Study BMB 460 Quiz 1 Flash Cards

 
Pile Management Card
BMB 460 Quiz 1

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fluoresence from which fluorophore is actually being measured
NBD (green)
higher when unquenched and not as close together
Which fluorophore does the quenching and which is quenched?
detects for NBD forescence (green)
quenched if too tight together
what are the two fluorophores used to do FRET in these experiments
NBD and rhodamine
SNARE energy
The SNAREs form a 4 stranded coiled-coil and a very tight “zipping up” of two membranes

where the energy for fusion comes from.
Name the t-SNAREs that act in synaptic
vesicle fusion
syntaxin and Snap25
Name the v-SNARE that acts in synaptic vesicle fusion.
synaptobrevin
If a protein wants to know whether it is in the nucleus what does it need to recognize?
??
Is [Ran-GTP] higher in the nucleus or the cytoplasm?
Is [Ran-GDP] higher in the nucleus or the cytoplasm?
•RanGTP is present in the nucleus (due to action
of GEF called RCC1)
What is the GAP specific for Ran?
Rna1p and RanBP1
causes Ran to hydrolyze GTP
What is the GEF specific for Ran? () where is it? What does it bind? Why is
this important?
Rcc1
-in the nucleus
-
which component of the dimeric receptor binds the nucleoporin protein in the nuclear
pore?
•Importin-α/β/ cargo complex binds nucleoporin
via importin-β
which component of the dimeric (alpha/beta) receptor binds the NLS?
•Importin-α/cargo binds importin-β
what does a classical NLS look like?
•4 to 8 amino acid
-rich in positively charged amino acids
•NLS can be situated anywhere in
the protein
mutation of a NLS can demonstrate that the NLS is necessary for nuclear localization
transfer of a NLS to a gold bead is an experiment that can demonstrate that a NLS is
sufficient for nuclear localization. Understand how this is not the same thing
importin alpha and beta
form the heterodimeric nuclear import receptor for proteins with
a classic NLS.
What proteins are capable of crossing the nuclear membranes?
-must have nuclear localization signal
-or a small protein, less than 60kD
nulear PM info
-double membrane
-perforated by nuclear pores
-lumen is continuous with the ER lumen
-phospholipid bilayer curves around at the nuclear pore
NaF
inhibits glycolysis.
Dinitrophenol
uncoupler - dissipates proton gradient and reduces ATP by >80%.
TCA precipitation assay protocol
-incubate cells in 3H-leucine, which will be incorporated into proteins.
-precipitate proteins using TCA.
• TCA precipitates proteins and nucleic acids but not amino acids and
nucleotides.
• Measure %radioactivity that is incorporated into proteins (precip/precip+sup)
• Compare this ratio in presence and absence of inhibitor

%TCA -%TCA w/ inhibitor / %TCA
Assaying potency of the inhibitors -
TCA precipitation
What do cyclohexamide and puromycin do? What idea is being tested by using these?
puromycin or
cyclohexamide (protein synthesis inhibitors).
Why do human antigens appear to be moving faster?
cause
mouse/human colors
Mouse: flourescen (green)
Human: rhodamein (red)
What proteins are recognized by the antibodies in the alloantiserum?
HUman: VA-2 cells
Mouse: MHC molecules
How were nonspecific antibodies removed from each population?
pre-clear
What is a cell line?
To maintain cells in culture requires that the cells be immortalized so that they can
proliferate almost indefinitely rather than dying out. We often call these
immortalized cells a “cell line”.
Where are sugar
modifications found on transmembrane proteins (outside of the plasma membrane or
inside, lumenal side of Golgi or cytoplasmic side)?
Glycosylated on the noncytoplasmic face
Where are Golgi membrane proteins integrated into the bilayer? What about
mitochondrial proteins?
Mitochondria and peroxisomes - posttranslational transport.
• ER - cotranslational transport (MBOC 12-44 and 12-45).
Does the ER lumenal or cytoplasmic portion of a protein
destined for the plasma membrane end up on the cell surface?
N-terminus in the ER lumen will end up on the cell surface
• N-terminus in the cytoplasm will end up in the cytoplasm
What organelles can insert proteins with an alpha-helical transmembrane protein into the
bilayer?
golgi, ER?
What is an integral membrane protein? (How could you dissociate an integral membrane protein from a lipid bilayer in an experiment?)
require detergent to dissociate
• Integral membrane proteins are attached through some kind of hydrophobic connection
with hydrophobic core of the bilayer.
What is a peripheral membrane protein? (How could you dissociate a peripheral membrane protein from a lipid bilayer in an experiment?
dissociate with high salt or pH changes.
• Associations involve ionic interactions but not covalent interactions.
Micelle?
sequestering the hydrophobic single tail regions in the micelle centre.
What effect will SDS have on a lipid bilayer? ... on a transmembrane protein?
SDS denatures and separates subunits - inactivates
Detergents are themselves amphipathic molecules, and this allows them to coat
hydrophobic surfaces
Where is most of the cellular cholesterol found?
What effect does it have on the membrane?
-PM
-thicker and less permeable
why can cholesterol "flip-flop" while phospholipids can't?
Because of the limited hydrophilicity of the head group,
cholesterol is the one lipid that can flip from one leaflet to
another without the requirement of a special transporter
Less abundant components are
cholesterol -
inositol phospholipids
Major membrane lipid components are
phosphatidylserine
phosphatidylcholine
phosphatidylethanolamine
sphingomyelin
What is a leaflet?
Each layer of a membrane bilayer is often called a leaflet
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