Protein Degradation and Protein Techniques Flashcards
Metabolic flexibility:
quick changes in concentrations of key regulatory enzymes, hormones, receptors, etc
Protine turnover
miss formed/folded - danger of forming bad protine aggregates - need to degrade them
made to be degraded and made over again
Numerous physiological processes are just as dependent on timely degradative reactions as they are on synthetic ones (ie cell cycle progression, synthesis and degradation of cyclin proteins)
Chaperones __
Proteasomes __
Birth
Death
Chaperones bind to __
exposed hydrophobic regions (should be on the inside if fully folded, if not shows that it is not fully folded, damaged, etc.)
Chaperones (many) are called
Many are called heat shock proteins (hsp): hsp60 and hsp70 family
Often many cycles of _____________required (energy requirement) to fold polypeptide correctly
ATP hydrolysis
hsp70
monomers
bind while being synthesized by binding to hydrophobic regions - ATP required
Chaperone functions
1 Assist - chaperone guides
2 Rescue - help refold that have miss folded
3 Protect - prevents exposed hydrophobic patches to start to aggregate - dangerous and toxic to cells
Hsp60
isolation chamber
Acts later, after a protein is fully synthesized
GroES cap (small chaperone)
symetrical - can bind to either side (not at same time)
in chamber so cant bind to other / issolated enviorment represents aqueous cell (can fold how it would in cell)
ATP - helps protine unfold and be encapuslated in chamber
another ATP binds, weakens cap
specific half life
protiens life span mesurement
All proteins turnover with a specific half-life depending on their function.
House-keeping proteins half life
structural - longer half life
House-keeping proteins typically have long half-lives (on the order of days)
Regulatory proteins half life
Regulatory proteins often have relatively short half-lives (on the order of min. to hrs.).
PEST rich sequences
high % of protines that have v half lives in cells
exibit short peptide sequence enriched in
Pro-Glu-Ser-Thr (indicator/red flag for degregation)
Degradative Signals:
flag protine to be degraded
1) PEST rich sequences
2) N terminous residues
3) hydrophobic patch on protines surrface
N terminous resudues
very basic or hydrophoic (bulky)
N-end rule
Ubiquitin Proteasomal System
most protines are degraded by the ubiquitin proteasomal system (UPS) (some others include proteolytic enzymes in the cytoplasm and lysosome(also degrades cell debre and waste))
Protine to be covalantaty modified with ubiquitin
ubiquitination
attachment of ubiquitin
Proteolysis via the UPS is
selective
(more types of E3 than E2&E1 because they interact directly with the target protine (and same for E2 than E1))
UPS steps
- Transfer ubiqidin to active site of enzime 1 (E1= uniqidin activating enzime) via ATP hydrolosis
- ubiquidin is Transfered to E2’s active site (E2=ubiquidin conjugating enzime)
- Degration signials on target protine (ex. hydropobic patches, PEST sequences, N-end rule) cause E3 (E3=ubiquidin ligase enzime) with E2/complex to bind to target protine - add ubiqudin to target protine. once ~4 added, reconized by protiesome.
Proteasome
milti protine, large complex with many enzimes
has two caps. ring in cap - lined with ATPases - unfold polypeptide
Cavity is lined with ptoteases (in central cylinder) - cut/cleave peptide bonds, now pwptide fragments (used in synthis of other protines)
Reconized protine baised on ubiquidin chain
Enzimes recycle ubiquidin
Regulation of Protine degradation (via E3)
E3 (ubiquidin ligase) is turend on by either E3 phosporlation ot by allosteric transition in E3 molecule
(control when E3 is active)
Regulation of Protein Degradation (via degradation signal)
phosporlation by protine kinase, reconized by E3
Unmask subunit blocking signal (noncovalant)
Removal of protine on peptide (covalant) - creation of destabalizing N terminous
Salting out
salt used to purify protines (issolate protine of intrest)
high concentration of salt, needed to make sure it does not denature the protines
Protines are hydrated (ionizeable groups interact with water) same water interactis with salt, dehydrating the protine, increasing protine-protine interactions
Aggregates (of protine-protin interactions)
(ex. ass high (NH4)2SO4)
Chromatography (what it is)
moble phase
stationary phase
seperate baised on charatraistics
modble phase - mixture of protines
stationary phase - cchartaristics of collum itself
diff. protines are eluted at differnt times.
Ion-Exchange Chromatography
Can be + or -
protines sperated baused odd of charge
pH can affect net charge.
Cation exchange resins
positive charg binds to stationary phase - matrix with neg charge
neg. protines out first than pos charge
Anion exchange resins
negative chare protines - bind to stationary phase/matrix with pos. charge
pos charge protines out first
Gel-Filtration Chromatography
seperate baised on size
smaller prtoeins get traped in tiny pourous beeds
large moledules remain in solution, flowing more rapidly and emerging first
Affinity Chromatography
beed with covalently attached substrate, enzime/protine interacts with substrate/ binds to ligand
results in better/more specific protine purfication
ex. bound substrates (specific enzimes) and bound antibodies (specific protine)
SDS Page -
– neg charge associates with hydrophobic regions
o SDS is an detergent (charged, ionic) apathic, disrupt stability of protein
o Gel is polyahramide – neg to pos
o Access the changes in MW (ex. covalent bonds, addition/subtraction of groups)
Beta Mercaptoethanol is
a reducing agent – reduces disulfide bonds/bridges
