Transcript CH 5 The Primary Level of Protein Structure

CH 5 The Primary Level of
Protein Structure
HW 2, 3, 4, 6, 7
Amino Acids and Peptides
• Bioimportance:
– Monomer units for proteins
– Participate in cellular functions such as nerve
transmissions
– Biosynthesis of porphyrins, purines,
pyrimidines, and urea
• While human proteins only contain Lamino acids, other organisms contain both
D and L
• 10 of the 20 amino acids commonly used
are essential nutrients, which mean they
must be in our diet, we can not synthesize
them
• There are over 300 naturally occurring
amino acids, but only 20 are used to make
proteins.
• These 20 are listed in table 5.3 page 129,
according to side chain properties
• Friday, we will have a quiz on the names,
1 letter abbreviation, and 3 letter
abbreviation
• Next Monday, we will have a quiz on the
name, structure, and pKa’s (Table 5.1)
• While we only use 20, some of these can
be altered in a peptide by adding or
removing a functional group to increase
diversity.
Chirality
• All amino acids, except Gycine, have an
alpha carbon that is chiral.
• This is the source of all chirality in
organisms
• The absolute configuration of all the alpha
carbons are L
Amino Acids properties
• Amino Acids may have a positive,
negative or zero net charge.
• Most amino acids are in the zwitterionic
state at physiologic pH
• An amino acid can not exist as COOH/NH2
because any pH low enough to protonate
the COO- group would as protonate the
NH2
• Some amino acids, histidine and arginine,
are resonance hybrids, but they still only
have a 1+ charge.
• By altering the net charge via pH, we can
create separation processes for amino
acids, peptides, and proteins
• The isoelectric species is the form of a
molecule that has an equal number of
positive and negative charges, thus it is
neutral.
• The isoelectric pH, also called pI, is the pH
midway between the pKa values on either
side of the isoelectric species
• Examples:
• This pI guides selection of separation
conditions
• The pKa of side chains varies slightly
– Nonpolar effects-
• Thus the pKa in peptides and proteins will
depend on unique local environments
• These changes in charge affect physical
properties of amino acids, peptides, and
proteins.
• Functional groups of the side chains typically
determine chemical properties
• When amino acids are in a protein or
peptide chain, they are called residues
• Peptides are usually written with the free
alpha amino group to the left and the free
alpha carboxyl group to the right
• The backbone will start with N, then the
alpha carbon, then the carbonyl carbon,
then repeat.
• The side chains are bonded to the alpha
carbon
• Lines are used with 3-letter abbreviations,
omitted with 1-letter abbreviations
• For some peptides, non-common amino
acids may be used or non-peptide bonds
my be used
• The peptide bond is not charged, however
you still have a + at the N-terminal, - at the
C-terminal, and any charges on side
chains
• Peptides are therefore classified as
Polyelectrolytes
Structure Feature of Peptide Bond
• See figure 5.12 page 138
• The peptide bond has some double bond
character and does not have rotation
about the C-N peptide bond
• The carbonyl O and C, the N,and the H on
the N, all lie in the same plane.
• Rotation only occurs between the alpha
carbon and N, and alpha carbon and
COO-
Determination of Primary Structure
• We know proteins are very important
• An important goal of molecular medicine is the
identification of proteins who presence,
absence, or deficiency is associated with
specific physiologic states or disease
• The primary structure of proteins, which is the
sequence of amino acids, provides both a
molecular finger print for its identification and
information that can be used to identify and
clone the gene or genes that encode it.
• In order to determine the amino acid
sequence, a protein or peptide must be
highly purified
• Because of the 1000’s of different proteins
in each cell, this is very difficult to do.
