Protein Degradation and Protein Techniques

Question 1

Metabolic flexibility:

Answer 1

quick changes in concentrations of key regulatory enzymes, hormones, receptors, etc

Question 2

Protine turnover

Answer 2

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)

Question 3

Chaperones __
Proteasomes __

Answer 3

Birth
Death

Question 4

Chaperones bind to __

Answer 4

exposed hydrophobic regions (should be on the inside if fully folded, if not shows that it is not fully folded, damaged, etc.)

Question 5

Chaperones (many) are called

Answer 5

Many are called heat shock proteins (hsp): hsp60 and hsp70 family

Question 6

Often many cycles of _____________required (energy requirement) to fold polypeptide correctly

Answer 6

ATP hydrolysis

Question 7

hsp70

Answer 7

monomers
bind while being synthesized by binding to hydrophobic regions - ATP required

Question 8

Chaperone functions

Answer 8

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

Question 9

Hsp60

Answer 9

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

Question 10

specific half life

Answer 10

protiens life span mesurement

All proteins turnover with a specific half-life depending on their function.

Question 11

House-keeping proteins half life

Answer 11

structural - longer half life

House-keeping proteins typically have long half-lives (on the order of days)

Question 12

Regulatory proteins half life

Answer 12

Regulatory proteins often have relatively short half-lives (on the order of min. to hrs.).

Question 13

PEST rich sequences

Answer 13

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)

Question 14

Degradative Signals:

Answer 14

flag protine to be degraded

1) PEST rich sequences
2) N terminous residues
3) hydrophobic patch on protines surrface

Question 15

N terminous resudues

Answer 15

very basic or hydrophoic (bulky)

N-end rule

Question 16

Ubiquitin Proteasomal System

Answer 16

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

Question 17

ubiquitination

Answer 17

attachment of ubiquitin

Question 18

Proteolysis via the UPS is

Answer 18

selective

(more types of E3 than E2&E1 because they interact directly with the target protine (and same for E2 than E1))

Question 19

UPS steps

Answer 19

1. Transfer ubiqidin to active site of enzime 1 (E1= uniqidin activating enzime) via ATP hydrolosis
2. ubiquidin is Transfered to E2’s active site (E2=ubiquidin conjugating enzime)
3. 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.

Question 20

Proteasome

Answer 20

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

Question 21

Regulation of Protine degradation (via E3)

Answer 21

E3 (ubiquidin ligase) is turend on by either E3 phosporlation ot by allosteric transition in E3 molecule

(control when E3 is active)

Question 22

Regulation of Protein Degradation (via degradation signal)

Answer 22

phosporlation by protine kinase, reconized by E3

Unmask subunit blocking signal (noncovalant)

Removal of protine on peptide (covalant) - creation of destabalizing N terminous

Question 23

Salting out

Answer 23

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)

Question 24

Chromatography (what it is)

moble phase

stationary phase

Answer 24

seperate baised on charatraistics

modble phase - mixture of protines

stationary phase - cchartaristics of collum itself

diff. protines are eluted at differnt times.

Question 25

Ion-Exchange Chromatography

Answer 25

Can be + or -

protines sperated baused odd of charge

pH can affect net charge.

Question 26

Cation exchange resins

Answer 26

positive charg binds to stationary phase - matrix with neg charge

neg. protines out first than pos charge

Question 27

Anion exchange resins

Answer 27

negative chare protines - bind to stationary phase/matrix with pos. charge

pos charge protines out first

Question 28

Gel-Filtration Chromatography

Answer 28

seperate baised on size

smaller prtoeins get traped in tiny pourous beeds

large moledules remain in solution, flowing more rapidly and emerging first

Question 29

Affinity Chromatography

Answer 29

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)

Question 30

SDS Page -

Answer 30

– 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)

Question 31

Beta Mercaptoethanol is

Answer 31

a reducing agent – reduces disulfide bonds/bridges