Chapter 3: Amino Acids, Peptides, and Proteins - PowerPoint PPT Presentation

1 / 55
About This Presentation
Title:

Chapter 3: Amino Acids, Peptides, and Proteins

Description:

Non-standard amino acids. Formation of Peptide Bonds. The building blocks of proteins ... Amphoteric. Amino group is protonated. Carboxyl group is deprotonated ... – PowerPoint PPT presentation

Number of Views:1991
Avg rating:3.0/5.0
Slides: 56
Provided by: Timb187
Category:

less

Transcript and Presenter's Notes

Title: Chapter 3: Amino Acids, Peptides, and Proteins


1
Chapter 3 Amino Acids, Peptides, and Proteins
  • Dr. Clower
  • Chem 4202

2
Outline (part I)
  • Sections 3.1 and 3.2
  • Amino Acids
  • Chemical structure
  • Acid-base properties
  • Stereochemistry
  • Non-standard amino acids
  • Formation of Peptide Bonds

3
Amino Acids
  • The building blocks of proteins
  • Also used as single molecules in biochemical
    pathways
  • 20 standard amino acids (a-amino acids)
  • Two functional groups
  • carboxylic acid group
  • amino group on the alpha (?) carbon
  • Have different side groups (R)
  • Properties dictate behavior of AAs

4
Zwitterions
  • Both the NH2 and the COOH groups in an amino
    acid undergo ionization in water.
  • At physiological pH (7.4), a zwitterion forms
  • Both and charges
  • Overall neutral
  • Amphoteric
  • Amino group is protonated
  • Carboxyl group is deprotonated
  • Soluble in polar solvents due to ionic character
  • Structure of R also influence solubility

5
Classification of Amino Acids
  • Classify by structure of R
  • Nonpolar
  • Polar
  • Aromatic
  • Acidic
  • Basic

6
Nonpolar Amino Acids
  • Hydrophobic, neutral, aliphatic

7
Polar Amino Acids
  • Hydrophilic, neutral, typically H-bond

8
Disulfide Bonds
  • Formed from oxidation of cysteine residues

9
Aromatic Amino Acids
  • Bulky, neutral, polarity depend on R

10
Acidic and Basic Amino Acids
  • Acidic
  • R group carboxylic acid
  • Donates H
  • Negatively charged
  • Basic
  • R group amine
  • Accepts H
  • Positively charged
  • His ionizes at pH 6.0

11
Acid-base Properties
  • Remember H3PO4 (multiple pKas)
  • AAs also have multiple pKas due to multiple
    ionizable groups

12
Table 3-1
Amino acid organization chart
13
pH and Ionization
  • Consider glycine
  • Note that the uncharged species never forms

14
Titration of Glycine
  • pK1
  • cation zwitterion
  • pK2
  • zwitterion anion
  • First equivalence point
  • Zwitterion
  • Molecule has no net charge
  • pH pI (Isoelectric point)
  • pI average of pKas ½ (pK1 pK2)
  • pIglycine ½ (2.34 9.60) 5.97
  • Animation

15
pI of Lysine
  • For AAs with 3 pKas, pI average of two
    relevant pKa values
  • Consider lysine (pK1 2.18, pK2 8.95, pKR
    10.53)
  • Which species is the isoelectric form?
  • So, pI ½ (pK2 pKR)
  • ½ (8.95 10.53) 9.74
  • Note pKR is not always higher than pK2 (see
    Table 3-1 and Fig. 3-12)

16
Learning Check
  • Would the following ions of serine exist at a pH
    above, below, or at pI?

17
Stereochemistry of AAs
  • All amino acids (except glycine) are optically
    active
  • Fischer projections

18
D and L Configurations
  • d dextrorotatory
  • l levorotatory
  • D, L relative to glyceraldehyde

19
Importance of Stereochemistry
  • All AAs found in proteins are L geometry
  • S enantiomer for all except cysteine
  • D-AAs are found in bacteria
  • Geometry of proteins affects reactivity (e.g
    binding of substrates in enzymes)
  • Thalidomide

20
Non-standard Amino Acids
  • AA derivatives
  • Modification of AA after protein synthesized
  • Terminal residues or R groups
  • Addition of small alkyl group, hydroxyl, etc.
  • D-AAs
  • Bacteria

21
CHEM 2412 Review
  • Carboxylic acid amine ?
  • Structure of amino acid

22
The Peptide Bond
  • Chain of amino acids peptide or protein
  • Amino acid residues connected by peptide bonds
  • Residue AA H2O

