Flashcards in Chapter 3: Nonenzymatic Protein Function and Protein Analysis Deck (125):
What do structural proteins compose?
The cytoskeleton, anchoring proteins, and much of the extracellular matrix
What are the 5 most common structural proteins?
Structural proteins are generally _____ by nature
What gives structural proteins their fibrous nature?
Highly repetitive secondary structure and supersecondary structure
What is supersecondary structure? What is it sometimes known as?
- Repetitive organization of secondary structural elements together
What is the structure of collagen?
Three a-helices woven together to form a secondary helix
_______ and _____ make up most of the extracellular matrix of connective tissue
Collagen and elastin
What is the primary role of elastin?
To stretch and then recoil like a spring, which restores the original shape of the tissue
In which cells are keratins found?
____ is the primary protein that makes up hair and nails
What is the primary role of keratin?
- Contributes to the mechanical integrity of the cell
- Functions as regulatory proteins
What is the most abundant protein in eukaryotic cells?
Which protein makes up microfilaments and thin filaments in myofibrils?
Actin has a + and a - side; this polarity allows for what?
Allows motor proteins to travel unidirectionally along an actin filament
Which protein makes up microtubules?
Which two structural proteins have polarity?
Actin and tubulin
What is the characteristic of motor proteins that allow them to move?
Have one or more heads capable of force generation through a conformational change
How do motor proteins have catalytic activity? What does that power?
Acting as ATPases to power movement
Name the most common applications of motor proteins.
- Muscle contraction
- Vesicle movement within cells
- Cell motility
What are the three common examples of motor proteins?
Motor proteins have transient interactions with either _____ or _____
actin or microtubules
What is the primary motor protein that interacts with actin?
Which protein is the thick filament in a myofibril?
What is responsible for the power stroke of sarcomere contraction?
Movement at the neck of myosin
What is myosin also involved in, apart from muscle contraction?
Which two motor proteins are associated with microtubules?
Kinesins and dyneins
How many heads and necks does myosin have?
- One head
- One neck
How many heads and necks do kinesins and dyneins have?
- Two heads, at least one remains attached to tubulin at all times
- 0 necks
____ play roles in aligning chromosomes during metaphase and depolymerizing microtubules during anaphase of mitosis.
____ are involved in the sliding movement of cilia and flagella.
Kinesins bring vesicles toward the _____ end of the microtubule, and dyneins bring vesicles toward the ____ end.
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
The oxyhemoglobin dissociation curve is an example of what?
A binding protein's affinity curve for its molecule of interest
Give examples of binding proteins.
Hemoglobin, calcium-binding proteins, DNA-binding proteins
Where are cell adhesion molecules (CAMs) found?
Proteins found on the surface of most cells
What is the primary role of CAMs?
Aid in the binding of the cell to the extracellular matrix or other cells
What kind of membrane proteins are CAMs?
Integral membrane proteins
What are the three major families of CAMs?
Cadherins are a group of ______ that mediate ____-dependent cell adhesion.
______ often hold similar cell types together.
______ are a group of proteins that all have two membrane-spanning chains called _ and _.
- a and B
What is the major role of integrins?
- Permit cells to adhere to proteins in the extracellular matrix
- Some also have signalling capabilities
Selectins are unique because they bind _________ molecules that project from other cell surfaces.
Which bonds are the weakest formed by the CAMs?
Where are selectins expressed?
White blood cells and endothelial cells that line blood vessels
What is the major role of selectins?
Host defense, including inflammation and white blood cell migration (immune system)
What is the most common type of protein found in the immune system?
What are antibodies also called?
What are antibodies produced by?
What is the structure of antibodies?
- Y-shaped proteins
- 2 identical heavy chains
- 2 identical light chains
What hold the heavy and light chains in antibodies together?
Disulfide linkages and noncovalent interactions
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
What are the two main regions on the antibody?
- Antigen-binding region
- Constant region
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)
What are the targets of antibodies called?
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
What is opsonization?
Marking the pathogen for destruction by other white blood cells immediately
What is agglutinating?
