protein purification Flashcards
the 3D structure in which the polypeptide is the most competent to fulfill its biological function
native polypeptide
elimination of all structural features, including disulfide bridges also called ______
no activity
denatured polypeptide
random coil
factors responsible for denaturation
heat (hydrogen bonds)
extreme pH –> alters net charge –> some hydrogen bonds
organic solvents, detergents, urea –> hydrophobic interactiolns
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:
chaotropic agents
dialysis
urea, guanidinium ion
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
reducing agents
DTT, B-Me
denaturation = _______
reduction =_______
chaotropic agents
reducing agents
a means to remove small molecules (salt, denaturing agents etc)
dialysis
a ________ is made of a material such as cellulose that will allow small molecules, but not _____, to diffuse in and out
dialysis membrane
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
christian B afinsen
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
RNA’ase
light absorption
denature
renature
the structure of RNAase A ncludes ___ disulfide bonds and numerous _____ interactions
4
noncovalent
remove urea
then remove DTT
result:
100% activity
remove DTT
then remove urea
1% activity
a small protein that contains 8 cysteines linked via four disulfide bonds
ribonuclease
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 _______
2 mercaptoethanol
spontaneously refolds
native confo0mration
_________ determines 3D conformation and biological activity, the disulfide bridges are not essential
primary structure
______ 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
non-covalent
weak bonding interactions
proteins fold to the ____ in the microsecond to second time scales
lowest energy
it is mathematically impossible for protein folding to occur by randomly trying every conformation until the lowest energy one is found
levinthal’s paradox
proteins folding follow a distinct path
completing critical folding steps accelerates subsequent folding
1,2,3,4
- local secondary structures formation
- certain alpha-helix and beta sheet are formed first
- ionic interaction can guide these early events
- local structures then guide long range interactions
prevent misfolding
they do not actively promote the folding of the substrate protein, but instead prevent aggregation of unfolded peptides
chaperones
______ are the most critical in the folding of a protein
hydrophobic interactions
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
exergonic (spontaneous)
-delta G
nonpolar groups
aqueous
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
molten globule
separation relies on differences in physical and chemical properties
charge size affinity for igand solubility hydrophobicity thermal stability
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
hydrogen bonding nonpolar phase ammonium sulfate denaturing soluble salting out
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 _______
salt conentration low enhancing salting in maximum precipitate salting out
to elute the proteins, the salt concentration is gradually _____, proteins _____ and elute out in the order of their ______ hydrophobicity
decreased,
resolubilize
increasing
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
gel filtration chromatography
separation based on size and shape
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 ____
highly porous structure
void volume
pores
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
molecules exlcuded from the pores
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 _______
molecules with partial access
decreasing sizes
will all move down the column at the same speed and remain unseparated from each other
molecules with full access
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
void volume
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
Vt
based on size
unparalled resolution
fast
denatures proteins
SDS page
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
negative
irrelevant
DTT
B-Me
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 ____
denaturation
shape, electric charge density
size
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)
affinity chromatography
antibodies are most commonly used in
affinity chromatogrophy
percent of the initial activity at the end of each purification step
yield
the total activity divided by the total amount of protein
specific activity
