Chapter 3: Nonenzymatic Protein Function and Protein Analysis Flashcards
1
Q
What do structural proteins compose?
A
The cytoskeleton, anchoring proteins, and much of the extracellular matrix
2
Q
What are the 5 most common structural proteins?
A
- Collagen
- Elastin
- Keratin
- Actin
- Tubulin
3
Q
Structural proteins are generally _____ by nature
A
fibrous
4
Q
What gives structural proteins their fibrous nature?
A
Highly repetitive secondary structure and supersecondary structure
5
Q
What is supersecondary structure? What is it sometimes known as?
A
- Motif
- Repetitive organization of secondary structural elements together
6
Q
What is the structure of collagen?
A
Three a-helices woven together to form a secondary helix
7
Q
_______ and _____ make up most of the extracellular matrix of connective tissue
A
Collagen and elastin
8
Q
What is the primary role of elastin?
A
To stretch and then recoil like a spring, which restores the original shape of the tissue
9
Q
In which cells are keratins found?
A
Epithelial cells
10
Q
____ is the primary protein that makes up hair and nails
A
Keratin
11
Q
What is the primary role of keratin?
A
- Contributes to the mechanical integrity of the cell
- Functions as regulatory proteins
12
Q
What is the most abundant protein in eukaryotic cells?
A
Actin
13
Q
Which protein makes up microfilaments and thin filaments in myofibrils?
A
Actin
14
Q
Actin has a + and a - side; this polarity allows for what?
A
Allows motor proteins to travel unidirectionally along an actin filament
15
Q
Which protein makes up microtubules?
A
Tubulin
16
Q
Which two structural proteins have polarity?
A
Actin and tubulin
17
Q
What is the characteristic of motor proteins that allow them to move?
A
Have one or more heads capable of force generation through a conformational change
18
Q
How do motor proteins have catalytic activity? What does that power?
A
Acting as ATPases to power movement
19
Q
Name the most common applications of motor proteins.
A
- Muscle contraction
- Vesicle movement within cells
- Cell motility
20
Q
What are the three common examples of motor proteins?
A
- Myosin
- Kinesin
- Dynein
21
Q
Motor proteins have transient interactions with either _____ or _____
A
actin or microtubules
22
Q
What is the primary motor protein that interacts with actin?
A
Myosin
23
Q
Which protein is the thick filament in a myofibril?
A
Myosin
24
Q
What is responsible for the power stroke of sarcomere contraction?
A
Movement at the neck of myosin
25
What is myosin also involved in, apart from muscle contraction?
Cellular transport
26
Which two motor proteins are associated with microtubules?
Kinesins and dyneins
27
How many heads and necks does myosin have?
- One head
| - One neck
28
How many heads and necks do kinesins and dyneins have?
- Two heads, at least one remains attached to tubulin at all times
- 0 necks
29
____ play roles in aligning chromosomes during metaphase and depolymerizing microtubules during anaphase of mitosis.
Kinesins
30
____ are involved in the sliding movement of cilia and flagella.
Dyneins
31
Kinesins bring vesicles toward the _____ end of the microtubule, and dyneins bring vesicles toward the ____ end.
Kinesins: +
Dyneins: -
32
What is the role of binding proteins?
Bind a specific substrate, either to sequester it in the body or hold its concentration at steady state
33
The oxyhemoglobin dissociation curve is an example of what?
A binding protein's affinity curve for its molecule of interest
34
Give examples of binding proteins.
Hemoglobin, calcium-binding proteins, DNA-binding proteins
35
Where are cell adhesion molecules (CAMs) found?
Proteins found on the surface of most cells
36
What is the primary role of CAMs?
Aid in the binding of the cell to the extracellular matrix or other cells
37
What kind of membrane proteins are CAMs?
Integral membrane proteins
38
What are the three major families of CAMs?
- Cadherins
- Integrins
- Selectins
39
Cadherins are a group of ______ that mediate ____-dependent cell adhesion.
- glycoproteins
| - calcium
40
______ often hold similar cell types together.
Cadherins
41
______ are a group of proteins that all have two membrane-spanning chains called _ and _.
- Integrins
| - a and B
42
What is the major role of integrins?
- Permit cells to adhere to proteins in the extracellular matrix
- Some also have signalling capabilities
43
Selectins are unique because they bind _________ molecules that project from other cell surfaces.
carbohydrates
44
Which bonds are the weakest formed by the CAMs?
Selectins
45
Where are selectins expressed?
White blood cells and endothelial cells that line blood vessels
46
What is the major role of selectins?
Host defense, including inflammation and white blood cell migration (immune system)
47
What is the most common type of protein found in the immune system?
Antibody
48
What are antibodies also called?
Immunoglobulins (Ig)
49
What are antibodies produced by?
B-cells
50
What is the structure of antibodies?
- Y-shaped proteins
- 2 identical heavy chains
- 2 identical light chains
51
What hold the heavy and light chains in antibodies together?
Disulfide linkages and noncovalent interactions
52
Where is the antigen-binding region on an antibody? What does it bind to?
