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205 Cards in this Set

  • Front
  • Back
Primary Structure
Polypeptides fold into natvie conformation.
Helix held together in backbone by
partial charges
Example of type of bonds in backbone
hydrogen bonds
Secondary Structures
folding by h-bonds along backbone
Most interactions are
noncovalent
backbone linked
covalently
Tertiary Structure
folding patterns based on a single poly. that results mainly from chemial properties of R-groups.
folding depends on 1) and 2)
1) environment 2)R-group interactions
Enviornment usually 1) and 2)
1) aqueous
Hydrophillic
polar
Hydrophobic
non-polar
Domains
distinct regions of a native structure usually have own subfunctions SINGLE POLYPEPTIDE
Different from Quat. structure b/c?
single vs. muti polypeptides
Quat. structures
result from non-covalent attractions b/w separate subunits (polypeptides)
associations are freely revesible
non-covalent interactions
Heat's affect on bonds?
breaks bonds, all proteins are heat sensitive
Ligand
molecule that binds to a protein
Structual Complementary
"fits"
LTP
Lipid Transfer protein; transfers polypeptides b/w molecules; phosolipid carrying protein
Chemical Complementary
pH can affect
Ex of Chemical Comp.
Glyercol enters RBC pH sensitive optimal 6.5
what residue is left at binding site?
Histidine
Aqueporin
structure by which glycerol enters RBC
all proteins pH sensitive 1) and 2)
1) usually b/c of small charge change at one or two spots 2) not due to gross change in shape
What about lysomomes and pH?
Low internal pH is used to turn on activity
Membranes are made of 3 things:
lipids, proteins, carbos
high activity= ________ proteins
more
low activity-__________ proteins
less
Intrinsic proteins solubalized ?
by detergents
which are water soluble?
peripheral
EDTA
chelator that shakes the peripheral proteins free from the membrane
Extrinisic Proteins-
bind usually through a intrinsic protein through non-covalent linking of subgroups.
Lipid Anchors
what int. use to bind to membrane
Lipid anchor on which face?
p-face
GPI anchor which face?
always on e face
most intrinsic proteins have which type of domains?
transmembrane domains
what are the most common membrane spreading protein
alpha helix
TMGP
Multipass Transmembrane glycoprotein
membrane spanning helices are _ AA long
20 Amino Acids
membrane spanning R-groups are hydro-
phobic (blocked by charges of backbon)
Bacteriorhodopsin hydropathy plot
transmembrane - multipass
aquaporin
protein used to transport glycerol into the cell
main residue at binding site
histidine
in ___ low pH is used to ____ on a cell
lysosome; turn on
detergents?
solublize membrane for intrinsic proteins
which type of protein is water soluble
peripheral
non covalent binding to intrinsic proteins through which association
quaternary
lipid anchors
sometimes holds intrinsic proteins
which face are lipid anchors located?
p-face
GPI anchor?
always on the E-face; more elaborate
what type of domain do integral proteins have?
transmembrane
most common transmembrane protein?
alpha-helix
TMGP
Multipass transmembrane glycoprotein
how long are typical membrane spanning proteins?
20 AA long
membrane spanning R-groups are hydro-
phobic
Hydropothy Plot
shows multipass or single pass protein
Bacteriorhodopsin
multipass transmembrane protein
least common protein
monotropic integral protein
RBC easily isolated why? 1 and 2
1) abundant 2) easily manupliated
SDS
detergent that dissolves whole membranes; then wraps around protein where membrane used to be; continues to be shielded
Coomassie Blue
general protein stain
EDTA
breaks away for peripherals
Spectins A and B
very large membrane proteins; characteristics of RBC
Spectins purpose?
form a network on the p-face; keeps unique concave shape in tact
Band 3- general
Major integral protein; lots of them in RBC;
Band 3 does what?
is a transmembrane chloride/carbonate anion exchanger; allows enterance and exit for these
Lectins- def.
highly specific carborhydrate binding proteins
Lectins- bind to what?
oligasacchrides in membrane glycoproteins
polypeptide stain shows?
all proteins
flourscent lectin shows?
only glycoproteins
PAS-1
the major RBC glycoprotein
How can you figure out topoolgoy?
differential effects of reacting agents on the two membrance faces
Partial Protelyase
releasing tyrpsin to one side of the membrane, and then examinig which proteins were affected, then those are on that side of the membrane.
Galactose Oxidase Tritation
labels galactose in oligosaccharides (labels carrot tops)
Lactoperoxidase Iodination
labels polypeptides (proteins) at tyrosines
RBC labeled by which mechanism? where?
