Chapter 3: Nonenzymatic Protein Function / Protein Analysis Flashcards
List the 5 primary structural proteins:
collagen, elastin, keratins, actin, and tubulin
Collagen
has a characteristic trihelical fiber
makes up most of the extracellular matrix of connective tissue
found throughout the body and provides STRENGTH AND FLEXIBILITY
Elastin
one of the primary structures of protein in the body
important component of the extracellular matrix of connective tissue
for STRETCH AND RECOIL to restore original shape of the tissue
Keratin
one of the primary structures of protein in the body
intermediate filament proteins found in epithelial cells
contribute to mechanical integrity of the call and also function as regulatory proteins
primary protein that makes up HAIR AND NAILS
Actin
makes up ** microfilaments** and **THIN FILAMENTS **in myofibrils
have ** polarity ** to allow motor proteins to travel unidirectionally along an actin filament
what is the most abundant protein in eukaryotic cells?
actin
Tubulin
makes up microtubules
3 microtubule functions
provide structure, chromosome separation in mitosis and meiosis, intracellular transport with kinesin and dynein
Motor proteins
responsible for muscle contraction and cellular movement (cilia / flagell)
may display enzymatic activity such as ATPases (power conformational change for motor function)
list 3 common motor proteins
myosin
kinesin
dynein
Myosin
- primary motor protein that interacts with actin (thick filament in myofibril)
- can be involved in cellular transport
- each subunit has a single head and neck
- movement at the neck is responsible for the power stroke of sarcomere contraction
Kinesins and dyneins
the motor proteins associated with microtubules
they have two heads; at least one remains attached to tubulin at all times
Kinesin moves towards..
the positive end of the microtubule / the outer membrane
Dyneins move toward
the negative end of the microtubule / toward the nucleus
Binding proteins
acts as an agent to bind two or more molecules together
??
Cell Adhesion Molecules (CAMs)
Allow cells to bind to other cells or surfaces
3 categories of cell adhesion molecules
cadherins
integrin
selectins
Cadherins
calcium dependent glycoproteins that hold similar cells together
Integrin
ADHERE A CELL TO A PROTEIN
play important role in cell signaling
Selectins
ADHERE A CELL TO A CARB
most commonly used in the immune system
Immunoglobulins (antibodies)
neutralize targets in the body, such as toxins and bacteria, and then recruit other cells to help eliminate the threat
Y-shaped proteins made up of 2 identical heavy chains and 2 identical light chains
Antigen binding region
a region on the tips of the Y with specific polypeptide sequences that will bind ONE and only one specific antigenic sequence
Antigen constant region
the part of the antigen that is NOT the binding region
involved in recruitment of other immune system cells (ex. macrophages)
What are the 3 possible outcomes once an antibody binds to its target antigen?
-
neutralize the antigen
- makes the pathogen unable to exert its effect on the body
- marks the pathogen for destruction by other white blood cells immediately (opsinization)
- agglutinate (clump together) the antigen and antibody into large insoluble protein complexes that can be phagocytized and digested by macrophages
Biosignalling
process in which cells receive and act on signals
Ion channels
transmembrane proteins that provide a pathway for ions to enter the cell
facilitated diffusion of molecules down a concentration gradient
3 main groups of ion channels
ungated
voltage gated
ligand-gated
Ungated ion channels
ion channels that are always open
Voltage-gated channels
open or close based on membrane potential charge near the channel
Ligand-gated Channels
binding of a specific ligand to the channel causes it to open or close
ex. neurotransmitters bind to post-synaptic channels (ex. GABA opens chloride channels)
Enzyme-linked receptors
membrane receptors that also display catalytic activity in response to ligand binding
often participate in cell signaling through initiation of second messenger cascades
3 primary protein domains of enzyme-linked receptors
membrane-spanning domain
ligand-binding domain
catalytic domain
Membrane-spanning domain of enzyme-linked receptors
anchors the receptor in the cell membrane
Ligand-binding domain of enzyme-linked receptors
stimulated by the appropriate ligand and induces a conformational change that activates the catalytic domain
Catalytic domain of enzyme-linked receptors
activates cellular enzymes; initiates second messenger cascade
One of the most common enzyme-linked (catalytic) receptors
receptor tyrosine kinases (RTKs)
composed of a monomer that dimerizes upon ligand binding
the dimer is the active form that phosphorylates additional cellular enzymes (included the receptor itself)
G protein-coupled receptors (GPCRs)
large family of integral membrane proteins involved in signal transduction
7 transmembrane alpha-helices
interact with heterotrimeric G proteins to transmit signals to effector cells
ligand binding increases affinity of the receptor for the G protein
binding of G protein switches to active state and affects intercellular signalling pathway
3 main types of G proteins:
Gs - stimulates adenylate cyclase which i_ncreases levels of cAMP_ in the cell
Gi - inhibits adenylate cyclase which decreases levels of cAMP in the cell
Gq - activates phospholipase C which cleaves a phospholipid from the membrane to form PIP2; PIP2 is cleaved to DAG and IP3; IP3 opens calcium channels in the ER to increase calcium levels in the cell
G protein structure
3 subunits: alpha, beta, and gamma
inactive form: alpha subunit binds GDP and is in a complex with beta and gamma subunits
active form: …
G protein activation:
- inactive form: alpha subunit binds GDP and is in a complex with B and Y subunits
- when ligand binds to GPCR, receptor becomes activated and engages the G protein
- GDP on G protein is replaced with GTP
- alpha subunit dissociate from B and Y subunits
- activated alpha subunit alters activity of adenylate cyclase (As vs Ai)
- Once GTP on A subunit is dephosphorylated to GDP, A subunit will bind to B and Y subunits, rendering the G protein inactive again
How are proteins isolated from body tissues / cell cultures?