• It usually requires many successive
purification techniques
Purification
• The classic approach exploits differences
in:
– Relative solubility of proteins as a function of
pH
– Polarity
– Salt concentration
– Chromatographic separtions
Chromatographic Separations
• Mobile phase vs stationary phase
– Paper chromatography
– TLC
– Column
• Types of stationary phase
– Size exclusion
– Absorption
– Ion exchange
Other types of Chromatography
• pH based chromatography
• Hydrophobic Interaction chromatography
Affinity Chromatography
• Exploits high selectivity of binding proteins
• This is what we did in lab
• All the chromatography mentioned so far,
is typically done slowly with low pressure
• The stationary phases involved are
somewhat “spongy” and their
compressibility limit flow rates
HPLC
• High Pressure Liquid Chromatography
uses incompressible silica or alumina as
stationary phase which allows much
higher flow rates and pressures
• This also helps limit diffusion thus
enhances the resolution
• This method is very effective on complex
mixtures of lipids or peptides with very
similar properties
HPLC
• The stationary phase is typically
hydrophobic and water miscible organic
solvents such as acetonitrile or methanol
are used as mobile phases
SDS-PAGE
• SDS- sodium dodecyl sulfate (anion
detergent)
• PAGE- poly acrylamide gel electrophoresis
• Electrophoresis separates biomolecules
based on their ability to move through a gel
matrix due to an applied electric field
• SDS denatures and binds to proteins at a
known ratio of 1 SDS for every 2 peptide
bonds
• 2-mercaptoethanol or dithiothreitol is used to
break disulfide linkages
• The charge on SDS,-1, overcomes and
negates charges on side chains
• This leads to a constant charge to mass ratio
which means the peptides are separated
purely by the resistance the matrix provides
• Larger peptides have more resistance,
therefore move slower
• The gel is then stained, usually with
Coomassie Blue, to visualize the movement
IEF
• IEF- Isoelectric Focusing
• Ionic buffers called ampholytes and applied
electric field are used to generate a pH
gradient with in a matrix
• The peptide/protein then migrate through the
matrix to an area where the pH=pI, so there is
no net charge on the peptide
• IEF can be used in conjunction with SDSPAGE to perform a 2-D analysis, separating
peptides first by pI, then by size
Sequencing Peptides
• Sanger was the first to determine the
sequence of a polypeptide
• Mature insulin consist of 2 chains, the A
chain has 21 residues, the B chain has 30
residues
• The chains are held together by disulfide
linkages
• Sanger first broke the linkages to separate
the chains
• He then broke the chains into smaller pieces
using trypsin, chymotrypsin, and pepsin
• These reagents cleave peptide bonds at
known locations (see table 5.4 p 139)
• These smaller fragments where the separated
and hydrolyzed to form even smaller peptide
chains
• Each was reacted with 1-fluoro-2,4dinitrobenzene, called Sanger’s reagent,
which derivatizes the exposed alpha amino
group
• The amino acid content of the peptide was
then determined.
• Working backwards, he was able to
determine the complete sequence of
insulin and win the Nobel Prize in 1958
Peptide Cleaving Agents
Reagent
Bond Cleaved
CNBr
Met-X
Trypsin
Lys-X and Arg-X
Chymotrypsin
Hydrophobic AA-X
Endoproteinase Lys-C Lys-X
Endoproteinase Arg-C Arg-X
Endoproteinase Asp-N X-Asp
Reagent
V8 protease
Hydroxylamine
o-Iodosobenzene
Mild Acid
Bond Cleaved
Glu-X particularly where
X is hydrophobic
Asn-Gly
Trp-X
Asp-Pro
Edman’s Reagent
• Pehr Edman introduced phenylisothiocyanate,
called Edman’s reagent, to selectivity label the
amino-terminal residue of a peptide
• Unlike Sanger’s, Edman’s derivative can be
removed under mild conditions with out
disrupting the rest of the peptide
• After removal, a new amino terminal is
produced and the process is repeated
• This allows for the direct sequencing of a
peptide
• However, due to the efficiency of the
reaction, this process is limited to peptides
no larger than 20-30 residues
• Process:
• Because of the limitations to Edman’s
process and since most polypeptides
contain several hundred residues, most
polypeptides must be broken into smaller
chains which are then identified.
• Sample problem on hand out.
Microbiology Impact
• Advances in Microbiology have led to a
new, simpler way to identify the primary
structure of proteins.
• Knowledge of the DNA sequence permits
deductions of the sequence of AA
• Sequencing DNA requires much less
sample
Example
• So by sequencing only a small portion of
protein, we can correlate it to DNA, find
the section of DNA that encodes the
protein, then deduce the whole sequence.
• Limitation: No information is provided for
post-translational modifications!