Lactoperoxidase Iodination; externally or internally
Band 3 labels by which type? where?
Equally external and internal Iodination
General Lesson #1
all membrane proteins are asymmetrically placed or orientated
General lesson #2:
Transmembrane proteins always glycoproteins
Which sides bears oligosacchride?
E-face
Are membranes flexible?
YES
Def. Fluidity
ability to lipids and molecules to move freely
Temperature and Fluidity
temp. sensitive
Phase Transition Point
Meliting Point
With more than type of protein
Broader melting point
Same type of protein (artifical)
Sharp melting point
How can you figure out topoolgoy?
differential effects of reacting agents on the two membrance faces
Partial Protelyase
releasing tyrpsin to one side of the membrane, and then examinig which proteins were affected, then those are on that side of the membrane.
How can you figure out topoolgoy?
differential effects of reacting agents on the two membrance faces
Galactose Oxidase Tritation
labels galactose in oligosaccharides (labels carrot tops)
Partial Protelyase
releasing tyrpsin to one side of the membrane, and then examinig which proteins were affected, then those are on that side of the membrane.
Lactoperoxidase Iodination
labels polypeptides (proteins) at tyrosines
Galactose Oxidase Tritation
labels galactose in oligosaccharides (labels carrot tops)
RBC labeled by which mechanism? where?
Lactoperoxidase Iodination; externally or internally
Lactoperoxidase Iodination
labels polypeptides (proteins) at tyrosines
Band 3 labels by which type? where?
Equally external and internal Iodination
RBC labeled by which mechanism? where?
Lactoperoxidase Iodination; externally or internally
General Lesson #1
all membrane proteins are asymmetrically placed or orientated
Band 3 labels by which type? where?
Equally external and internal Iodination
General lesson #2:
Transmembrane proteins always glycoproteins
Which sides bears oligosacchride?
E-face
General Lesson #1
all membrane proteins are asymmetrically placed or orientated
Are membranes flexible?
YES
General lesson #2:
Transmembrane proteins always glycoproteins
Def. Fluidity
ability to lipids and molecules to move freely
Which sides bears oligosacchride?
E-face
Temperature and Fluidity
temp. sensitive
Are membranes flexible?
YES
Phase Transition Point
Meliting Point
Def. Fluidity
ability to lipids and molecules to move freely
With more than type of protein
Broader melting point
Temperature and Fluidity
temp. sensitive
Same type of protein (artifical)
Sharp melting point
Phase Transition Point
Meliting Point
With more than type of protein
Broader melting point
Same type of protein (artifical)
Sharp melting point
Indirect Evidence #1: that proteins move
made an artifical protein; optimum temperature was at 23 C- put in cAMP cycle- when below 23 (proteins didn't move) nothing worked- when above 23 (proteins able to move) worked great!
What about fish that live in the cold?
Are able to regulate their lipid content of their membranes for different temperatures.
Direct Evidence #1: that proteins move
Antibodies locate lymphocyte receptor proteins, initally all over the protein (almost evenly); then eventually caps (all on one side)
Antibodies locate what?
lymphocyte receptors proteins
Capping or patching
eventually all antibodies are on one side of the protein
Capping no go when?
membranes are gelled ( can't move)
Patching occurs because of what phenomeum
cross-linking of proteins by antibodies
The Rapid intermixing of cell surface antigens after formation of mouse-human heterokaryons
L.D. Frye and M. Edidin; demonstrated that proteins free to diffuse naturally
Fab
differently labeled antibody fragments
Human and Mouse Experiment
Intitally goes to separate size of the cell; then if you wait they will freely diffuse around cell; obviously allowed to move around the cell
What does blending rate depend on?
Temperature
FLIP- def.
Fluorescence Loss in Photobleaching
FRAP- def.
Fluorescence Recovery After Photobleaching
FRAP what happens?
start out will proteins; bleach some to make white; there is a white patch; eventually patch becomes covered with original proteins diffusing across "hole"; therefore proteins are able to diffuse
Integrin
highly localized protein
Why is integrin so restricted?
Because is bound to cytoplasmic actin network
Band 3 restricted why?
sometimes acts as though its coralled
Band 3 unrestricted why?
Tryposin cleaves P-Face band 3 protein allowing movement
Network for band 3
spectrin a and b
Answer 1: why proteins restricted
mobility restricted by cytoplamic networks
Answer 2: why proteins restricted
sometimes mobility is restricted because of interactions with other membrane proteins
Tight Junctions- def.
