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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Fundamentals of Protein Structure
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Our life is maintained by molecular network
systems
Molecular network system in a cell
(From ExPASy Biochemical Pathways
http//www.expasy.org/cgi-bin/show_thumbnails.pl?2
)
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Proteins play key roles in a living system

• Three examples of protein functions
• CatalysisAlmost all chemical reactions in a
  living cell are catalyzed by protein enzymes.
• TransportSome proteins transports various
  substances, such as oxygen, ions, and so on.
• Information transferFor example, hormones.

Alcohol dehydrogenase oxidizes alcohols to
aldehydes or ketones
Haemoglobin carries oxygen
Insulin controls the amount of sugar in the blood
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Amino acid Basic unit of protein
Different side chains, R, determin the properties
of 20 amino acids.
Amino group
Carboxylic acid group
An amino acid
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20 Amino acids
Leucine (L)
Isoleucine (I)
Glycine (G)
Valine (V)
Alanine (A)
Methionine (M)
Asparagine (N)
Tryptophan (W)
Phenylalanine (F)
Proline (P)
Tyrosine (Y)
Threonine (T)
Serine (S)
Glutamine (Q)
Cysteine (C)
Histidine (H)
Glutamic acid (E)
Asparatic acid (D)
Lysine (K)
Arginine (R)
White Hydrophobic, Green Hydrophilic, Red
Acidic, Blue Basic
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Proteins are linear polymers of amino acids
R2
R1
COO?
COO?
NH3

NH3

C
C
H
H
A carboxylic acid condenses with an amino group
with the release of a water
H2O
H2O
R1
R2
R3
C
CO
C
CO
NH3
NH
NH
C
CO
Peptide bond
Peptide bond
H
H
H
The amino acid sequence is called as primary
structure
D
F
T
A
A
S
K
G
N
S
G
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Amino acid sequence is encoded by DNA base
sequence in a gene
DNA molecule
DNA base sequence

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Amino acid sequence is encoded by DNA base
sequence in a gene
Second letter Second letter Second letter Second letter Second letter Second letter Second letter Second letter
T T C C A A G G
First letter T TTT Phe TCT Ser TAT Tyr TGT Cys T Third letter
First letter T TTC Phe TCC Ser TAC Tyr TGC Cys C Third letter
First letter T TTA Leu TCA Ser TAA Stop TGA Stop A Third letter
First letter T TTG Leu TCG Ser TAG Stop TGG Trp G Third letter
First letter C CTT Leu CCT Pro CAT His CGT Arg T Third letter
First letter C CTC Leu CCC Pro CAC His CGC Arg C Third letter
First letter C CTA Leu CCA Pro CAA Gln CGA Arg A Third letter
First letter C CTG Leu CCG Pro CAG Gln CGG Arg G Third letter
First letter A ATT Ile ACT Thr AAT Asn AGT Ser T Third letter
First letter A ATC Ile ACC Thr AAC Asn AGC Ser C Third letter
First letter A ATA Ile ACA Thr AAA Lys AGA Arg A Third letter
First letter A ATG Met ACG Thr AAG Lys AGG Arg G Third letter
First letter G GTT Val GCT Ala GAT Asp GGT Gly T Third letter
First letter G GTC Val GCC Ala GAC Asp GGC Gly C Third letter
First letter G GTA Val GCA Ala GAA Glu GGA Gly A Third letter
First letter G GTG Val GCG Ala GAG Glu GGG Gly G Third letter
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Gene is proteins blueprint, genome is lifes
blueprint
DNA
Genome
Gene
Protein
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Gene is proteins blueprint, genome is lifes
blueprint
Glycolysis network
Genome
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In 2003, Human genome sequence was deciphered!

• Genome is the complete set of genes of a living
  thing.
• In 2003, the human genome sequencing was
  completed.
• The human genome contains about 3 billion base
  pairs.
• The number of genes is estimated to be between
  20,000 to 25,000.
• The difference between the genome of human and
  that of chimpanzee is only 1.23!

3 billion base pair gt 6 G letters 1 letter
gt 1 byte The whole genome can be recorded in
just 10 CD-ROMs!
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Each Protein has a unique structure
Amino acid sequence NLKTEWPELVGKSVEEAKKVILQDKPEAQI
IVLPVGTIVTMEYRIDRVRLFVDKLDNIAEVPRVG
Folding!
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Basic structural units of proteins Secondary
structure
a-helix
ß-sheet
Secondary structures, a-helix and ß-sheet, have
regular hydrogen-bonding patterns.
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Three-dimensional structure of proteins
Tertiary structure
Quaternary structure
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Hierarchical nature of protein structure

• Primary structure (Amino acid sequence)
• ?
• Secondary structure (a-helix, ß-sheet)
• ?
• Tertiary structure (Three-dimensional structure
  formed by assembly of secondary structures)
• ?
• Quaternary structure (Structure formed by more
  than one polypeptide chains)

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Close relationship between protein structure and
its function
Hormone receptor
Antibody
Example of enzyme reaction
substrates
A
enzyme
enzyme
B
Matching the shape to A
Digestion of A!
enzyme
A
Binding to A
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Protein structure prediction has remained elusive
over half a century

• Can we predict a protein structure from its
  amino acid sequence?
• Now, impossible!

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Summary

• Proteins are key players in our living systems.
• Proteins are polymers consisting of 20 kinds of
  amino acids.
• Each protein folds into a unique
  three-dimensional structure defined by its amino
  acid sequence.
• Protein structure has a hierarchical nature.
• Protein structure is closely related to its
  function.
• Protein structure prediction is a grand challenge
  of computational biology.