Chapter 3: Nonenzymatic Protein Function / Protein Analysis Flashcards

Question

Biosignalling

Answer 1

process in which cells receive and act on signals

Answer 2

transmembrane proteins that provide a pathway for ions to enter the cell facilitated diffusion of molecules down a concentration gradient

Answer 3

ungated voltage gated ligand-gated

Answer 4

ion channels that are always open

Answer 5

open or close based on membrane potential charge near the channel

Answer 6

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)

Answer 7

membrane receptors that also display catalytic activity in response to ligand binding often participate in cell signaling through initiation of second messenger cascades

Answer 8

membrane-spanning domain ligand-binding domain catalytic domain

Answer 9

anchors the receptor in the cell membrane

Answer 10

stimulated by the appropriate ligand and induces a conformational change that activates the catalytic domain

Answer 11

activates cellular enzymes; initiates second messenger cascade

Answer 12

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)

Answer 13

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

Answer 14

**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

Answer 15

3 subunits: alpha, beta, and gamma inactive form: alpha subunit binds GDP and is in a complex with beta and gamma subunits active form: ...

Answer 16

* 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

Answer 17

Cell lysis followed by homogenization

Answer 18

crushing, grinding, or blending the tissues of interest into an evenly mixed solution

Answer 19

subjects compounds to an electric field, which moves them according to their net charge and size

Answer 20

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)

Answer 21

**v = Ez/f** * v = velocity of molecule * E = electric field strength * z = net charge of molecule * f = frictional coefficient

Answer 22

polyacrylamide gel

Answer 23

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

Answer 24

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

Answer 25

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

Answer 26

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

Answer 27

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

Answer 28

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

Answer 29

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

Answer 30

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)

Answer 31

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

Answer 32

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)

Answer 33

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

Answer 34

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)

Answer 35

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)