Chapter 3: Nonenzymatic Protein Function and Protein Analysis Flashcards
1
Q
A repetitive organization of secondary strucutral elements together sometimes referred to as a ________.
A
motif
2
Q
____________ has a characteristic trihelical fiber (three left-handed helices woven together to form a secondary right-handed helix) and makes up most of the extracellular matrix of connective tissue.
A
Collagen
3
Q
________ is another important component of the extracellular matrix of connective tissue. Its primary role is to stretch and then recoil like a spring.
A
Elastin
4
Q
____________ are intermediate filament proteins found in epithelial cells. Contribute to mechanical integrity of the cell and also function as regulatory proteins. Makes up hair and nails.
A
Keratins
5
Q
________ is a protein that makes up microfilaments and the thin filaments in myofibrils. Most abundant protein in eukaryotic cells.
A
Actin
6
Q
What allows motor proteins to travel unidirectionally on actin?
A
They have polarity; has a positive and negative side.
7
Q
____________ is the protein that makes up microtubules.
A
Tubulin
8
Q
4 functions
What do microtubules do?
A
- provide structure
- separate chromosomes in cell cyle
- intracellular transport with kinesin and dynein
9
Q
Does tubulin have polarity?
A
Yes!
10
Q
What other activity do motor proteins also exhibit? What kind?
A
Enzymatic activity; ATPases
11
Q
____________ power the conformational change necessary for motor function.
A
ATPases
12
Q
____________ is the primary motor protein that interacts with actin. It is involved in cellular transport and has a head/neck.
A
Myosin
13
Q
The movement at the ________ of myosin is responsible for the power stroke of sarcomere contraction.
A
neck
14
Q
____________ and ____________ are the motor proteins associated with microtubules.
A
Kinesins and dyneins
15
Q
How many heads do kinesin/dynein have? What are they attached to?
A
2; at least one is attached to tubulin at all times
16
Q
What role do kinesins play?
A
Align chromosomes during metaphase and depolymerase microtubules during anaphase
17
Q
What do dyneins do?
A
Sliding movement of cilia and flagella
18
Q
What do both kinesin and dynein do?
A
Vesicle transport
19
Q
Which way does kinesin go and which way does dynein go? What is an example of where this occurs?
A
Kinesin goes towrad the positive end of the MT, and dynein goes toward the negative end
occurs when kinesins bring neurotransmitter vesicles to the positive end of axonal microtubules, dynein brings vesicles of waste/recycled neurotransmitter back toward the negative end of the microtubule
20
Q
____________ ____________ include hemoglobin, calcium-binding proteins, DNA-binding proteins (often transcription factors), and others. These have the role of stabilizing functions and transporting/sequestering molecules by binding to them.
A
Binding proteins
21
Q
Each binding protein has an ____________ ________ for its molecule of interest.
A
affinity curve
22
Q
What is unique about the affinity curves of transport proteins?
A
They have varying affinity in different conditions so they can bind or unbind their targets
23
Q
________ ____________ ____________ are proteins found on the surface of most cells and aid in binding the cell to the extracellular matrix or other cells.
A
cell adhesion molecules (CAMs)
24
Q
What are the 3 major families of CAMs?
CIS
A
- Cadherins
- Integrins
- Selectins
25
____________ are a group of glycoproteins that mediate calcium-dependent cell adhesion.
Cadherins
26
Cadherins often hold similar cell types together and have type-specific cadherins. What are some examples?
E-cadherin for epithelial cells, N-cadherin for nerve cells
27
____________ are a group of proteins that all have 2 membrane-spanning chains called α and β. These chains are important in binding to and communicating with the extracellular matrix.
Integrins
28
Integrins also play a role in ____________ ____________ and can greatly impact cellular function by promoting what processes?
cellular signaling; cell division, apoptosis, others
29
____________ bind to carbohydrate molecules that project from other cell surfaces.
Selectins
30
Which CAM forms the weakest bonds?