Clumping together the antigen and the antibody into large insoluble protein complexes that can be phagocytized and digested by macrophages
Which two proteins function in cellular motility?
Cytoskeletal (structural) and motor proteins
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
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
Which CAM does this description correspond to: two cells of the same or similar type using calcium
Which CAM does this description correspond to: one cell to proteins in the extracellular matrix
Which CAM does this description correspond to: one cell to carbohydrates, usually on the surface of other cells
Process in which cells receive and act on signals
Acting as extracellular ligands, transporters for facilitate diffusion, receptor proteins, and second messengers are example of what process?
Can biosignaling have functions in both substrate binding and enzymatic activity?
What are ion channels?
Proteins that create specific pathways for charged molecules
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
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
Name the three types of ion channels.
What are ungated ion channels?
- Always open
- Net efflux through these channels unless substrate is at equilibrium
What are voltage-gated channels?
- Open within a range of membrane potentials
- ex: membrane depolarization in neurons allows voltage-gated channels to open
How are ligand-gated channels opened?
Open in the presence of a specific binding substance
What kind of substances can open a ligand-gated channel?
Hormone or neurotransmitter
In terms of kinetics of transport, what is Km?
Solute concentration at which the transporter is functioning at half of its maximum activity
How do enzyme-linked receptors participate in cell signalling?
Through extracellular ligand binding and initiation of second messenger cascades
What are the three primary domains of enzyme-linked receptors?
- Membrane-spanning domain
- Ligand-binding domain
- Catalytic domain
What does the membrane-spanning domain do in enzyme-linked receptors?
Anchors the receptor in the cell membrane
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
When the catalytic domain of an enzyme-linked receptor is activated, what does this result in?
Second messenger cascade
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
How are GPCRs characterized?
By their seven membrane-spanning a-helices
How do GPCRs differ?
In specificity of the ligand-binding area found on the extracellular surface of the cell
In order for GCPRs to transmit signals to an effector in the cell, what do they utilize?
Heterotrimeric G protein
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
What are the three main types of G proteins?
What is the role of Gs?
Stimulates adenylate cyclase, which increases levels of cAMP in the cell
What is the role of Gi?
Inhibits adenylate cyclase, which decreases levels of cAMP in the cell
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
What are the similarities between enzyme-linked receptors and G protein-coupled receptors?
- Extracellular domain
- Transmembrane domain
- Ligand binding
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
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
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)
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
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
What is the standard medium for protein electrophoresis?
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
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
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
What is PAGE useful for?
Compare the size or the charge of proteins KNOWN to be similar in size from other analytical methods
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
What is pI?
The pH at which the protein or amino acid is electrically neutral, with an equal number of positive and negative charges
What is isoelectric focusing?
- Protein placed in a gel with a pH gradient (acidic +, neutral, basic -)
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
How do chromatography techniques separate protein mixtures?
On the basis of their affinity for a stationary or a mobile phase
Which chromatography technique is not about the affinity of a substance for the mobile and stationary phases?
The amount of time a compound spends in the stationary phase is referred to as what?
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
Column chromatography separates on the basis of what?
Size and polarity
What is ion-exchange chromatography?
Uses a charged column and a variably saline eluent. The column will bind opposite charged compounds
What is size-exclusion chromatography?
Relies on porous beads; larger molecules elute first because they are not trapped in the small pores
What is affinity chromatography?
Uses a bound receptor or ligand and an eluent with free ligand or a receptor for the protein of interest
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
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%
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
How can amino acid composition be determined?
By simple hydrolysis and subsequent chromatographic analysis
How can amino acid sequencing be determined?
Sequential degradation, such as the Edman degradation
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
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
How can protein concentration be determined?
- Almost exclusively through spectroscopy (UV spectroscopy - aromatic side chains)
- Colorimetric changes
Which protein concentration method is the most common? Name other methods.
- Bradford protein assay
- Lowry reagent assay, BCA assay
What is the colour change in the Bradford protein assay? Which colour is associated with deprotonation and protonation?
to Blue (deprotonation)
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