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Ch.5 Proteins: Primary structure Polypeptide diversity Protein purification and analysis Protein sequencing Protein evolution Ch.5 Proteins: Primary structure Polypeptide diversity Primary structure = the amino acid sequence of its polypeptide chain(s) 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 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 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 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 Ion exchange Hydrophobic interaction Gel filtration Affinity Ch.5 Proteins: Primary structure Electrophoresis separates molecules according to charge and size SDS-PAGE Capillary electrophoresis 2D-electrophoresis pI, pH, acidic & basic proteins 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 Separate subunits; (identification of N-terminal residues) Cleave polypeptide chains (using at least two methods to generate distinct sets of peptide fragments) Sequence fragments Chemically (by Edman degradation) Mass spectrometry Assemble sequence data to yield primary structure Ch.5 Proteins: Primary structure Protein sequencing See Problems 14-18 Generation of peptide fragments: Endopeptidases Trypsin See Table 5-3, p.107 Cleaves on the C-terminal side of Arg or Lys residues High specificity; does not cleave if following residue is Pro Chymotrypsin Cleaves on the C-terminal side of Phe, Trp, Tyr Less specificity; does not cleave if following residue is Pro Generation of peptide fragments: Chemical methods Cyanogen bromide (CNBr) 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 A three-stage reaction that labels and removes the N-terminal residue of a polypeptide 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 Ch.5 Proteins: Primary structure Protein sequencing by Edman degradation 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 Tandem mass spectrometry (MS/MS) can be used to sequence short peptides (<25 residues) Ch.5 Proteins: Primary structure Protein sequencing by mass spectrometry Advantages: 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 Limitation: Inability to distinguish Ile and Leu, difficulty in distinguishing Gln and Lys Ch.5 Proteins: Primary structure Mass spectrometry of proteins 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 Protein sequences reveal evolutionary relationships Proteins evolve by the duplication of genes or gene segments Protein families can arise through gene duplication 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 Terms to be familiar with: Homologous proteins Distinguish between orthologous and paralogous. Domains With respect to residues in multiple sequence alignments: 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