Selectins
31
Selectins are expressed on which cells?
white blood cells, endothelial cells that line blood vessels
32
Antibodies are also called ________________, and they are proteins produced by ____-cells that function to neutralize targets in the body, like toxins and bacteria, and then recruit other cells to help eliminate the threat.
immunoglobulins (Ig); B-cells
33
What shape are antibody proteins?
Y
34
What are antibodies made up of?
2 identical heavy chains and 2 identical light chains
35
What holds heavy and light chains together?
disulfide linkages and noncovalent interactions
36
Each antibody has an ____________-____________ region at the tips of the Y.
antigen-binding region
37
What is in the antigen-binding region?
Specific polypeptide sequences that will bind one, and only one, specific antigenic sequence.
38
The remaining part of the antibody molecule (aside from the antigen-binding region) is known as the ____________ region.
constant
39
What is the constant region of an antibody responsible for?
Recruitment and binding of other cells of the immune system, such as macrophages
40
What are the targets of antibodies called?
Antigens
41
What 3 things can happen when an antibody binds to an antigen?
1. Antigen is neutralized, making the pathogen or toxin unable to exert its effect on the body
2. The pathogen is marked for destruction by other white blood cells
3. The antigen and antibody are clumped together into large insoluble protein complexes that can be phagocytized and digested by macrophages
42
When antibodies mark pathogens for destruction by WBCs, what is this process called?
opsonization
43
When the antigen and antibody are clumped together into large insoluble protein complexes, this process is called?
Agglutinating
44
____________ is a process in which cells receive and act on signals.
biosignaling
45
________ ____________ are proteins that create specific pathways for charged molecules.
Ion channels
46
____________ ____________, a type of passive transport, is the difficusion of molecules down a concentration gradient through a pore in the membrane created by this transmembrane protein.
Facilitated diffiusion
47
What 3 types of molecules is facilitated diffusion used for?
Molecules that are impermeable to the membrane:
1. Large
2. Polar
3. Charged
48
What are the 3 main types of ion channels?
1. ungated
2. voltage-gated
3. ligand-gated
49
____________- ________ channels are regulated by the membrane potential change near the channel.
voltage-gated
50
________-________ channels open or close due to the binding of a specific substance or ligand to the channel
ligand-gated
51
What parameters that apply to enzyemes are also applicable to transporters like ion channels?
Km and vmax
52
____________-________ receptors are membrane receptors that may also display catalytic activity in response to ligand binding.
enzyme-linked
53
What are the 3 primary protein domains of enzyme-linked receptors?
1. membrane-spanning domain
2. ligand-binding domain
3. catalytic domain
54
The ____________-____________ domain anchors the receptor in the cell membrane.
membrane-spanning
55
The ________-________ domain is stimulated by the appropriate ligand and induces a conformation change that activates the ________ domain.
ligand-binding; catalytic
56
After the catalytic domain of an enzyme-linked receptor is activated, a ________ ____________ cascade is initiated.
second messenger
57
What is an example of enzyme-linked receptor?
receptor tyrosine kinase
58
____ ________-________ receptors are a large family of integral membrane proteins involved in signal transduction.
G protein-coupled (GPCRs)
59
What characterizes GPCRs?
7 membrane-spanning alpha-helices
60
In order for GPCRs to transmit signals to an effector in the cell, they uitilize a ________________ ____ protein.
heterotrimeric G
61
G proteins are named for their intracellular link to ____________ ____________.
guanine nucleotides (GDP, GTP)
62
The binding of a ligand increases the affinity of a GPCR for what?
G protein
63
When a G protein binds to a GPCR, what happens?
The GPCR is switched to the active state and affects the intracellular signaling pathway
64
What are the 3 main types of G proteins?
Gs, Gi, and Gq
65
What does Gs do?
Stimulates adenylate cyclase, which increases levels of cAMP in the cell
| G**s** stimulates
66
What does Gi do?
Inhibits adenylate cyclase, which decreases levels of cAMP in the cell
67
What does Gq do?
