Biochemistry I - Lectures 1 _ 2 by malj

VIEWS: 2 PAGES: 29

									Ch.5 Proteins: Primary structure
n Polypeptide diversity
n Protein purification and analysis
n Protein sequencing
n Protein evolution
 Ch.5 Proteins: Primary structure

 Polypeptide diversity
 n Primary structure = the amino acid sequence of
   its polypeptide chain(s)
    q What is the importance of primary structure?
    q 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
n Purifying a protein requires a strategy
n Salting out separates proteins by their solubility
n Chromatography involves interaction with
  mobile and stationary phases
n Electrophoresis separates molecules according to
  charge and size
Ch.5 Proteins: Primary structure
Purifying a protein requires a strategy
n Source
n Conditions such as pH, temperature, ionic
  strength must be controlled and optimized
n Assay of protein activity
n Spectrophotometric and other methods used for
  determination of concentration
n 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
n Protein solubility generally increases with
  increasing ionic strength, reaches a maximum,
  then decreases with further increase in ionic
  strength
n 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

n Ion exchange
n Hydrophobic interaction
n Gel filtration
n Affinity
Ch.5 Proteins: Primary structure
Electrophoresis separates molecules according to
charge and size
n SDS-PAGE
n Capillary electrophoresis
n 2D-electrophoresis
pI, pH, acidic & basic proteins
n The pI, or isoelectric point, of a protein is the pH
  at which the net charge on the molecule is zero
n If pH > pI, then the protein is negatively charged
  (acidic proteins have pI < 7)
n 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
n Separate subunits; (identification of N-terminal residues)
n Cleave polypeptide chains (using at least two
  methods to generate distinct sets of peptide
  fragments)
n Sequence fragments
   qChemically (by Edman degradation)
   qMass spectrometry
n Assemble sequence data to yield primary
  structure
Ch.5 Proteins: Primary structure
Protein sequencing
n See Problems 14-18
Generation of peptide fragments: Endopeptidases
n Trypsin                                    See Table 5-3, p.107
  q Cleaves on the C-terminal side of Arg or Lys residues
  q High specificity; does not cleave if following residue is Pro
n Chymotrypsin
  q Cleaves on the C-terminal side of Phe, Trp, Tyr
  q Less specificity; does not cleave if following residue is Pro
Generation of peptide fragments: Chemical methods
n Cyanogen bromide (CNBr)
  q Cleaves on the C-terminal side of Met residues
  q Generates an N-terminal fragment that is a peptidyl
    homoserine lactone
Ch.5 Proteins: Primary structure
Protein sequencing by Edman degradation
n A three-stage reaction that labels and removes the N
  -terminal residue of a polypeptide
n Reaction can be run repetitively to sequence up to 100
  residues in favorable cases
n Automated in modern instrumentation and can be performed
  on small amounts of a peptide (5-10 pmol or <0.1μg
Ch.5 Proteins: Primary structure
Protein sequencing by Edman degradation
n Problem: Method requires a free N-terminus, and
  therefore polypeptides with blocked N-termini (e.g.
  acetylation) cannot be sequenced by Edman
  degradation
n Despite automation, the process takes a
  considerable amount of time
Ch.5 Proteins: Primary structure
Protein sequencing by mass spectrometry
n Tandem mass spectrometry (MS/MS) can be used to
  sequence short peptides (<25 residues)
Ch.5 Proteins: Primary structure
Protein sequencing by mass spectrometry
n Advantages:
   q Method can deal with blocked N-termini
   q Rapid acquisition of sequence data
   q Enables characterization of common post-translational
     modifications
Ch.5 Proteins: Primary structure
Protein sequencing by mass spectrometry
n Limitation: Inability to distinguish Ile and Leu,
  difficulty in distinguishing Gln and Lys
Ch.5 Proteins: Primary structure
Mass spectrometry of proteins
n Mass spectrometry measures mass-to-charge ratio
  (m/z) for ions in gas phase
n 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
n Protein sequences reveal evolutionary relationships
n Proteins evolve by the duplication of genes or gene
  segments
   q Protein families can arise through gene duplication
       n e.g. globin family
   q The rate of sequence divergence varies
   q Many proteins contain domains that occur in other
     proteins
Ch.5 Proteins: Primary structure

Protein evolution
n Terms to be familiar with:
   q Homologous proteins
       n Distinguish between orthologous and paralogous.
       n Domains
   q With respect to residues in multiple sequence alignments:
       n Invariant
       n Conservative substitutions
       n 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

								
To top