23
The Peptide Bond
  • Non-basic and non-acidic in pH 2-12 range due to
    delocalization of N lone pair
  • Amide linkage is planar, NH and CO are anti

24
Polypeptides
  • Linear polymers (no branches)
  • AA monomers linked head to tail
  • Terminal residues
  • Free amino group (N-terminus)
  • Draw on left
  • Free carboxylate group (C-terminus)
  • Draw on right
  • pKa values of AAs in polypeptides differ slightly
    from pKa values of free AAs

25
Naming Peptides
  • Name from the free amine (NH3)
  • Use -yl endings for the names of the amino acids
  • The last amino acid with the free carboxyl group
    (COO-) uses its amino acid name

(GA)
26
Amino Acid Ambiguity
  • Glutamate (Glu/E) vs. Glutamine (Gln/Q)
  • Aspartate (Asp/D) vs. Asparagine (Asn/N)
  • Converted via hydrolysis
  • Use generic abbreviations for either
  • Glx/Z
  • Asx/B
  • X undetermined or nonstandard AA

27
Learning Check
  • Write the name of the following tetrapeptide
    using amino acid names and three-letter
    abbreviations.

28
Learning Check
  • Draw the structural formula of each of the
    following peptides.
  • A. Methionylaspartic acid
  • B. Alanyltryptophan
  • C. Methionylglutaminyllysine
  • D. Histidylglycylglutamylalanine

29
Outline (part II)
  • Sections 3.3 and 3.4
  • Separation and purification
  • Protein sequencing
  • Analysis of primary structure

30
Protein size
  • In general, proteins contain gt 40 residues
  • Minimum needed to fold into tertiary structure
  • Usually 100-1000 residues
  • Percent of each AA varies
  • Proteins separated based on differences in size
    and composition
  • Proteins must be pure to analyze, determine
    structure/function

31
Factors to control
  • pH
  • Keep pH stable to avoid denaturation or chemical
    degradation
  • Presence of enzymes
  • May affect structure (e.g. proteases/peptidase)
  • Temperature
  • Control denaturation (0-4C)
  • Control activity of enzymes
  • Thiol groups
  • Reactive
  • Add protecting group to prevent formation of new
    disulfide bonds
  • Exposure to air, water
  • Denature or oxidize
  • Store under N2 or Ar
  • Keep concentration high

32
General Separation Procedure
  • Detect/quantitate protein (assay)
  • Determine a source (tissue)
  • Extract protein
  • Suspend cell source in buffer
  • Homogenize
  • Break into fine pieces
  • Cells disrupted
  • Soluble contents mix with buffer
  • Centrifuge to separate soluble and insoluble
  • Separate protein of interest
  • Based on solubility, size, charge, or binding
    ability

33
Solubility
  • Selectively precipitate protein
  • Manipulate
  • Concentration of salts
  • Solvent
  • pH
  • Temperature

34
Concentration of salts
  • Adding small amount of salt increases Protein
  • Salt shields proteins from each other, less
    precipitation from aggregation
  • Salting-in
  • Salting out
  • Continue to increase salt decreases protein
  • Different proteins salt out at different salt

35
Other Solubility Methods
  • Solvent
  • Similar theory to salting-out
  • Add organic solvent (acetone, ethanol) to
    interact with water
  • Decrease solvating power
  • pH
  • Proteins are least soluble at pI
  • Isoelectric precipitation
  • Temperature
  • Solubility is temperature dependent

36
Chromatography
  • Mobile phase
  • Mixture dissolved in liquid or solid
  • Stationary phase
  • Porous solid matrix
  • Components of mixture pass through the column at
    different rates based on properties

37
Types of Chromatography
  • Paper
  • Stationary phase filter paper
  • Same theory as thin layer chromatography (TLC)
  • Components separate based on polarity
  • High-performance liquid (HPLC)
  • Stationary phase small uniform particles, large
    surface area
  • Adapt to separate based on polarity, size, etc.
  • Hydrophobic Interaction
  • Hydrophobic groups on matrix
  • Attract hydrophobic portions of protein

38
Types of Chromatography
  • Ion-exchange
  • Stationary phase chemically modified to include
    charged groups
  • Separate based on net charge of proteins
  • Anion exchangers
  • Cation groups (protonated amines) bind anions
  • Cation exchangers
  • Anion groups (carboxylates) bind cations

39
Types of Chromatography
  • Gel-filtration
  • Size/molecular exclusion chromatography
  • Stationary phase gels with pores of particular
    size
  • Molecules separate based on size
  • Small molecules caught in pores
  • Large molecules pass through