Activates phospholipase C
| Mind your p's and q's - Gq activates phospholipase C
68
What does phospholipase C do when it is activated by Gq?
It cleaves a phospholipid from the membrane to form PIP2
69
What happens to PIP2?
It is cleaved into DAG and IP3
70
What does IP3 do?
It opens calcium channels in the endosplasmic reticulum, which increases calcium levels in the cell
71
What are the 3 subunits that comprise the G protein?
alpha, beta, and gamma
72
When the ____ subunit of a G protein is inactive, it binds GDP and is in a complex with the beta and gamma subunits.
alpha
73
When a ligand binds to the GPCR, the receptor becomes activated and engages what?
The corresponding G protein
74
Once GDP is replaced with GTP, the alpha subunit ________________ from the beta and gamma subunits.
dissociates
75
An activated alpha subunit can then alter the activity of ____________ ____________.
adenylate cyclase
76
Once GTP on an activated alpha subunit is dephosphorylated to GDP, it does what?
Rebinds to alpha and gamma subunits and the G protein is inactive
77
Proteins and other biomolecules are isolated by using cell lysis and ________________. Then ____________________ can be used to isolate proteins from much smaller molecules before other isolation techniques must be employed.
homogenization, centrifugation
78
________________ is a method of protein separation which works by subjecting compounds to an electric field, which moves them according to their net charge and size.
Electrophoresis
79
How does elecrophoresis work?
Negatively charged compounds migrate toward the positive anode, and the positively charged compounds migrate toward the negative cathode
80
The velocity of a molecule undergoing electrophoresis is called ________________ ________________.
migration velocity
81
Migration velocity is directly proportional to what?
Electric field (E) and charge (z)
82
What is migration velocity inversely proportional to?
frictional coefficient, f, which dpeends on mass and shape of migrating molecules
83
What is the equation for migration velocity?
v = Ez/f
84
What is the standrd medium for protein electrophoresis?
polyacrylamide gel
85
A smaller molecules moves (faster/slower?) through gel.
faster
86
Polyacrylamide gel electrophoresis (PAGE) is limited by what?
The varying mass to charge and mass to size ratios because multiple different proteins may experience the same level of migration
87
What is PAGE most useful for?
Comparing the molecular size or the charge of proteins known to be similar in size from other analytic methods like SDS-PAGE or size-exclusion chromatography
88
____________ ____________ ____________-PAGE electrophoresis is a useful tool because it separates proteins on the basis of relative molecular mass alone.
sodium dodecyl sulfate
89
What is SDS?
A detergent that disrupts all noncvalent interactions. It binds to proteins and creates large chains with net negative charges, which neutralizes a protein's original charged and denaturing the protein.
90
How does SDS-PAGE work?
Since SDS removes charge, the proteins move through the gel and are only affected by E and f, which depends on mass.
91
Proteins can also be separated on the basis of their ________________ ________, which is the pH at which the protein or amino acid is electrically neutral.
isoelectric point
92
For most amino acids, the zwitterion form occurs when the amino group is ____________, the carboxyl group is ____________, and any side chain is electrically ____________. What are the exceptions?
protonated, deprotonated, neutral
arginine and lysine - they have basic side chains
93
For arginine and lysine, they have side chains with (higher/lower?) pKa values than their amino groups. The zwitterion form occurs when the amino group is ________________, side chain is ____________, and carboxyl group is ____________.
higher; deprotonated, protonated, deprotonated
94
____________ ____________ exploits the acidic and basic properties of amino acids by separating on the basis of isoelectric point.
isoelectric focusing
95
How does isoelectric focusing work?
| read p102
proteins are placed in a gel with a pH gradient, where acidic is at positive, basic is at negative, and neutral is in the middle.
An electric field is generated. Proteins that are positively charged will migrate toward the cathode and proteins that are negative will migrate toward the anode (A+, b/c H+ rich).
As the protein reaches the portion of the gel where pH is equal to pI, the protein will become neutral and stop moving.
