Peptides and Proteins

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Peptides and Proteins
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Learning Objectives
Describe with structures a small peptide (given
the amino acid sequence), and indicate the
predominant state of ionization of the ionizable
groups at a given pH.
Identify and describe the four levels of protein
structure.
Describe the following types of chromatography
and how they are used to separate and purify
proteins ion exchange, size exclusion,
hydrophobic, and affinity chromatography.
Describe acrylamide gel electrophoresis with and
without the addition of SDS.
Describe isoelectric focusing and two-dimensional
gel electrophoresis.
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Formation of a peptide bond
At physiological pH, this reaction does not occur
to any appreciable extent.
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The pentapeptide ser-gly-tyr-ala-leu
Peptides are named beginning with the amino
terminal residue, which by convention is always
placed at the left.
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Levels of structure in proteins
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Levels of structure in proteins
Primary structure a description of the covalent
backbone of a polymer (macromolecule), including
the sequence of monomeric subunits and any
interchain and intrachain covalent
bonds. Secondary structure the
residue-by-residue conformation of the backbone
of a polymer. Tertiary structure the
three-dimensional conformation of a polymer in
its native folded state. Quaternary structure
the three-dimensional structure of a multisubunit
protein particularly the manner in which the
subunits fit together.
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Working with proteins (enzymes)
assay the method used to detect the presence of
the protein
activity and specific activity
crude extract fractionation precipitation
(ammonium sulfate, acetone) dialysis column
chromatography preparative gel electrophoresis pre
parative isoelectric focusing
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Column Chromatography
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Ion-exchange chromatography
Column material negatively charged polymer
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Ion-exchange chromatography
Column material polymer beads with negatively
charged groups
Proteins
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Size exclusion chromatography
Column material porous polymer beads
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Size exclusion chromatography
Protein molecules separate on the basis of size
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Affinity Chromatography
Column material ligand specific to protein of
interest is covalently liked to polymer.
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Affinity Chromatography
Wash column to remove proteins that do not bind
to ligand
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Affinity Chromatography
Solution of ligand
Elute protein of interest with ligand
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Polyacrylamide gel electrophoresis
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Native gel (stained with Coomassie Blue)
Purification stages of RNA polymerase from E.
coli. First lane crude extract Last lane
purified protein indicating four non-identical
subunits.
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SDS gel electrophoresis
SDS binds to most proteins (probably by
hydrophobic interactions) in amounts roughly
proportional to the molecular weight of the
protein.
The bound SDS (about one molecule of SDS per
every two amino acid residues) contributes a
large net negative charge, rendering the
intrinsic charge of the protein insignificant.
Each protein has a similar charge-to-mass
ratio. Electrophoresis in the presence of SDS
separates proteins almost exclusively on the
basis of mass (molecular weight), with the
smaller proteins running faster in the gel.
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Sample preparation for SDS gel electrophoresis
usually involves heating the sample in a boiling
water bath for 5 minutes in the presence of SDS
and 2-mercaptoethanol. This effectively denatures
(unfolds) most proteins and allows the SDS to
bind efficiently. The 2-mercaptoethanol reduces
any disulfides present in the native protein and
prevents disulfide formation during the heating
step.
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SDS gel electrophoresis
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SDS gel electrophoresis
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Two-dimensional gel electrophoresis
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Learning Objectives
Describe the chemistry involved in amino acid
compositional analysis, N-terminal analysis and
the Edman degradation.
Describe protein homology and the terms variable,
invariant (conserved) and conservative
substitution.
Define proteome, and describe the field of
proteomics.
Describe the chemistry of disulfide reduction and
alkylation (acetylation) with iodoacetamide.
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The Covalent Structure of Proteins
Genome projects
DNA sequences
amino acid sequences
function splicing
variants post-translational
modification cell-specific expression
cell cycle expression (gene regulation)
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Proteomics
proteome the entire protein complement encoded
by an organisms DNA.
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Protein Homology
homologous proteins proteins having similar
sequences and similar functions in different
species. invariant residues the same amino acid
appearing in the same position in sequences from
different species these are often said to be
conserved. variable residues variation in amino
acid sequences between species in homologous
proteins. conservative substitutions similar,
but not identical, residues located in the same
sequence position in homologous proteins.
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Amino acid sequence of human cytochrome c
trimethyllysine
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Amino Acid Analysis
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N-Terminal Analysis
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Edman Degradation
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reduction of disulfide bonds
alkylation with iodoacetamide
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N-Terminal Protein Sequence Analysis Proteins
are sequenced on an Applied Biosystems Model 492
N-terminal protein sequencer using N-terminal
Edman degradation chemistry. PTH amino acids are
identified with an Applied Biosytems Model 140
PTH Analyzer and a Model 785A Programmable
detector set at wavelength 269 nm. Sample
Requirements l. Size Sample must be 20 picomole
minimum. More (60 pmole) is better.
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2. State(a) The purer the better. Remember that
if there is more than one amino terminus, the
Edman degradation chemistry will show this (e.g.,
if the protein is only 80 pure and the other 20
is another protein, two sequences will be seen in
approximately an 80/20 split). (b) In a liquid
sample, if you have salts, urea, glycerol, etc.,
we can apply the sample to a Prosorb cartridge
(extra charge) to get rid of these components
before sequencing. (c) SDS Detergents
interfere with sequencing. (d) Use freshly
prepared reagents of good quality in your sample
preparation.
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3. Amino Acids Detected If you need to see
cysteines, we can reduce and alkylate the sample
(extra fee) with iodoacetamide, otherwise
cysteine is not detected. Phosphorylated amino
acids are not detected. Blocked N-terminii can
not be sequenced. 4. Sample Submission Submit
sample as an aqueous solution in an
Eppendorf-type tube or electroblotted onto PVDF
(polyvinylidene difluoride) membrane such as
Millipore's Immobilon P or Applied Biosystems
Problott. The quantity of membrane that can be
fitted into the cartridge is approximately four 3
x 5 mm pieces of PVDF (four lanes off of a gel).
Contact Deb for electroblotting conditions if you
have not done this before
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5. Report The major and minor amino acid calls
at each cycle will be reported with the yield in
pmoles at each position. PTH chromatograms are
also given to the client. Data will be kept on
file by the Facility for 5 years after date of
obtaining the data. 6. Acknowledgement of
Service When publishing experimental results
please credit the Genomics and Proteomics
Facility in the Institute of Molecular Biology at
the University of Oregon, Eugene OR 97403.
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MALDI MS
Matrix-assisted laser desorption/ionization mass
spectrometry
Electrospray Mass Spectroscopy Tandem Mass
Spectroscopy