40
Types of Chromatography
  • Affinity
  • Matrix chemically altered to include a molecule
    designed to bind a particular protein
  • Other proteins pass through

41
UV-Vis Spectroscopy
  • Absorbance used to monitor protein concentrations
    of each fraction
  • l 280 nm
  • Absorbance of aromatic side groups

42
Electrophoresis
  • Migration of ions in an electric field
  • Electrophoretic mobility (rate of movement)
    function of charge, size, voltage, pH
  • The positively charged proteins move towards the
    negative electrode (cathode)
  • The negatively charged proteins move toward the
    positive electrode (anode)
  • A protein at its pI (neutral) will not migrate in
    either direction
  • Variety of supports (gel, paper, starch,
    solutions)

43
Protein Sequencing
  • Determination of primary structure
  • Need to know to determine 3D structure
  • Gives insight into protein function
  • Approach
  • Denature protein
  • Break protein into small segments
  • Determine sequences of segments
  • Animation

44
End group analysis
  • Identify number of terminal AAs
  • Number of chains/subunits
  • Identify specific AA
  • Dansyl chloride/dabsyl chloride
  • Sanger method (FDNB)
  • Edman degradation (PITC)

Bovine insulin
45
Dansyl chloride
  • Reacts with primary amines
  • N-terminus
  • Yields dansylated polypeptides
  • Dansylated polypeptides hydrolyzed to liberate
    the modified dansyl AA
  • Dansyl AA can be identified by chromatography or
    spectroscopy (yellow fluorescence)
  • Useful method when protein fragmented into
    shorter polypeptides

46
Dabsyl chloride and FDNB
  • Same result as dansyl chloride
  • Dabsyl chloride
  • 1-Fluoro-2,4-dinitrobenzene (FDNB)
  • Sanger method

47
Edman degradation
  • Phenylisothiocyanate (PITC)
  • Reacts with N-terminal AA to produce a
    phenylthiocarbamyl (PTC)
  • Treat with TFAA (solvent/catalyst) to cleave
    N-terminal residue
  • Does not hydrolyze other AAs
  • Treatment with dilute acid makes more stable
    organic compound
  • Identify using NMR, HPLC, etc.
  • Sequenator (entire process for proteins lt 100
    residues)

48
Fragmenting Proteins
  • Formation of smaller segments to assist with
    sequencing
  • Process
  • Cleave protein into specific fragments
  • Chemically or enzymatically
  • Break disulfide bonds
  • Purify fragments
  • Sequence fragments
  • Determine order of fragments and disulfide bonds

49
Cleaving Disulfide Bonds
  • Oxidize with performic acid
  • Cys residues form cysteic acid
  • Acid can oxidize other residues, so not ideal

50
Cleaving Disulfide Bonds
  • Reduce by mercaptans (-SH)
  • 2-Mercaptoethanol
  • HSCH2CH2OH
  • Dithiothreitol (DTT)
  • HSCH2CH(OH)CH(OH)CH2SH
  • Reform cysteine residues
  • Oxidize thiol groups with iodoacetete (ICH2CO2-)
    to prevent reformation of disulfide bonds

51
Hydrolysis
  • Cleaves all peptide bonds
  • Achieved by
  • Enzyme
  • Acid
  • Base
  • After cleavage
  • Identify using chromatography
  • Quantify using absorbance or fluorescence
  • Disadvantages
  • Doesnt give exact sequence, only AAs present
  • Acid and base can degrade/modify other residues
  • Enzymes (which are proteins) can also cleave and
    affect results

52
Enzymatic and Chemical Cleavage
  • Enzymatic
  • Enzymes used to break protein into smaller
    peptides
  • Endopeptidases
  • Catalyze hydrolysis of internal peptide bonds
  • Chemical
  • Chemical reagents used to break up polypeptides
  • Cyanogen bromide (BrCN)

53
An example
54
Another example
  • A protein is cleaved with cyanogen bromide to
    yield the following sequences
  • Arg-Ala-Tyr-Gly-Asn
  • Leu-Phe-Met
  • Asp-Met
  • The same protein is cleaved with chymotrypsin to
    yield the following sequences
  • Met-Arg-Ala-Tyr
  • Asp-Met-Leu-Phe
  • Gly-Asn
  • What is the sequence of the protein?

55
Suggested Problems, Chapter 3
  • 1-5, 7, 10-13, 15, 18
Write a Comment
User Comments (0)
About PowerShow.com