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    BSE321A_160125.pptx

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    BSE321

    Structural Biology
    Non-natural amino acids
    Hydroxyproline is produced by hydroxylation of the amino acid proline by the enzyme prolyl
    hydroxylase following protein synthesis (as a post-translational modification). The enzyme
    catalyzed reaction takes place in the lumen of the endoplasmic reticulum. Although it is not
    directly incorporated into proteins, hydroxyproline comprises roughly 4% of all amino acids
    found in animal tissue, an amount greater than seven other amino acids that are translationally
    incorporated.
    Non-natural amino acids
    Steps and methods for structural analysis of
    proteins at different resolutions
    Probing protein structure, function and
    interactions
    High-resolution methods: X-ray crystallography
    Nuclear Magnetic Resonance
    spectroscopy
    Cryogenic electron microscopy

    Low resolution methods: Resonance energy transfer


    methods
    Surface Plasmon Resonance and
    Isothermal Calorimetry
    Hydrogen/Deuterium
    exchange mass spectrometry
    Before you can probe structure, function and
    interaction of proteins

    Consistent supply of mg quantities of highly pure and stable protein

    Recombinant protein expression


    Structural studies of proteins
    Recombinant expression and purification

    - Biophysical methods to investigate protein structures typically require consistent


    supply of mg quantities of highly pure and stable protein samples.

    - Levels of proteins in native tissues are typically suboptimal for large-scale isolation.

    - Therefore, the first step towards structural studies of proteins is recombinant


    protein expression.

    Expression in a suitable host


    Cloning in a plasmid vector
    system

    Structural studies e.g. X-ray Purification of the recombinant


    crystallography, cryo-EM, NMR proteins
    Critical factors for recombinant protein expression

    Expression host for recombinant Bacterial (E. coli) or eukaryotic (yeast,


    insect cells, mammalian cells), cell-free
    proteins
    expression system etc.

    Expression construct design for Promoter, tags for detection and


    recombinant proteins purification, antibiotic resistance etc.

    Expression conditions for Culture conditions (e.g. temperature,


    recombinant proteins time, cell density, induction),
    virus-based, transient vs. stable etc.
    Plasmid vectors for recombinant expression

    Which tag to use?


    N- or C-terminus?
    Expression systems for recombinant proteins
    Expression systems for recombinant proteins
    Expression systems for recombinant proteins
    Recombinant protein purification
    Affinity chromatography
    Affinity chromatography
    Purification of a biomolecule with respect to biological function or individual chemical
    structure
    Examples of affinity tags and ligands

    • His-tag • Transition metal ion


    • FLAGTM peptide • Monoclonal antibody
    • Strep-tag • Biotin
    • GST tag • Glutathione
    • Maltose binding protein fusion • Amylose
    • Calmodulin binding protein fusion • Ca2+
    Ion exchange chromatography (IEC)
    Cation exchange chromatography Anion exchange chromatography

    Positively charged molecules are attracted to a Negatively charged molecules are attracted to
    negatively charged solid support. a positively charged solid support.
    Cation exchange chromatography

    ---positively charged molecules are attracted to a


    negatively charged solid support. Commonly used
    cation exchange resins are S-resin, sulfate
    derivatives; and CM resins, carboxylate derived
    ions
    Anion exchange chromatography

    ---negatively charged molecules is attracted to a


    positively charged solid support. Commonly used
    anion exchange resins are Q-resin, a Quaternary
    amine; and DEAE resin, DiEthylAminoEthane
    Size exclusion chromatography (SEC)
    Size exclusion chromatography (SEC)
    Assessment of protein purity
    Poly-acrylamide gel electrophoresis

    Further confirmation of purified protein


    - Mass spectrometry
    - N-terminal sequencing
    - Functional assay
    Next Lecture: Structural basis of
    Protein-Ligand interaction

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