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proteins_1_primary_structure__purificati

proteins_1_primary_structure__purificati - Ch Chemistry 237...

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Ch i t 237 Chemistry 237 Proteins 1: Purification and Proteins 1: Purification and Primary Sequence Determination
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Learning Goals • Learn the basics of protein purification Learn the basics of protein purification Learn the techniques of protein sequencing Discuss some of the uses of sequences
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Peptides and proteins Linear sequence of amino acids. One free N One free N terminus and one free C terminus. Held together by peptide bonds Sequences are written from the N written from the N to the C terminus.
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Why we bother Proteins are the agents of biological function Enzymes - Ribonuclease Regulatory proteins - Insulin Transport proteins - Hemoglobin Structural proteins - Collagen • Contractile proteins - Actin, Myosin Contractile proteins Actin, Myosin Exotic proteins - Antifreeze proteins in fish, green fluorescent protein
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Why we bother C t l i Catalysis: –enolase (in the glycolytic pathway) –DNA polymerase (in DNA replication) Transport: –hemoglobin (transports O 2 in the blood) –lactose permease (transports lactose across the cell membrane) • Structure: collagen (connective tissue) –collagen (connective tissue) –keratin (hair, nails, feathers, horns) • Motion: –myosin (muscle tissue) –actin (muscle tissue, cell motility)
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What to Study about Peptides and Proteins? Wh t i it d iti ? What is its sequence and composition? What is its three-dimensional structure? H d i fi d i i f ld? How does it find its native fold? How does it achieve its biochemical role? How is its function regulated? How does it interacts with other macromolecules? How is it related to other proteins? Where is it localized within the cell? What are its physico-chemical properties?
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Covalent Structures of Proteins Sequence determination techniques: 1) Gene sequence. Each amino acid is coded for by 3 nucleotides DNA coded for by 3 nucleotides. DNA sequence can be translated into protein sequence. 2) Chemical and enzymatic sequencing of 2) Chemical and enzymatic sequencing of purified protein. Mass spectrometry sequencing.
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Protein Purification Know gene sequence. Know protein sequence Know protein sequence. Know size charge maybe something about the Know size, charge, maybe something about the structure and function. Design a purification scheme based on those properties
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Protein Purification Know protein function. Don’t know protein sequence Don t know protein sequence. Don’t know size charge maybe something about Don t know size, charge, maybe something about the structure Try different separation techniques and monitor protein concentration and function protein concentration and function.
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Protein Purification • Separation relies on differences in Separation relies on differences in physico-chemical properties – Size Size – Charge – Affinity for a ligand – Solubility – Hydrophobicity – Thermal stability Chromatography is commonly used for preparative separation
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