Quiz 5 Flashcards
the primary structure of a protein
provides information about its function
in cells, this potential variety
is limited by the efficiency of protein synthesis and by the ability of the polypeptide to fold into a functional structure
the peptide backbone
-N-Ca-Co
N
the amide nitrogen of the amino acid
Ca
is the alpha carbon of the amino acid
Co
is the carbonyl carbon of the amino acid
peptide formation is the creation of an
amide bond between the carboxyl group of the first amino acid and the amino group of the next amino acid
condensation reaction
results in a low of water
the peptide bond
- adopts a trans conformation
- due to the partial double bond character of the peptide bond, the six atoms of the peptide-bond group define a plane the AMIDE PLANE
environmental conditions that affect a proteins stability during purification
- pH
- temperature
an assay based on a proteins chemical or binding properties
may be used to quantify a protein during purification
fractionation procedures
take advantage of a proteins unique structure and chemistry in order to separate it from other molecules
” salting out”
- increasing the salt concentration causes selective salting out [precipitation ] of proteins with different solubilities
what influences a proteins chromatographic behavior
- charge
- polarity size
- ligand-binding
ultracentrifugation
what the overall size and shape of macromolecules and larger assemblies can be assessed through.
salting out [1]
separates based on protein solubility in high ammonium acetate concentration
chromatography [1]
separates based on a proteins ionic charge, polarity, size or ligand binding ability.
gel electrophoresis [1]
and its variations can separate proteins according to charge, size, and isoelectric point
ultracentrifugation [1]
separates based on the overall size and shape of macromolecules and larger assemblies
below the isoelectric point or PI
[the pH at which there is a net charge of 0] the protein will carry a net positive charge
above the pl
a net negative charge
positively charged proteins
bind cation exchange columns
negatively charged proteins
bind anion exchange columns
proteins are eluted [released] by
high salt concentration
dialysis
semipermeable membrane bag is immersed in beaker of solution, diffusible solutes in the dialysis bag equilibrate across the membrane, while your protein stays inside
small proteins are impeded
by access to the inside of the gel beads, delaying travel through the column
large proteins
have no access to the matrix of the gel beads and flow easily around gel beads
proteins are eluted in the order
largest to smallest
small-molecule targets (ligands, black)
are immobilized through covalent attached to a solid matrix (tan) column
absorbance
dated the presence of proteins, but it does not distinguish between different proteins
proteins in a small sample can be separated by
electrophoresis, based on their size, using a semi-solid phase gel.
after separation, proteins are detected by staining with
coomassie brilliant blue for visualization of “bands” representing the proteins of different sizes
SDS page
sodium dodecyl sulfate poly acrylamide gel electrophoresis.
small samples are taken from fraction tubes, SDS is added, samples are boiled to denature and coat all of the amino acids with negative charges and separated by electrophoresis on a gel
260 nm is problematic, why?
at 280nm there is a strong absorbance by Tyr and Top but not Phe
A reducing agent, such as DTT or BMe
is needed to disrupt structure but does not break disulfide bonds
a proteins mobility in the gel
is inversely proportional to the molecular weight
separation is based on
size, without irreversibly denaturing the protein
to be sequenced
a protein must be separated into individual polypeptides that can be cleaved into sets of overlapping fragments
the amino acid sequence can be determined by
Edman degradation, a procedure for removing N-terminal residues at one time
mass spectromy
can identify amino acid sequences from the mass-to-charge ration of gas-phase protein fragments
proteins can be sequenced in two ways
- direct amino acid sequencing
- sequencing the corresponding dna of the gene
today, gene databases provide these deduced protein sequences
acid hydrolysis
liberates the amino acids of a protein
- some amino acids are partially or completely destroyed by acid hydrolysis especially, trp
chromatography is used
to separate amino acids
the amino acid compositions
of different proteins are different
the sequence of amino acids in a protein is
distinctive but composition is not
subunit interactions depend on weak forces, which can
- be denatured
denaturation can be achieved with
- extremes of pH
- high urea concentration
- high guanidine HCL concentration
- high salt concentration
disulfide reducing agents
-2-mercaptoethanol, B-me
- dithiothreitol, DTT
to prevent disulfide bridges from reforming, follow with
an irreversible alkylating agent, like iodoacetate, IOAc
n terminal analysis, uses
edmans reagent
- derivatives are PTH
c-terminal analysis is enzymatic with carboxypeptidases
- carboxypeptidase A cleaves any residue except PRO, ARG, AND LYS
- carboxypeptidase B cleaved only works on arg and lys
enzymatic fragmentation
- trypsin
- chymotrypsin
- staphylococcal protease
chemical fragmentation
cyanogen bromide
trypsin
cleavage on the C side of Lys, Arg [basic]
chymotrypsin
C side of Phe , Tyr, Trp, less so Leu [large nonpolar]
staphylococcal protease
- c side of glu, asp[acidic] in phosphate buffer pH7
- specific for glu, in acetate [pH 5] and bicarbonate. buffer [pH 10]
cyanogen bromide
- CNBr acts only on methionine residues
- CNBr useful because proteins usually have only a few Met residues
- a peptide with a C-terminal homoserine lactone
compare and align overlapping peptide sequences
to learn the sequence of the original polypeptide chain
mass spec [MS]
mass spectrometry separates particles on the basis of mass-to-charge ratio
