CHAPTER 3 Amino Acids, Peptides, Proteins - PowerPoint PPT Presentation

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CHAPTER 3 Amino Acids, Peptides, Proteins

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Amino Acids, Peptides, Proteins Structure and naming of amino acids Structure and properties of peptides Ionization behavior of amino acids and peptides – PowerPoint PPT presentation

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Title: CHAPTER 3 Amino Acids, Peptides, Proteins


1
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

2
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)

3
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

4
Amino Acids Atom Naming
  • Organic nomenclature start from one end
  • Biochemical designation start from
  • ?-carbon and go down the R-group

5
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

6
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)

7
Aliphatic Amino Acids
  • http//en.wikipedia.org/wiki/FileAa.svg

8
Aromatic Amino Acids
  • http//en.wikipedia.org/wiki/FileAa.svg

9
Charged Amino Acids
  • http//en.wikipedia.org/wiki/FileAa.svg

10
Polar Amino Acids
  • http//en.wikipedia.org/wiki/FileAa.svg

11
Special Amino Acids
  • http//en.wikipedia.org/wiki/FileAa.svg

12
(No Transcript)
13
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
14
The Genetic Code is organized by Amino Acid
Properties
15
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.

16
Substituent effects on pKa Values
?-carboxy group is much more acidic than in
carboxylic acids ?-amino group is slightly less
basic than in amines
17
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.
18
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
  • At this point, the net charge is zero
  • AA is least soluble in water
  • AA does not migrate in electric field

19
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?

20
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

21
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-)

22
Peptide Ends are Not the Same
Numbering starts from the amino terminus
AA1 AA2 AA3 AA4
AA5
23
The Three Letter Code
  • Naming starts from the N-terminus
  • Sequence is written as
  • Ala-Glu-Gly-Lys
  • Sometimes the one-letter code is used
  • AEGK

24
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)

25
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

26
Polypeptide Size in Some Proteins
27
Classes of Conjugated Proteins
28
Peptides and Proteins- Burning 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?

29
Purification Fractionation of Protein Mixtures
  • Separation relies on differences in
    physico-chemical 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

30
Protein Fractionation
http//www.salinesystems.org/content/figures/1746-
1448-4-1-2-l.jpg
31
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

32
Separation by Size
  • Size exclusion (Gel Filtration) Chromatography
  • Loading vol. lt5 of column volume
  • Samples diluted
  • Dialysis or Centrifugal concentrators

33
Separation by Affinity
  • Affinity Chromatography
  • Free Ligand-Beads -- centrifugation
  • Ligand-Magnetic-Beads
  • Immuno-assays on solid supports

34
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

35
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

36
Protein Sequencing
37
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

38
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

39
Nonpolar, Aliphatic R Groups
40
Aromatic R Groups
Also Hydrophobic These amino acid side chains
absorb UV light at 270-280 nm
41
Polar, Uncharged R Groups
These amino acids side chains can form hydrogen
bonding Cysteine can form disulfide bonds
42
Basic R Groups
43
Acidic R Groups
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