96
____________ uses physical and chemical properties to separate and identify compounds from a complex mixture.
chromatography
97
How does chromatography work?
The more similar the compound is to its surroundings, the more it will stick to and move slowly through its surroundings.
98
Chromatography works by placing the sample onto a solid medium called the ________________ ____________ or ____________.
stationary phase, adsorbent
99
After placing a sample onto the stationary phase, the ____________ phase is run through the staionary phase, which allows the sample to run through the stationary phase and ____________.
mobile; elute
100
The amount of time that a compound spends in the stationary phase is referred to as ____________ ________.
retention time
101
Varying retenting times of each compound in the solution results in separation of the components within the stationary phase, or ________________.
partitioning
102
In ________ ________________, a column is filled with silica or alumina beads as an adsorbent, and gravity moves the solvent and compounds down the column.
column chromatography
103
How does column chromatography work?
The less polar a compound, the faster it elutes. Solvent polarity, pH, or salinity can be changed to help elute the protein of interest.
104
In ____-________ chromatography, the beads are coated with charged substances, so they attract/bind compounds that have an opposite charge.
ion-exchange
105
What is used to elute charged molecules that have stuck to an ion-exchange column?
salt gradient
106
In ________-____________ chromatography, the beads used in the column contain tiny pores of varying sizes.
size-exclusion
107
In size-exclusion chromatography, what happens to small compounds?
they get stuck in the pores of beads and are slowed down, meanwhile large compounds move around these beads
108
In ____________ chromatography, columns can be customized to bind any protein of interest by creating a column with high affinity for that protein.
affinity
109
How is affinity chromatography accomplished?
Coating beads with a receptor that binds the protein or a specific antibody to the protein; the protein is retained in the column and then eluted by washing the column with a free receptor (or target or antibody), which competes with the bead-bound receptor to free the protein
110
Protein structure can be determined by what two processes?
X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy
111
Before crystallographic analysis, what must be done?
The protein must be isolated and crystallized
112
How does crystallography work?
It measures electron density on an extremely high-resolution scale and can also be used for nucleic acids. This generates an X-ray diffraction pattern, which has dots that can be interpreted to determine protein structure
113
Amino acids that compose a protein can be determined how?
Complete protein hydrolysis and subsequent chromatographic analysis
114
To determine the primary structure of a protein, what must occur?
Sequential digestion of the protein with specific cleavage enzymes
115
Small proteins are best analyzed with the ________ ________________, which uses cleavage to sequence proteins of up to 50 to 70 amino acids.
Edman degradation
116
Edman degradation works by doing what?
Selectively and sequentially removing the N-terminal amino acid of the protein, which can be analyzed via mass spectroscopy
117
For larger proteins, a ____________ ____________ may be used.
synthetic
118
In larger proteins, amino acid composition can be done by using a synthetic reagent to digest, which cleaves proteins at specific amino acid residues, which does what?
Creates smaller fragments that can then be analyzed by electrophoresis or Edman degradaation
119
Protein activity is generally determined how?
by monitoring a known reaction with a given concentration of substrate and comparing it to a standard; activity is correlated with concentration
120
Concentration is determined almost exclusively through ________________
spectroscopy
121
Because proteins contain ________ side chains, they can be analyzed with UV spectroscopy without any treatment. This type of analysis is particularly sensitive to sample contaminants.
aromatic
122
Proteins can also cause colorimetric changes with specific reactions/ Give examples of assays.
1. bicinchoninic acid (BCA) assay,
2. Lowry reagent assay
3. Bradford assay
123
The ____________ protein assay mixes a protein in solution with Coomassie brilliant blue dye.
Bradford
124
How does Coomassie work?
It gives up protons upon binding to amino acid groups, turning blue in the process. Ionic attractions between dye and protein stabilize the blue form.
Increased protein concentrations correspond to a larger concentration of blue dye!
125
How is concentration determined in Bradford assay?
Create a standard curve with known concentration reagent, then determine concentration based on standard.