- At the tips of the "Y"
| - Polypeptide sequences will bind to ONE specific antigenic sequence
53
What are the two main regions on the antibody?
- Antigen-binding region
| - Constant region
54
What is the role of the constant region in an antibody?
Involved in recruitment and binding of other cells of the immune system (ex: macrophages)
55
What are the targets of antibodies called?
Antigens
56
When antibodies bind to their antigens, what are the three possible outcomes?
1) Neutralizing the antigen, making the pathogen or toxin unable to exert its effect on the body
2) Opsonization
3) Agglutinating
57
What is opsonization?
Marking the pathogen for destruction by other white blood cells immediately
58
What is agglutinating?
Clumping together the antigen and the antibody into large insoluble protein complexes that can be phagocytized and digested by macrophages
59
Which two proteins function in cellular motility?
Cytoskeletal (structural) and motor proteins
60
Are motor proteins enzymes?
Yes, they can be
Motor function is generally considered nonenzymatic, but the ATPase functionality of motor proteins indicates that these molecules do have catalytic activity
61
What could permit a binding protein involved in sequestration to have a low affinity for its substrate and still have a high percentage of substrate bound?
If the binding protein is present in sufficiently high quantities relative to the substrate, nearly all the substrate will be bound despite a low affinity
62
Which CAM does this description correspond to: two cells of the same or similar type using calcium
Cadherin
63
Which CAM does this description correspond to: one cell to proteins in the extracellular matrix
Integrin
64
Which CAM does this description correspond to: one cell to carbohydrates, usually on the surface of other cells
Selectin
65
Define biosignaling.
Process in which cells receive and act on signals
66
Acting as extracellular ligands, transporters for facilitate diffusion, receptor proteins, and second messengers are example of what process?
Biosignaling
67
Can biosignaling have functions in both substrate binding and enzymatic activity?
Yes
68
What are ion channels?
Proteins that create specific pathways for charged molecules
69
What is facilitated diffusion?
- Type of passive transport
- Diffusion of molecules down a concentration gradient through a pore in the membrane created by integral membrane proteins, which serve as channels for these substrates
70
What kind of substrates pass through facilitated diffusion?
- Molecules that are impermeable to the membrane
- Large, polar, or charged
- Avoid the hydrophobic FA tails of the phospholipid bilayer
71
Name the three types of ion channels.
1) Ungated
2) Voltage-gated
3) Ligand-gated
72
What are ungated ion channels?
- Always open
| - Net efflux through these channels unless substrate is at equilibrium
73
What are voltage-gated channels?
- Open within a range of membrane potentials
| - ex: membrane depolarization in neurons allows voltage-gated channels to open
74
How are ligand-gated channels opened?
Open in the presence of a specific binding substance
75
What kind of substances can open a ligand-gated channel?
Hormone or neurotransmitter
76
In terms of kinetics of transport, what is Km?
Solute concentration at which the transporter is functioning at half of its maximum activity
77
How do enzyme-linked receptors participate in cell signalling?
Through extracellular ligand binding and initiation of second messenger cascades
78
What are the three primary domains of enzyme-linked receptors?
- Membrane-spanning domain
- Ligand-binding domain
- Catalytic domain
79
What does the membrane-spanning domain do in enzyme-linked receptors?
Anchors the receptor in the cell membrane
80
What does the ligand-binding domain stimulated by in enzyme-linked receptors? What does it induce?
- Stimulated by the appropriate ligand
| - Induces a conformational change that activates the catalytic domain
81
When the catalytic domain of an enzyme-linked receptor is activated, what does this result in?
Second messenger cascade
82
What are G protein-coupled receptors (GPCRs)?
- Large family of integral membrane proteins, involved in signal transduction
- Have a membrane-bound protein associated with a trimeric G protein
83
How are GPCRs characterized?
By their seven membrane-spanning a-helices
84
How do GPCRs differ?
In specificity of the ligand-binding area found on the extracellular surface of the cell
85
In order for GCPRs to transmit signals to an effector in the cell, what do they utilize?
Heterotrimeric G protein
86
Explain the trimeric G protein cycle.
- Ligand binding engages the G protein
- GDP is replaced with GTP; the a subunit dissociates from the B and g subunits
- The activated a subunit alters the activity of adenylate cyclase or phospholipase C
- GTP is dephosphorylated to GDP; the a subunit rebinds to the B and g subunits
87
What are the three main types of G proteins?
- Gs
- Gi
- Gq
88
What is the role of Gs?
Stimulates adenylate cyclase, which increases levels of cAMP in the cell
89
What is the role of Gi?
Inhibits adenylate cyclase, which decreases levels of cAMP in the cell
90
What is the role of Gq?
- Activates phospholipase C, which cleaves a phospholipid from the membrane to from PIP2
- PIP2 is then cleaved into GAD and IP3
- IP3 can open calcium channels in the endoplasmic reticulum, increasing calcium levels in the cell
91
What are the similarities between enzyme-linked receptors and G protein-coupled receptors?
