protein purification

Question 1

the 3D structure in which the polypeptide is the most competent to fulfill its biological function

Answer 1

native polypeptide

Question 2

elimination of all structural features, including disulfide bridges also called ______
no activity

Answer 2

denatured polypeptide

random coil

Question 3

factors responsible for denaturation

Answer 3

heat (hydrogen bonds)
extreme pH –> alters net charge –> some hydrogen bonds
organic solvents, detergents, urea –> hydrophobic interactiolns

Question 4

small molecules that perturb (distort) the 3 dimensional structure by interfering with non covalent interactions that stabilize the proteins conformation
these agents can be removed from the solution by _____ and their effect is canceled
examples:

Answer 4

chaotropic agents
dialysis
urea, guanidinium ion

Question 5

small molecules that reduce disulfide bridges, leaving the cysteines with their original sulfhydryl groups
their actions can be reversed by their removal and introduction of oxidizing conditions
examples

Answer 5

reducing agents

DTT, B-Me

Question 6

denaturation = _______

reduction =_______

Answer 6

chaotropic agents

reducing agents

Question 7

a means to remove small molecules (salt, denaturing agents etc)

Answer 7

dialysis

Question 8

a ________ is made of a material such as cellulose that will allow small molecules, but not _____, to diffuse in and out

Answer 8

dialysis membrane

Question 9

wanted to know what are the forces that determine the correct folding of a protein
he designed a model system to address this question using the enzyme RNA’ase A

Answer 9

christian B afinsen

Question 10

a small protein, easy to denature and renature
particularly convenient because the degradation of an RNA substrate can be monitored by changes in ________ and because the protein can be easily made to ______ and _____ in vitro

Answer 10

RNA’ase
light absorption
denature
renature

Question 11

the structure of RNAase A ncludes ___ disulfide bonds and numerous _____ interactions

Answer 11

4

noncovalent

Question 12

remove urea
then remove DTT
result:

Answer 12

100% activity

Question 13

remove DTT

then remove urea

Answer 13

1% activity

Question 14

a small protein that contains 8 cysteines linked via four disulfide bonds

Answer 14

ribonuclease

Question 15

urea in the presence of _________ fully denatures ribonuclease

when urea and 2-mercaptoethanol are removed, the protein _________ and the correct disulfide bonds are reformed
the sequence alone determines the _______

Answer 15

2 mercaptoethanol

spontaneously refolds
native confo0mration

Question 16

_________ determines 3D conformation and biological activity, the disulfide bridges are not essential

Answer 16

primary structure

Question 17

______ interactions of the functional groups on the polypeptide have the major role in how a polypeptide folds

random distribution of disulfide bonds was achieved when these bonds were allowed to be formed in the presence of denaturant (urea) -> indicate that ______ are required for correct positioning of disulfide bonds and restoration of native structure

Answer 17

non-covalent

weak bonding interactions

Question 18

proteins fold to the ____ in the microsecond to second time scales

Answer 18

lowest energy

Question 19

it is mathematically impossible for protein folding to occur by randomly trying every conformation until the lowest energy one is found

Answer 19

levinthal’s paradox

Question 20

proteins folding follow a distinct path
completing critical folding steps accelerates subsequent folding
1,2,3,4

Answer 20

1. local secondary structures formation
2. certain alpha-helix and beta sheet are formed first
3. ionic interaction can guide these early events
4. local structures then guide long range interactions

Question 21

prevent misfolding

they do not actively promote the folding of the substrate protein, but instead prevent aggregation of unfolded peptides

Answer 21

chaperones

Question 22

______ are the most critical in the folding of a protein

Answer 22

hydrophobic interactions

Question 23

because the folding of a protein into its organized, native form is a spontaneous process, we can say that the folding is a ______ and have a _____

hydrogen bonds and ionic interactions exist to a very similar level, whether the protein is folded or not
hydrophobic interactions exist mostly when the protein folds, allowing the _____ to aggregate inside, away from the ______ environment
this is the main energetic drive to protein folding

