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    Amino Acids, Peptides, Proteins

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    lordniklaus
    This chapter discusses amino acids, peptides, and proteins. It covers the structure and naming of amino acids, properties of peptides, ionization behavior, and purification and analysis methods. The main functions of proteins are also summarized, including catalysis, transport, structure, and motion. Key topics include the building blocks of proteins from amino acids, classifications of amino acids, ionization properties, and methods for separating and analyzing peptides and proteins.

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    Amino Acids, Peptides, Proteins

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    lordniklaus
    72 views43 pages
    This chapter discusses amino acids, peptides, and proteins. It covers the structure and naming of amino acids, properties of peptides, ionization behavior, and purification and analysis methods. The main functions of proteins are also summarized, including catalysis, transport, structure, and motion. Key topics include the building blocks of proteins from amino acids, classifications of amino acids, ionization properties, and methods for separating and analyzing peptides and proteins.

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    Amino Acids Tutorial

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    ch03

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    This chapter discusses amino acids, peptides, and proteins. It covers the structure and naming of amino acids, properties of peptides, ionization behavior, and purification and analysis methods. The main functions of proteins are also summarized, including catalysis, transport, structure, and motion. Key topics include the building blocks of proteins from amino acids, classifications of amino acids, ionization properties, and methods for separating and analyzing peptides and proteins.

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    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    72 views43 pages

    Amino Acids, Peptides, Proteins

    Uploaded by

    lordniklaus
    This chapter discusses amino acids, peptides, and proteins. It covers the structure and naming of amino acids, properties of peptides, ionization behavior, and purification and analysis methods. The main functions of proteins are also summarized, including catalysis, transport, structure, and motion. Key topics include the building blocks of proteins from amino acids, classifications of amino acids, ionization properties, and methods for separating and analyzing peptides and proteins.

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    of 43

    CHAPTER 3 Amino Acids, Peptides, Proteins

    Structure and naming of amino acids Structure and properties of peptides Ionization behavior of amino acids and peptides Purification and assay methods Peptide sequencing and chemical synthesis Protein sequence analysis

    Proteins: Main Agents of Biological Function


    Catalysis:
    enolase (in the glycolytic pathway) DNA polymerase (in DNA replication)

    Transport:
    hemoglobin (transports O2 in the blood) lactose permease (transports lactose across the cell membrane)

    Structure:
    collagen (connective tissue) keratin (hair, nails, feathers, horns)

    Motion:
    myosin (muscle tissue) actin (muscle tissue, cell motility)

    Amino Acids: Building Blocks of Protein


    Proteins are heteropolymers of -amino acids Amino acids have properties that are well suited to carry out a variety of biological functions:
    Capacity to polymerize Useful acid-base properties Varied physical properties Varied chemical functionality

    Amino Acids: Atom Naming


    Organic nomenclature: start from one end Biochemical designation: start from -carbon and go down the R-group

    Most -Amino Acids are Chiral


    The -carbon has always four substituents and is tetrahedral All (except proline) have an acidic carboxyl group, a basic amino group, and an alpha hydrogen connected to the -carbon Each amino acid has an unique fourth substituent R In glycine, the fourth substituent is also hydrogen

    Amino Acids: Classification


    Common amino acids can be placed in five basic groups depending on their R substituents:
    Nonpolar, aliphatic (7) Aromatic (3) Polar, uncharged (5)

    Positively charged (3)


    Negatively charged (2)

    Aliphatic Amino Acids

    http://en.wikipedia.org/wiki/File:Aa.svg

    Aromatic Amino Acids

    http://en.wikipedia.org/wiki/File:Aa.svg

    Charged Amino Acids

    http://en.wikipedia.org/wiki/File:Aa.svg

    Polar Amino Acids

    http://en.wikipedia.org/wiki/File:Aa.svg

    Special Amino Acids

    http://en.wikipedia.org/wiki/File:Aa.svg

    Uncommon Amino Acids in Proteins

    Not incorporated by ribosomes Arise by post-translational modifications of proteins Reversible modifications, esp. phosphorylation is important in regulation and signaling

    The Genetic Code is organized by Amino Acid Properties

    Ionization
    At acidic pH, the carboxyl group is protonated and the amino acid is in the cationic form At neutral pH, the carboxyl group is deprotonated but the amino group is protonated. The net charge is zero; such ions are called Zwitterions

    At alkaline pH, the amino group is neutral NH2 and the amino acid is in the anionic form.

    Substituent effects on pKa Values


    -carboxy group is much more acidic than in carboxylic acids -amino group is slightly less basic than in amines

    Amino Acids Can Act as Buffers


    Amino acids with uncharged side-chains, such as glycine, have two pKa values: The pKa of the -carboxyl group is 2.34 The pKa of the -amino group is 9.6

    It can act as a buffer in two pH regimes.

