cell interior
13
Q
Although chaperones make the folding process more efficient and reliable, the final
3-D shape of the protein is specified
A
only by its amino acid sequence
14
Q
What proteins can assist with protein folding
A
chaperone
15
Q
Two common secondary structures for folding proteins are
A
the alpha helix and beta sheet
16
Q
Amino acid side chains are not or are involved in secondary structure formation
A
are not
17
Q
The alpha helix is formed by
A
by hydrogen bonds in the polypeptide backbone as it
twists, amino acid side groups project out
18
Q
How many amino acids can form an alpha helix
A
many
19
Q
20 __ amino
acids can span a lipid
bilayer, interact with fatty
acids in phospholipids
A
hydrophobic
20
Q
Hydrophobic side chains in __ position every __
amino acids form a hydrophobic “stripe”
A
1st and 4th, seven
21
Q
Two or more alpha-helices with hydrophobic stripes can
interact to form a
A
coiled coil
22
Q
Beta sheets are formed by
A
by hydrogen bonds
between polypeptide
backbones of adjacent strands
23
Q
In the beta sheet, the amino acid side chains project…
A
above & below the
b-sheet
24
Q
regions of 40-350 amino acids that can fold independently into a stable structure
A
Protein domains
25
There are more than __ recognized protein domains
2000
26
what is the first protein level of organization, primary structure?
amino acid sequence
27
what is the second protein level of organization, secondary structure?
simple folding patterns (a-helix, b-sheet)
28
what is the third protein level of organization, tertiary structure?
3-dimensional shape of the polypeptide
29
what is the fourth protein level of organization, quaternary structure?
multiple polypeptide subunits in a protein complex
30
Where are protein domains located, what structure?
tertiary
31
Protein subunits can assemble to form
larger macromolecules
32
Depending on the location of __ on
the surface of a protein, a single protein subunit
can assemble into a wide variety of shapes
binding sites
33
Fibrous, elongated proteins are common in the
Extracellular matrix
34
Elastin and collagen are
ECM proteins
35
Fibrous, elongated proteins are often stabilized by
disulfide bonds
36
The binding sites of antibodies are
versatile
37
All proteins bind to
ligands
38
powerful and highly specific catalysts
enzymes
39
bind substrates and hold them in conformations that promote a particular reaction
enzymes
40
Binding sites for proteins are usually a pocket near
the surface of the folded protein
41
Binding usually involves many __ bonds
non-covalent
42
Heme group binds
iron
43
Heme group facilitates
O2 binding to hemoglobin
44
Aberrant conformation causes
polymerization
45
Enzymes are often regulated by other molecules
that bind at a
second site, allosteric control
46
controls the location,
assembly, stability and activity of protein complexes
covalent modification
47
can control protein activity by
triggering a conformational change
phosphorylation
48
regulated by cyclic
gain and loss of a phosphate group
GTP binding proteins
49
allows motor proteins to
produce large movements in cells
Nucleotide hydrolysis
50
Z binds to a __ on enzyme and exerts __ control
second, allosteric
51
As concentration of Z increases, it feeds back to the enzyme that
converts B to X to shut down the pathway
52
Allosteric control can also provide what feedback
positive feedback
53
When the concentration of ATP is low, the concentration of ADP is
high
54
can bind the enzyme, changing its conformation so it favors the
active form
ADP
55
What protein use ATP to phosphorylate amino acid side chains
Kinases
56
remove the phosphate group (Pi)
Phosphatases
57
Pi adds what charge
negative
58
The __ charge on Pi results in a
conformational change
59
Phosphorylation can either
activate or inactivate a protein
60
Ubiquitination stimulates
degradation
61
Palmitoylation on cysteine targets...
the protein to a membrane
62
G-proteins with bound
GTP are
active
63
G-proteins with bound GDP
are
inactive
64
GTP hydrolysis switches
G-protein
off
65
GDP-GTP exchange
switches G-protein
on
66
Many cell signaling
pathways use what protein
G-proteins
67
what drives
directional movement
NTP (nucleotide hydrolysis)
68
NTP is observed with
motor proteins
69
s enables the protein to
bias its conformational
changes in one direction
Nucleotide (NTP) hydrolysis
70
Amino acids only differ by their
Side chains
71
breaks holes in cell membranes
Homogenization
72
Spin the homogenate to enrich for your protein or cellular
component of interest
Centrifugation
73
2 centrifugation techniques:
Velocity Sedimentation and
Equilibrium Sedimentation
74
Take advantage of your protein’s unique size, shape, charge or binding properties
Column chromatography
75
3 chromatography methods:
Gel filtration, Ion exchange and Affinity chromatography
76
Overview of protein purification
1. Centrifugation
2. Column chromatography
3. Analysis
77
Assess the purity and identity of your protein sample
Analysis
78
Assesses the number and sizes of protein in your sample
Gel electrophoresis
79
Bigger organelles sediment more __ at lower speeds
quickly
80
1st step differential Centrifugation allows one to make a
Crude, enriched fraction
81
separates components based on the rate at which they
move through a gradient
Velocity sedimentation
82
Used to isolate large protein complexes like ribosomes
Velocity sedimentation
83
separates components based on buoyant density
Equilibrium sedimentation
84
In equilibrium sedimentation, Components migrate in gradient until their
Density matches that of the surrounding medium
85
2nd step of Centrifugation
Velocity vs equilibrium sedimentation
86
Often used to purify DNA, RNA, different membranes
Equilibrium sedimentation
87
Most soluble proteins are too __ to purify by Centrifugation
Small
88
Column chromatography can separate
Protein mixtures
89
Gel filtration separates proteins based on
Size
90
Beads for gel filtration contain
Small proes
91
In gel filtration,
__ proteins flow around beads and out the column first.
__ proteins enter the beads and flow out more slowly.
Larger, Smaller
92
Ion exchange separates proteins based on
Charge
93
What do yo use for ion exchange
Positively or negatively charged beads and add proteins
94
In ion exchange,
Positively charged beads will bind
Negatively charged proteins
95
Positively charged proteins in ion exchange will flow
Through without binding
96
Increase __ of buffer to elute the protein
ionic strength
97
In ion exchange, negative ions in buffer….
Displace negatively charged proteins
98
Affinity chromatography separates proteins
based on
Specific binding
99
In affinity chromatography, buy beads with a
Covalently attached substrate
100
Add a protein mixture, wash away proteins that can’t bind t substrate occurs in
affinity chromatography
101
Analyze protein mixtures by
Gel electrophoresis (SDS PAGE)
102
How to analyze proteins by gel electrophoresis
1. Treat the protein sample with two chemicals
2. Load treated protein sample onto gel with electric field
3. Proteins migrate towards positively charged anode
4. Smaller proteins migrate through the gel faster than larger ones
103
What two chemicals are used to treat the protein sample in gel electrophoresis
Beta-mercaptoethanol and SDS
104
What chemical breaks disulfide bonds
Beta mercapotethanol
105
What chemical denatures proteins & coats them with negative charges
SDS
106
SDS-PAGE separates proteins by
Size
107
In SDS PAGE, larger proteins are at the
Top, smaller at bottom
108
Protein complexes composed of four subunits (2 Heavy Chains, 2 Light Chains)
Antibodies
109
Small variable __ in antigen binding site recognize and bind antigens in antibodies
loops
110
In an antibody, Each light chain and heavy chain contains
One variable domain and one or more constant domains
111
Each antibody contains __ antigen binding sites, which are made up of __
that have variable amino acid sequences
2, loops
112
These loops in antibodies are located in the ___ of the heavy chain and the light chain on each side of the antibody
variable domains,
113
To make an antibody, Host animal degrades protein and presents fragments to
antibody-producing cells called
B cells
114
Binding of antigens stimulates
B cells to divide and secrete antibodies
115
Use for protein purification
Immunoaffinity chromatography
116
Use antibodies to detect a particular protein
within a mixture of proteins
Biochemical chromatography
117
Use antibodies to locate a protein inside a cell
Microscopic detection
118
In biochemical detection, antigen A is separated from other molecules by
Electrophoresis
119
In biochemical detection, Incubation with labeled antibodies that bind to antigen A allows…
the position of the antigen to be determined
120
Flourescent tags of DNA, microtubules, and Actin
DNA= blue
Microtubules= green
Actin=red
121
Smaller and less dense components
Supernatant
122
Larger and more dense components
Pellet
123
1st step differential Centrifugation can’t make what into a pellet
Most proteins
124
After running the protein through the gel in SDS PAGE..
Stain the gel with dye specific for proteins
125
common assay for analyzing column fractions
SDS PAGE
126
Separate denatured proteins according to their size (molecular weight)
SDS PAGE
127
Separate native (folded) proteins according to their size and shape
Gel filtration chromatography
128
Separate native proteins according to their ability to bind a specific substrate
Affinity chromatography
129
Separate native proteins according to their charge
Ion exchange chromatography
130
Separate proteins according to the rate at which they move through a gradient
Velocity sedimentation
131
Separate proteins according to their buoyant density
Equilibrium sedimentation
132
Flourescent microscopy allows you to
Detect specific components
133
What parts of amino acids are involved in peptide bonds
Amino group on one amino acid and carboxyl group on the other
134
How do most motor proteins make their movements unidirectional
They couple a conformational change to hydrolysis of an ATP molecule
135
Binding site of protein structure is located
Inside cavity on protein surface
136
Variable domains are located near the
Antigen binding site
