Chapter 3: Amino Acids, Peptides, and Proteins

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Chapter 3 Amino Acids, Peptides, and Proteins

• Dr. Clower
• Chem 4202

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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

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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

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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

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Classification of Amino Acids

• Classify by structure of R
• Nonpolar
• Polar
• Aromatic
• Acidic
• Basic

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Nonpolar Amino Acids

• Hydrophobic, neutral, aliphatic

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Polar Amino Acids

• Hydrophilic, neutral, typically H-bond

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Disulfide Bonds

• Formed from oxidation of cysteine residues

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Aromatic Amino Acids

• Bulky, neutral, polarity depend on R

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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

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Acid-base Properties

• Remember H3PO4 (multiple pKas)
• AAs also have multiple pKas due to multiple
  ionizable groups

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Table 3-1
Amino acid organization chart
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pH and Ionization

• Consider glycine
• Note that the uncharged species never forms

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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

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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)

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Learning Check

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

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Stereochemistry of AAs

• All amino acids (except glycine) are optically
  active
• Fischer projections

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D and L Configurations

• d dextrorotatory
• l levorotatory
• D, L relative to glyceraldehyde

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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

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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

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CHEM 2412 Review

• Carboxylic acid amine ?
• Structure of amino acid

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The Peptide Bond

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

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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

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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

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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)
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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

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Learning Check

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

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Learning Check

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

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Outline (part II)

• Sections 3.3 and 3.4
• Separation and purification
• Protein sequencing
• Analysis of primary structure

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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

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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

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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

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Solubility

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

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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

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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

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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

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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

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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

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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

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Types of Chromatography

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

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UV-Vis Spectroscopy

• Absorbance used to monitor protein concentrations
  of each fraction
• l 280 nm
• Absorbance of aromatic side groups

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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)

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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

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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
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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

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Dabsyl chloride and FDNB

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

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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)

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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

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Cleaving Disulfide Bonds

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

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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

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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

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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)

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An example
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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?

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Suggested Problems, Chapter 3

• 1-5, 7, 10-13, 15, 18