    Amino Acids Carry a Net Charge of Zero at a Specific pH


    Zwitterions predominate at pH values between the pKa values of amino and carboxyl group
    For amino acid without ionizable side chains, the Isoelectric Point (equivalence point, pI) is
    pK1 pK 2 pI 2

    At this point, the net charge is zero AA is least soluble in water AA does not migrate in electric field

    Ionizable Side Chains Can Show Up in Titration Curves


    Ionizable side chains can be also titrated Titration curves are now more complex pKa values are discernable if two pKa values are more than two pH units apart Why is the side-chain pKa so much higher?

    How to Calculate the pI When the Side-chain is Ionizable?


    Identify species that carries a net zero charge Identify pKa value that defines the acid strength of this zwitterion: (pK2)

    Identify pKa value that defines the base strength of this zwitterion: (pKR)
    Take the average of these two pKa values

    Peptides and Peptide bonds


    Peptide bond in a di-peptide Peptides are small condensation products of amino acids They are small compared to proteins (di, tri, tetra oligo-)

    Peptide Ends are Not the Same


    Numbering starts from the amino terminus
    AA1 AA2 AA3 AA4 AA5

    The Three Letter Code


    Naming starts from the N-terminus Sequence is written as: Ala-Glu-Gly-Lys Sometimes the oneletter code is used: AEGK

    Peptides: A Variety of Functions


    Hormones and pheromones:
    insulin (think sugar) oxytocin (think childbirth) sex-peptide (think fruit fly mating)

    Neuropeptides
    substance P (pain mediator)

    Antibiotics:
    polymyxin B (for Gram - bacteria) bacitracin (for Gram + bacteria)

    Protection, e.g. toxins


    amanitin (mushrooms) conotoxin (cone snails) chlorotoxin (scorpions)

    Proteins are:
    Polypeptides (covalently linked -amino acids) + possibly cofactors, coenzymes, prosthetic groups, other modifications
    Cofactor is a general term for functional non-amino acid component
    Metal ions or organic molecules

    Coenzyme is used to designate an organic cofactors


    NAD+ in lactate dehydrogenase

    Prosthetic groups are covalently attached cofactors


    Heme in myoglobin

    Polypeptide Size in Some Proteins

    Classes of Conjugated Proteins

    Peptides and ProteinsBurning Questions


    Sequence and composition? Three-dimensional structure? Folding Mechanism? Biochemical role? Functional regulation? Molecular interactions with small and macro-molecules?

    Structural and sequence relatives?


    Cellular and sub-cellular localization? Physical and chemical properties?

    Purification Fractionation of Protein Mixtures


    Separation relies on differences in physicochemical properties
    Solubility Selective Precipitation (Centrifugation) Thermal stability -Charge --Electrophoresis, Isoelectric Focusing, IEC Size Dialysis, Sedimentation (Centrifugation), GFC Affinity for a ligand Pull down assays (Centrifugation), AC Hydrophobicity (HIC)

    Chromatography is commonly used for preparative separation

    Protein Fractionation

    http://www.salinesystems.org/content/figures/1746-1448-4-1-2-l.jpg

    Separation by Charge
    Ion Exchange Chromatography
    Anion exchange
    Matrix positive Proteins negative Displaced by anions

    Cation exchange Opposite pH determines net charge on Proteins Salt concentration gradient

    Native gel electrophoresis Iso-electric Focusing

    Separation by Size
    Size exclusion (Gel Filtration) Chromatography
    Loading vol. <5% of column volume Samples diluted

    Dialysis or Centrifugal concentrators

    Separation by Affinity
    Affinity Chromatography Free Ligand-Beads -centrifugation Ligand-MagneticBeads Immuno-assays on solid supports

    Electrophoresis for Protein Analysis


    Separation in analytical scale is commonly done by electrophoresis
    Electric field pulls proteins according to their charge Gel matrix hinders mobility of proteins according to their size and shape

    SDS PAGE: Molecular Weight


    SDS sodium dodecyl sulfate a detergent SDS micelles binds to, and unfold all the proteins
    SDS gives all proteins an uniformly negative charge The native shape of proteins does not matter Rate of movement will only depend on size: small proteins will move faster
    -

    Protein Sequencing

    Spectroscopic Detection of Aromatic Amino Acids


    The aromatic amino acids absorb light in the UV region Proteins typically have UV absorbance maxima around 275-280 nm Tryptophan and tyrosine are the strongest chromophores Concentration can be determined by UV-visible spectrophotometry using Beers law: A = cl

    Chapter 3: Summary
    In this chapter, we learned about: The many biological functions of peptides and proteins The structures and names of amino acids found in proteins The ionization properties of amino acids and peptides The methods for separation and analysis of proteins

    Nonpolar, Aliphatic R Groups

    Aromatic R Groups
    Also Hydrophobic
    These amino acid side chains absorb UV light at 270-280 nm

    These amino acids side chains can form hydrogen bonding Cysteine can form disulfide bonds

    Polar, Uncharged R Groups

    Basic R Groups

    Acidic R Groups

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