Ex of how membrane proteins restrict motion
Tight Junction how do they work?
for connective seals between ephitheial cells
Claudins
their motion is restricted by intramembrane and intermembrane interactions with other claudins
Answer 3: why protiens restricted
because of lipids
Lipid Rafts
some patches stay together after mild detergent treaments
Lipids Rafts high in
Spingolipids and cholestrol; membrane proteins (protestic groups with fatty acids)
Rafts are thick! why?
have proteins that are long than membrane spanning proteins (sometimes others get stuck); longer one like being covered by membrane (stay longer)
Filipin
a stain for rafts (because marks cholestrol)
In liver and muscle cells what does adrenaline stimulate?
glycogen catabolism over anabolism
2 types of protein function
1) Allosteric Modification 2) Covalent Modification
Allosteric- def.
protein with two permissible native conformations (active or inactive)
Allosteric effector
"key" to the lock that turns protein on
Covalent
only imput of energy can proteins change conformation (function)
Protein Kinase
puts on a phosophate group(turns on protein)
Protein Phosphatase
takes off a phosphate group (turns off protein)
Allosteric v. Covalent
A- faster, shorter lasting c- longer lasting, less prone to flucations
Can they be in same pathway?
yes!
Protein Kinase A
PKA; determines what the pathway makes
Two options for PKA to make:
1)phosphorylase kinase 2)Glycogen Synthase
Where is PKA found?
All Eukaryotic Cells
How is PKA turned on?
cAMP binds splitting pieces, turning them on
What is the rise in cAMP before PKA is turned on?
10-7 M to 10-6 M
what makes PKA; what degrades it?
Adenaline Cyclase (makes); phosphodiesterase
Two ways to make more cAMP?
increase- adenylate cyclase; decrease phosodiesterase
Caffeine significance with cAMP?
inhibits phosodiesterase (increases production of cAMP)
Dibutyryl cAMP - def.
lipid-soluble cAMP analogue;
Dibutyryl cAMP- purpose
diffuses through membrane and raises intracellular cAMP
Second Messanger-
small, diffusable intracellular molecule, whose concentration reflects extracellular events
Where is cAMP a second messager?
Liver and muscle cells
Signal Transduction- def.
relaying a singal by conversion from one physical form to another
How to Make cAMP process? (graph)
Adrenaline->Adenylate Cyclase-> cAMP
Receptor-G-Protein-effector
proteins involved in production of cAMP
G-Protein for cAMP?
trimeric (a b and g)
What G protein is being used for production of cAMP?
Gs
What anchors G-proteins
fatty acid prosthetic groups
What is also bound to a G-Protein
GDP
GDP and Gs
makes inactive
Process to turn on Effecor with Gs
Extracellular ligand stimulate Active conformation-> receptor activates G protein-> GDP leaves in comes GTP->Ga leaves-> Ga with GTP stimulate Adenylate Cyclase->makes cAMP
How does GTP eventually turn off?
Ga hydrolyzes GTP to GDP- self inactivates
Ga is also known as a what? why?
GTP-ase; because cuts off GTP to turn off protein
What good is the g-protein?
static filter- helps with constancy over time, even when signal isn't there
GTP how many binding sites
4 binidng sites
Chromaffin Cell source of what?
adrenaline
calcium iononphore
chemical that can stimulate secretion
How do ionophores work?
insert into membrane and produce unregulated ion-specific pathways
membranes pereability to Ca++?
limited
The Phosphoinositide Pathway
PIP2
PIP2 cleaved by what?
Phosolipase C
How do you activate Phosolipase C (plc)?
signal tranduction; Gq interactions and active PLC
DAG from where?
cleaved end of PIP2
DAG actives who? and how?
Protein Kinase C; allosterically
Phorbol Esters
mimic effects of DAG-> bind to PKC
IP3 regulates what? how?
permeability of Ca++ channel in ER; allosterically
IP3 comes from where?
PIP2 minus the fatty acid anchor
Ca in Cytosol....
very low 10^-7
ER is a CSC
Calcium Sequesting Compartment
Where is there a 10,000 fold difference in Ca?
between the cytosol and ER
Ca++ in ER (value)
10-3
Ca++ in cytosol (value)
10-7
Ca natually diffuses which way?
out into cytosol
Aequorin
determines free calcium in cell; glows in the presence of free calcium; can be vectored into cells
Fura-2 Dye
lipid soluble probe
Calcium allosteric effector to?
Calcium-binding proteins
CaM
Calmodulin (CaM) most important calcium binding protein
How does CaM work?
Small protein that binds to other proteins and effects their functions
Ip3 rise in what?
calcium
T-Tubules
protein in contat with ER Ca++ channels
Neurons and Ca++
have volate sensitive Ca++ channels