Cell lysis followed by homogenization
Homogenization
crushing, grinding, or blending the tissues of interest into an evenly mixed solution
Centrifugation
Electrophoresis
subjects compounds to an electric field, which moves them according to their net charge and size
Electrophoresis
uses a gel matrix to observe the migration of proteins in response to an electric field
(proteins move according to their net charge and size)
Equation relating electrophoresis factors
v = Ez/f
- v = velocity of molecule
- E = electric field strength
- z = net charge of molecule
- f = frictional coefficient
What is a standard medium for protein electrophoresis?
polyacrylamide gel
Native PAGE
polyacrylamide gel electrophoresis (PAGE) a method for analyzing proteins in their native states
maintains the proteins shape
results are difficult to compare because the mass-to-charge and mass-to-size ratios differ for each cellular protein
the functional native protein may be recovered from the gel, but only if the gel has not been stained (most stains denature proteins)
most useful for: compare analytic methods like SDS-PAGE or size-exclusion chromatography
SDS Page
sodium dodecyl sulfate (SDS) PAGE
SDS denatures the proteins and masks the native charge
allows for comparison of size alone
the functional protein cannot be recaptured from the gel
Isoelectric Focusing
separates proteins by their isoelectric point (pI)
the protein migrates toward and electrode until it reaches a region of the gel where pH = pI of the protein
recall: PI = pH at which the protein is electrically neutral (zwitterion)
acidic gel @ positive anode; basic gel @ negative cathode
Chromatography
separates protein mixtures on the basis of their affinity for a stationary phase or a mobile phase
the more similar the compound is to its surroundings (polarity, charge, etc.) the more it will stick to its surroundings (slower movement)
preferred over electrophoresis when large amounts of protein are being separated
Column Chromatography
uses beads of a polar compound, like silica or alumina (stationary phase) with a non polar solvent (mobile phase)
the less polar a compound, the faster it will elute down the column through the stationary phase
Ion-exchange chromatography
the beads in the column are coated with charged substances, so they attract or bind compounds with opposite charge
uses a charged column and a variably saline eluent
Size-exclusion chromatography
the beads in the column contain tiny pores of varying sizes, which allows small compounds to enter the beads (slowing them down)
large compounds can’t fit into the pores, so they travel around the beads and through the column faster
Affinity chromatography
the columns can be customized to have high affinity for a specific protein of interest
ex. coating beads with receptors that bind the proteins (the protein is retained in the column)
How is protein structure determined?
primarily by X-ray crystallography: after the protein is isolated and crystallized; measures electron density on an very high resolution scale; can also be used for nucleic acids
Nuclear magnetic resonance (NMR) spectroscopy may also be used
How is amino acid composition determined?
complete protein hydrolysis and subsequent chromatographic analysis
BUT amino acid sequencing requires sequential degradation, such as the Edman degradation (sequentially removes the N-terminal amino acid of the protein)
How is protein activity analyzed?
protein activity is generally determined by monitoring a known reaction with a given concentration of substrate and comparing it to a standard
reactions with a colour change have a particular applicability because microarrays can rapidly identify the samples from a chromatographic analysis that contains the compound of interest
How is protein concentration determined?
colorimetrically, either by UV spectroscopy (b/c proteins contain aromatic side chains) or through a colour change reaction (BCA assay, Lowry reagent assay, Bradford protein assay)
Bradford protein assay
mixes a protein in solution w/ a dye that is protonated and green-brown prior to mixing
the dye turns blue as it gives up protons to the amino acid groups
increased protein concentrations = larger concentration of blue dye in solution
(samples with known concentrations are first used to create and absorbance standard curve)
