Download PEPTIDE BONDS AND POLYPEPTIDES OLIGOPEPTIDE

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PEPTIDE BONDS AND POLYPEPTIDES
OLIGOPEPTIDE:
--chain containing only a few amino acids (see tetrapaptide, Fig 5.9)
POLYPEPTIDE CHAINS:
--many amino acids joined together
--not necessarily a protein! (all proteins are polypeptides, but the converse is not true)
•a protein has a specific amino acid sequence that is defined by a gene
amino acids in a polypeptide are called amino acid residues
polypeptides (and proteins) have a front end (amino terminus or N-terminus) and
a back end (carboxyl terminus or C-terminus)
•most proteins contain 50-2000 amino acids
•mean molecular weight of an amino acid is 110 (see Problem #1) so MW of proteins
could be 5500 to220,000 (ball park numbers)
Polypeptides as polyampholytes (substance whose groups have both acidic and
basic groups)
•they have many weakly acidic and basic groups (Fig 5.11)
•even a small shift in pH can significantly affect the structure and interactions of a
protein molecule
Molecular weight does not have units
mass has units called daltons
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mass of above proteins would be 5500 daltons to 220,000 daltons (how many
kD?)
•PEPTIDE BONDS:
•amide bond between α-amino and α-carboxyl groups
•structure solved by Pauling and Corey
•No rotation around peptide bond...a rigid, planer unit (Fig 5.12) so the -C=O and -N-H
bonds are nearly parallel (O, C, N, H usually coplanar)
1. x-ray crystallographic studies of synthetic peptides indicated that distance of C-N
bond was shorter that would be expected if it had only single bond character, so
must have double bond properties
2. rotations are permitted only around the α carbon
3. depending on the R group, a constraint can be imposed on these rotations by steric
hindrance
4. Therefore, trans form usually favored (-X-Pro can be cis althought trans still favored;
prolyl cis-trans isomerases an important new class of regulatory enzymes
STABILITY AND FORMATION OF THE PEPTIDE BOND
•formed by dehydration (loss of H2O) (Fig 5.8)
•requires input of free energy, about +10 kJ/mol (hydrolysis favored but very slow w/o
catalyst, so peptides are stable, similar to nucleic acids)
•can be hydrolyzed in hot 6N HCl (see below) or by proteolytic enzymes or proteases
(see Table 5.4) that often cleave at specific residues
see CNBr reaction , Fig 5.13
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•since synthesis unfavorable, like with nucleic acids, amino acids are activated by an
ATP-driven reaction before incorporation into proteins = coupling each amino acid
to the 3’ end of its tRNA to yield the aminoacyl-tRNA, catalyzed by aminoacyl-tRNA
synthetases, and ATP is hydrolyzed to AMP (Fig 5.19)
PRIMARY STRUCTURE, proteins of defined sequence
•Primary structure is the specific sequence of amino acids occuring in a protein
defined by genes
•Fred Sanger sequenced the first protein in 1953: bovine insulin
1st Nobel, because it showed that:
the sequence is precisely defined
only L-amino acids are found
they are linked by peptide bonds
•thousands of proteins have now been sequenced
each is unique
•Why is the primary structure important?
helpful in elucidating mechanism of action
determines the three dimensional structure, which confers biological function
•rules that govern protein folding are being discovered by studying the relationship
between primary structure and three dimensional structure
very important to an emerging area of medicine: molecular pathology
•one amino acid change in the primary structure can lead to abnormal function and
disease
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examples: sickle cell anemia and cancer
CHARACTERIZATION OF PRIMARY STRUCTURE
•Examples of primary structure:
Mb myoglobin (3D Fig 5.1, primary Fig 5.14) and see Table 5A.1 for how to isolate
and purify Mb)
Insulin (Fig 5.15 and 5.21)
Insulin has disulfide bonds:
•disulfide bonds
cross-links between chains or within a chain
formed through oxidation between pairs of cys side chains
product of the oxidation called cystine
PROTEIN PURIFICATION
For example, from < 0.1% of starting material (total protein) to 98% pure
1. Requirements:
Assay
Good supply of starting material
Overexpession in E coli or other organism not without problems...not as easy
as the book makes it sound
Patience
2. What kinds of proteins are easy to purify?
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Abundant proteins
Proteins that have some unique property
General procedures
1. Stabilization:
Use buffers at appropriate pH to prevent denaturation and/or degradation
Purification often done in cold because proteases less active...not so important if
using HPLC which is very fast
Protease inhibitors are often used to prevent degradation
Minimize foaming
Keep concentrated, proteins usually are more stable in a concentrated solution.
Especially important for long-term storage.
Usually stored at -20°C to -80°C
Assays
1. Use a direct assay if available, as for an enzyme, or a coupled reaction if a
detectable product is not made by a direct assay
Amount of product formed is proportional to the amount of enzyme present
2. Use antibodies to detect purification, if can be made through reverse genetics
Separation Techniques
Homogenation
Subcellular fractionation
Differential centrifugation
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Separation on the basis of charge, polarity, size, binding specificity, temperature
stability and other properties
Solubility
Solubilize appropriate fraction:
Salt step ("salting out")
Different proteins precipitate at different salt concentrations "ammonium sulfate
cut"
Chromatography
Ion exchange
DEAE cellulose (or agarose) = anion exchange, CM cellulose = cation exchange
Reverse Phase = hydrophobic interaction chromatography, nonpolar alkyl groups
attached to matrix
Affinity
Gel filtration (Fig 5A.5)
Silica beads with pores
HPLC
Gel electrophoresis
See one band on two different kinds of gels!!
PAGE (Native)
SDS-PAGE (Denaturing)
Isoelectric Focusing
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Determination of amino acid compostition
•hydrolysis of protein (usually in 6N HCl at 110°)
•hydrolysate (amino acids) is separated by ion exchange chromatography
a column of beads that separates molecules on the basis of charge
there are cation exchange (-) columns and anion exchange (+) columns
•amount of each amino acid is then quantitated by reaction with ninhydrin (Fig
5B.2) (darkness of resulting blue color is proportional to the amount of amino
acid that is present); Or Fig 5B.1 use a single column amino acid analyzer, can
be done by HPLC
•identification of amino and carboxy terminal residues
•amino terminal residue:
rxn with dabsyl or dansyl chloride
forms dabsylated or dansylated derivative which
can be identified
chromatography after cleavage in 6N HCl
destroys peptide so only amino term. amino acid can be identified
•carboxy terminal residue:
rxn with carboxypeptidase A
removes carboxy terminal residue
composition can then be redetermined to see what's missing
Cut up the protein and sequence the pieces...
•cut up the protein:
by
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all cut after the indicated amino acid(s)
trypsin: lys or arg
clostripain: arg
chymotrypsin: phe, trp, tyr
thermolysin: leu, ile, val
CNBr: met
more in your book
•Sequence the pieces:
Edman degradation (Fig 5C.1)
done these days by machine
react with PITH and cleave
does not detroy protein so can do over and over
analyze PTH-derivative
also analyze what's left
see example of sequencing of the B chain of insulin in Fig 5C.2 and locating the
disulfide bonds in Fig 5C.4
MS-MS
tandem mass spectroscopy
inject peptides into first sector
vaporize in vacuum by FAB or electrospray
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moves into second sector
smashed into random, different smaller pieces by collisions with inert gas
pieces move to detector via a strong magnetic field which allows the determination
of the mass of each piece
analyze data and put puzzle together