Chapter 5: Proteins: Primary Structure Flashcards
the composition of a polypeptide chain (sequence) is limited by the ability of the
polypeptide to fold into a what
functional structure
If the pI is greater than the pH what is the charge
positive
If the pI is less than the pH what is the charge
negative
contain subunits
Multisubunit proteins
identical and/or non-identical chains
subunits
A given protein is frequently obtained from a source chosen primarily for convenience
protein source
produces large amounts of a foreign protein often sequesters it in what
inclusion bodies.
A protein’s ionic charge, polarity, size, and ligand-binding ability influenced its
chromatographic behavior
is not necessarily to minimize the loss
of the desired protein, but to eliminate selectively the other components of the mixture so that only the required substance
remains.
purification technique
what pH do cells have to be to go from positive to neutral
high PH and PI
what pH do cells have to be to go from negative to neutral
low PH and PI
how to separate proteins by charge
Ion exchange chromatography
Electrophoresis
Isoelectric focusing
how to separate proteins by size
size exclusion
SDS-PAGE
Gel filtration chromatography
how to separate proteins by polarity
Hydrophobic interaction chromatography
how to separate proteins by binding specificity
Affinity chromatography
how to separate proteins by ligand-binding
affinity
A protein in a complex mixture can be detected by its binding to its
corresponding antibodies
Enzymes coupled to antibodies include alkaline phosphatase, and β-galactosidase. The amount of antigen is quantified by the
formation of a colored reaction product produced by the enzyme.
Direct Enzyme-Linked Immunosorbent Assay (ELISA)
this absorbance is commonly used to measure the concentration of proteins.
Trp
A protein with a what, absorbs in the
visible region of the spectrum
chromophore
to change the solubility of a protein what can you do?
adjust the pH to approximate the isoelectric point (pI) of the desired protein
charged molecules bind to oppositely charged
groups that are chemically linked to a matrix
ion exchange chromatography
Anions bind to cationic groups on
anion exchanger
most frequently used anion exchanger
diethylaminoethyl (DEAE) groups
most frequently used cation exchanger
carboxymethyl (CM) groups
depends on the presence of other
ions that compete with the protein for binding to the ion exchanger and on the pH of the solution
binding affinity of a particular protein
As the column is washed, proteins with relatively low affinities for the ion
exchanger move through the column faster than proteins that bind with higher
affinities
what to know about ion exchange
influences the net charge of the protein
pH of the solution
proteins that bind tightly to the ion exchanger can be
eluted
washed through the column
eluted
has a higher salt concentration or a pH that reduces the affinity with which the matrix binds the protein
eluant
buffer
eluant
proteins are separated by their pI values.
ion exchange chromatography
molecules are separated according to their size and shape.
gel filtration chromatography
size exclusion
molecular sieve chromatography
The stationary phase consists of gel beads containing pores that span a
relatively narrow size range.
gel filtration chromatography
size exclusion
molecular sieve chromatography
These what molecules therefore
traverse the column more rapidly than what molecules that pass through the pores
large
small
In this technique, a molecule a ligand that
specifically binds to the protein of interest
is covalently attached to an inert matrix
affinity chromatography
a divalent metal ion such as Zn 2+ or Ni 2+ is
attached to the chromatographic matrix so that proteins bearing metal-chelating groups can be retained.
metal chelate affinity chromatography
what stains the gel for SDS-PAGE
coomassie blue
Separated bands may be
visualized in the gel by soaking
the gel in a solution of a stain
(such as coomassie blue) that
binds tightly to proteins.
SDS-PAGE
how does electrophoresis differs from
gel filtration
electrophoretic mobility of smaller molecules is greater than the mobility of larger molecules
why is the pH of a SDS-PAGE usually high
so that nearly all proteins have net negative charges and move toward the positive electrode when the current is switched on
what shape do proteins assume in the presence of SDS.
rodlike
In SDS-PAGE, the relative mobility of proteins
vary approximately linearly with what?
the logarithm of their molecular masses
If a mixture of proteins is electrophoresed through a solution or gel that has a stable pH gradient in which the pH increases smoothly from anode to cathode, each protein will migrate to the position in the pH gradient
corresponding to its
pI
IEF can be combined with SDS-PAGE in an extremely powerful separation
technique named
two-dimensional (2D) gel electrophoresis
First, a sample of
proteins is subjected to IEF in one direction, and then the separated proteins are
subjected to SDS-PAGE in what direction
perpendicular direction
is a biochemical method for separating
complex mixtures of proteins into individual species.
2-dimensional electrophoresis (2DE)
what is included in protein sequencing
2 subunits
2 different amino acids
To be sequenced, a protein must be
separated into what
individual polypeptides
a procedure for removing N-terminal
residues one at a time
Edman degradation
the amino acid sequence can be
determined by what
Edman degradation
a procedure that liberates amino acids one at a time from the N-terminus of a polypeptide
first step of Edman degradation
evolutionarily related proteins
homologous proteins
indicate which of the protein’s residues are
essential to its function, which are less significant, and which have little specific
function
homologous proteins
Finding the same residue at a particular position makes it an
invariant residue
Changes in other amino acids with similar side chains
conservatively substituted
A particular amino acid position that tolerates many different amino acid residues, indicating that the position is
hypervariable