Answer 23

exergonic (spontaneous)
-delta G
nonpolar groups
aqueous

Question 24

folding occurs by spontaneous collapse of the polypeptide into a compact strucure by hydrophobic interactions between nonpolar residues (hydrophobic collapse) and state is known as

Answer 24

molten globule

Question 25

separation relies on differences in physical and chemical properties

Answer 25

``` charge size affinity for igand solubility hydrophobicity thermal stability ```

Question 26

hydrophobic chromatogrophy partitioning based on ________ use of salts some very hygroscopic salts occupy a substational solvation shell, reducing the availibilty of water molecule for ________ salt is ________ these salts then induce some soluble molecules to partition into a _______, because oft he reduced ability to hydrogen bond with water reducing the number of hydrogen bonds to a protein can maintain with water, will cause some proteins to begin ______ and become less ______ these proteins will be much more likely to partition into the stationary non polar phase under these conditions called ________ at low

Answer 26

``` hydrogen bonding nonpolar phase ammonium sulfate denaturing soluble salting out ```

Question 27

hydrophobic chromatogrophy the solubility of a protein depends on the ______ in the solution at ___ concentrations, the presence of salt stabilizes the various charged groups on a protein molecule, thus attracting protein into the solution and _______ the solubility of protein this is known as ________ however, as the salt concentration is increased, a point of _____ protein solubility is usually reached further increase in the salt concentration implies that there is less and less water available to solubilize a protein finally a protein starts to ______, when there are not sufficent water molecules to interact with protein molecules. this phenomenon of protein precipitation in the presence of exces salt is known as _______

Answer 27

``` salt conentration low enhancing salting in maximum precipitate salting out ```

Question 28

to elute the proteins, the salt concentration is gradually _____, proteins _____ and elute out in the order of their ______ hydrophobicity

Answer 28

decreased, resolubilize increasing

Question 29

``` porous beads made of an insouble but highly hydrated polymer such as dextran or agarose separation based on _________ leaves proteins intact good resultion not efficient with crude solution relatively slow flow rateds ```

Answer 29

gel filtration chromatography | separation based on size and shape

Question 30

the ability of gel filtration to separate molecules according to size resides with the _______ of gel filtration media and is basically a question of accessible volumes in a column all molecules have access to the liquid between the beads. this volume is called the _____ resin contains ____ allowing the sample molecules to penetrate into the gel filtration beads to different degrees depending on size. size, together with the volme of these pores determines the ____

Answer 30

highly porous structure void volume pores

Question 31

move down the column at the same speed as the mbile phase | they will consequently leave the column after one void volume of mobile pase has passed through the column

Answer 31

molecules exlcuded from the pores

Question 32

to the pores will be retarded in relation to their respective degree of access to the pores: in other worlds they will elute from the column in order of _______

Answer 32

molecules with partial access | decreasing sizes

Question 33

will all move down the column at the same speed and remain unseparated from each other

Answer 33

molecules with full access

Question 34

the earliest elution off a gel filtration column is at the ______which will include all proteins that are to large to enter any portion of the beads

Answer 34

void volume

Question 35

the latest elution volume could be the ___ which will include all the proteins that are small enough to utilize all the pore volume of the beads

Answer 35

Vt

Question 36

based on size unparalled resolution fast denatures proteins

Answer 36

SDS page

Question 37

samples are treated with SDS which binds with protein and imparts ___ charge to all proteins partially denature proteins hence the structure is ____ during electro phoresis samples are treated with _____ or _____ a reducing agent

Answer 37

negative irrelevant DTT B-Me

Question 38

the combination of DTT and B Me plus heating to 100 degrees C results in a complete _____ of polypeptides (proteins) all polypeptides are separated from one another have the same ____ and _____ so the differences in the migration is based on ____

Answer 38

denaturation shape, electric charge density size

Question 39

separation based on activity/function leaves proteins intact (native) requires prior knowledge of biological properties and availibility of an interacting species (ligand, substrate, analog, antibody)

Answer 39

affinity chromatography

Question 40

antibodies are most commonly used in

Answer 40

affinity chromatogrophy

Question 41

percent of the initial activity at the end of each purification step

Answer 41

yield

Question 42

the total activity divided by the total amount of protein

Answer 42

specific activity