Ch.5

Report
Ch.5 Proteins: Primary structure
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Polypeptide diversity
Protein purification and analysis
Protein sequencing
Protein evolution
Ch.5 Proteins: Primary structure
Polypeptide diversity
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Primary structure = the amino acid sequence of
its polypeptide chain(s)
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What is the importance of primary structure?
Are there limits to the size and composition of
naturally occurring proteins?
A representation of the primary structure of insulin (bovine)
Ch.5 Proteins: Primary structure
Polypeptide diversity
Ch.5 Proteins: Primary structure
Protein purification and analysis
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Purifying a protein requires a strategy
Salting out separates proteins by their solubility
Chromatography involves interaction with
mobile and stationary phases
Electrophoresis separates molecules according to
charge and size
Ch.5 Proteins: Primary structure
Purifying a protein requires a strategy
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Source
Conditions such as pH, temperature, ionic
strength must be controlled and optimized
Assay of protein activity
Spectrophotometric and other methods used for
determination of concentration
Purification is a stepwise process
Ch.5 Proteins: Primary structure
Purifying a protein requires a strategy
Ch.5 Proteins: Primary structure
Salting out separates proteins by their solubility
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Protein solubility generally increases with
increasing ionic strength, reaches a maximum,
then decreases with further increase in ionic
strength
Ammonium sulfate is the ionic compound of
choice for salting
out proteins
Ch.5 Proteins: Primary structure
Chromatography involves interaction with
mobile and stationary phases
The most common types of chromatography
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Ion exchange
Hydrophobic interaction
Gel filtration
Affinity
Ch.5 Proteins: Primary structure
Electrophoresis separates molecules according to
charge and size
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SDS-PAGE
Capillary electrophoresis
2D-electrophoresis
pI, pH, acidic & basic proteins
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The pI, or isoelectric point, of a protein is the pH
at which the net charge on the molecule is zero
If pH > pI, then the protein is negatively charged
(acidic proteins have pI < 7)
If pH < pI, the protein is positively charged (basic
proteins have pI > 7)
Charge vs. pH for a protein
Ch.5 Proteins: Primary structure
Protein sequencing
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Separate subunits; (identification of N-terminal residues)
Cleave polypeptide chains (using at least two
methods to generate distinct sets of peptide
fragments)
Sequence fragments
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Chemically (by Edman degradation)
Mass spectrometry
Assemble sequence data to yield primary
structure
Ch.5 Proteins: Primary structure
Protein sequencing
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See Problems 14-18
Generation of peptide fragments: Endopeptidases
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Trypsin
See Table 5-3, p.107
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Cleaves on the C-terminal side of Arg or Lys residues
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High specificity; does not cleave if following residue is Pro
Chymotrypsin
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Cleaves on the C-terminal side of Phe, Trp, Tyr
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Less specificity; does not cleave if following residue is Pro
Generation of peptide fragments: Chemical methods
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Cyanogen bromide (CNBr)
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Cleaves on the C-terminal side of Met residues
Generates an N-terminal fragment that is a peptidyl
homoserine lactone
Ch.5 Proteins: Primary structure
Protein sequencing by Edman degradation
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A three-stage reaction that labels and removes the
N-terminal residue of a polypeptide
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Reaction can be run repetitively to sequence up to 100
residues in favorable cases
Automated in modern instrumentation and can be performed
on small amounts of a peptide (5-10 pmol or <0.1μg
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Ch.5 Proteins: Primary structure
Protein sequencing by Edman degradation
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Problem: Method requires a free N-terminus, and
therefore polypeptides with blocked N-termini (e.g.
acetylation) cannot be sequenced by Edman
degradation
Despite automation, the process takes a
considerable amount of time
Ch.5 Proteins: Primary structure
Protein sequencing by mass spectrometry
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Tandem mass spectrometry (MS/MS) can be used to
sequence short peptides (<25 residues)
Ch.5 Proteins: Primary structure
Protein sequencing by mass spectrometry
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Advantages:
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Method can deal with blocked N-termini
Rapid acquisition of sequence data
Enables characterization of common post-translational
modifications
Ch.5 Proteins: Primary structure
Protein sequencing by mass spectrometry
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Limitation: Inability to distinguish Ile and Leu,
difficulty in distinguishing Gln and Lys
Ch.5 Proteins: Primary structure
Mass spectrometry of proteins
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Mass spectrometry measures mass-to-charge ratio
(m/z) for ions in gas phase
Electrospray ionization (ESI) mass spectrometry is an
accurate method for determining mass of intact
polypeptides
Ch.5 Proteins: Primary structure
Protein sequence analysis and databases
Ch.5 Proteins: Primary structure
Protein
sequence analysis and databases
NCBI: http://www.ncbi.nlm.nih.gov/
BLAST
Ch.5 Proteins: Primary structure
Protein evolution
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Protein sequences reveal evolutionary relationships
Proteins evolve by the duplication of genes or gene
segments
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Protein families can arise through gene duplication
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e.g. globin family
The rate of sequence divergence varies
Many proteins contain domains that occur in other
proteins
Ch.5 Proteins: Primary structure
Protein evolution
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Terms to be familiar with:
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Homologous proteins
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Distinguish between orthologous and paralogous.
Domains
With respect to residues in multiple sequence alignments:
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Invariant
Conservative substitutions
Hypervariable positions
Ch.5 Proteins: Primary structure
Protein evolution
Ch.5 Proteins: Primary structure
Protein evolution
Ch.5 Proteins: Primary structure
Protein evolution
Ch.5 Proteins: Primary structure
Protein evolution

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