Protein Degradation and Protein Techniques Flashcards

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1
Q

Metabolic flexibility:

A

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

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2
Q

Protine turnover

A

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)

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3
Q

Chaperones __
Proteasomes __

A

Birth
Death

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4
Q

Chaperones bind to __

A

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

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5
Q

Chaperones (many) are called

A

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

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6
Q

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

A

ATP hydrolysis

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7
Q

hsp70

A

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

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8
Q

Chaperone functions

A

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

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9
Q

Hsp60

A

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

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10
Q

specific half life

A

protiens life span mesurement

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

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11
Q

House-keeping proteins half life

A

structural - longer half life

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

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12
Q

Regulatory proteins half life

A

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

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13
Q

PEST rich sequences

A

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)

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14
Q

Degradative Signals:

A

flag protine to be degraded

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

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15
Q

N terminous resudues

A

very basic or hydrophoic (bulky)

N-end rule

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16
Q

Ubiquitin Proteasomal System

A

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

17
Q

ubiquitination

A

attachment of ubiquitin

18
Q

Proteolysis via the UPS is

A

selective

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

19
Q

UPS steps

A
  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.
20
Q

Proteasome

A

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

21
Q

Regulation of Protine degradation (via E3)

A

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

(control when E3 is active)

22
Q

Regulation of Protein Degradation (via degradation signal)

A

phosporlation by protine kinase, reconized by E3

Unmask subunit blocking signal (noncovalant)

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

23
Q

Salting out

A

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)

24
Q

Chromatography (what it is)

moble phase

stationary phase

A

seperate baised on charatraistics

modble phase - mixture of protines

stationary phase - cchartaristics of collum itself

diff. protines are eluted at differnt times.

25
Q

Ion-Exchange Chromatography

A

Can be + or -

protines sperated baused odd of charge

pH can affect net charge.

26
Q

Cation exchange resins

A

positive charg binds to stationary phase - matrix with neg charge

neg. protines out first than pos charge

27
Q

Anion exchange resins

A

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

pos charge protines out first

28
Q

Gel-Filtration Chromatography

A

seperate baised on size

smaller prtoeins get traped in tiny pourous beeds

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

29
Q

Affinity Chromatography

A

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)

30
Q

SDS Page -

A

– 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)

31
Q

Beta Mercaptoethanol is

A

a reducing agent – reduces disulfide bonds/bridges