- Extracellular domain
- Transmembrane domain
- Ligand binding
92
What are the differences between enzyme-linked receptors and G protein-coupled receptors?
- Enzyme-linked receptors: autoactivity, enzymatic activity
- G protein-coupled receptors: two-protein complex, dissociation upon activation
- Trimer
93
How do transport kinetics differ from enzyme kinetics?
- Transport kinetics display both Km and vmax values
- They can also be cooperative, like some binding proteins
- Transporters do not have analogous Keq values for reactions because there is no catalysis
94
How are proteins and other biomolecules isolated from body tissues or cell cultures?
By cell lysis and homogenization (crushing, grinding, or blending the tissue of interest into an evenly mixed solution)
95
How does electrophoresis work? What does it separate on the basis of?
- Subjecting compounds to an electric field
| - Moves them according to their net charge and size
96
What is the velocity of migration equation for electrophoresis?
v = Ez/f
where E is the electric field strength
z is the net charge on the molecule
and f is the frictional coefficient
97
What is the standard medium for protein electrophoresis?
Polyacrylamide gel
98
Which compounds move faster in electrophoresis? Which compounds move slower?
Faster: small, highly charged, or placed in a large electric field
Slower: bigger, more convoluted, or electrically neutral
99
What is the advantage and disadvantage of PAGE electrophoresis?
- Maintains the proteins shape
| - Results are difficult to compare because the mass-to-charge ratio differs for each protein
100
What is the advantage and disadvantage of SDS-PAGE electrophoresis?
- Denatures the protein and masks the native charge so that comparison of SIZE is more accurate
- Functional protein cannot be recaptured
101
What is PAGE useful for?
Compare the size or the charge of proteins KNOWN to be similar in size from other analytical methods
102
What does SDS do to proteins? What is the only variable affecting their velocity?
- Denatures them, and creates large chains with net negative charges
- Frictional coefficient, which is only affected by mass
103
What is pI?
The pH at which the protein or amino acid is electrically neutral, with an equal number of positive and negative charges
104
What is isoelectric focusing?
- Protein placed in a gel with a pH gradient (acidic +, neutral, basic -)
105
In isoelectric focusing, where do positively charged proteins migrate? Where do negatively charged proteins migrate?
Positive: cathode (-)
Negative: anode (+)
* A+ : Anode has acidic (H+ rich) gel and a (+) charge
106
How do chromatography techniques separate protein mixtures?
On the basis of their affinity for a stationary or a mobile phase
107
Which chromatography technique is not about the affinity of a substance for the mobile and stationary phases?
Size-exclusion chromatography
108
The amount of time a compound spends in the stationary phase is referred to as what?
Retention time
109
What is the stationary phase in column chromatography? What is the mobile phase?
Stationary: beads of a polar compound (silica or alumina)
Mobile: nonpolar solvent
110
Column chromatography separates on the basis of what?
Size and polarity
111
What is ion-exchange chromatography?
Uses a charged column and a variably saline eluent. The column will bind opposite charged compounds
112
What is size-exclusion chromatography?
Relies on porous beads; larger molecules elute first because they are not trapped in the small pores
113
What is affinity chromatography?
Uses a bound receptor or ligand and an eluent with free ligand or a receptor for the protein of interest
114
What are the two potential drawbacks of affinity chromatography?
1) Protein of interest may not elute from the column because its affinity is too high
2) Can be permanently bound to the free receptor in the eluent
115
Protein structure is primarily determined through ____________ after the protein is isolated, though ____ can also be used
- X-ray crystallography - 75%
| - Nuclear Magnetic Resonance (NMR) - 25%
116
How does X-ray crystallography work?
- Protein must be isolated and crystallized
- Measures electron density
- X-ray diffraction pattern is generated; the dots can be interpreted to determine the protein's structure
117
How can amino acid composition be determined?
By simple hydrolysis and subsequent chromatographic analysis
118
How can amino acid sequencing be determined?
Sequential degradation, such as the Edman degradation
119
How can amino acid composition be determined for larger proteins?
Digestion with a synthetic reagent (ex: chymotrypsin) creates smaller fragments which can then be analyzed by electrophoresis
120
How is protein activity determined?
Monitoring a known reaction with a given concentration of substrate and comparing it to a standard, often accompanied by a colour change
121
How can protein concentration be determined?
- Almost exclusively through spectroscopy (UV spectroscopy - aromatic side chains)
- Colorimetric changes
122
Which protein concentration method is the most common? Name other methods.
- Bradford protein assay
| - Lowry reagent assay, BCA assay
123
What is the colour change in the Bradford protein assay? Which colour is associated with deprotonation and protonation?
```
Brown-green (protonation)
to Blue (deprotonation)
```
124
What factors would cause an activity assay to display lower activity than expected after concentration determination?
- Contamination of the sample with detergent or SDS could yield an artificially increased protein level
- Enzyme could have been denatured during isolation and analysis
125
T or F: The Edman degradation proceeds from the carboxyl terminus
False. The Edman degradation proceeds from the amino terminus
