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Ch.5 Proteins: Primary structure Polypeptide diversity Protein purification and analysis Protein sequencing Protein evolution
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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? Ch.5 Proteins: Primary structure A representation of the primary structure of insulin (bovine)
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Polypeptide diversityCh.5 Proteins: Primary structure
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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
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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
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Purifying a protein requires a strategy Ch.5 Proteins: Primary structure
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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
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Chromatography involves interaction with mobile and stationary phases Ion exchange Hydrophobic interaction Gel filtration Affinity Ch.5 Proteins: Primary structure The most common types of chromatography
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Electrophoresis separates molecules according to charge and size SDS-PAGE Capillary electrophoresis 2D-electrophoresis Ch.5 Proteins: Primary structure
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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 7)
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Charge vs. pH for a protein
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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
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Protein sequencing Ch.5 Proteins: Primary structure See Problems 14-18
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Generation of peptide fragments: Endopeptidases Trypsin 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 See Table 5-3, p.107
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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
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Protein sequencing by Edman degradation Ch.5 Proteins: Primary structure 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
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Protein sequencing by Edman degradation Ch.5 Proteins: Primary structure 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
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Protein sequencing by mass spectrometry Ch.5 Proteins: Primary structure Tandem mass spectrometry (MS/MS) can be used to sequence short peptides (<25 residues)
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Protein sequencing by mass spectrometry Ch.5 Proteins: Primary structure Advantages: Method can deal with blocked N-termini Rapid acquisition of sequence data Enables characterization of common post-translational modifications
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Protein sequencing by mass spectrometry Ch.5 Proteins: Primary structure Limitation: Inability to distinguish Ile and Leu, difficulty in distinguishing Gln and Lys
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Mass spectrometry of proteins Ch.5 Proteins: Primary structure 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
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Protein sequence analysis and databases Ch.5 Proteins: Primary structure
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Protein sequence analysis and databases Ch.5 Proteins: Primary structure NCBI: http://www.ncbi.nlm.nih.gov/BLAST
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Protein evolution Ch.5 Proteins: Primary structure 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
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Protein evolution Ch.5 Proteins: Primary structure 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
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Protein evolution Ch.5 Proteins: Primary structure
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Protein evolution Ch.5 Proteins: Primary structure
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Protein evolution Ch.5 Proteins: Primary structure
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Protein evolution Ch.5 Proteins: Primary structure
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