Proteins

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Proteins

• Bioanalytical Chemistry

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Chemical Composition

• Linear chains formed by the reaction of L-amino
  acids with each other
• Backbone
• Identical
• trans-peptide (amide) bonds
• less sterically hindered
• favored by 10001 over cis
• Side chains

3
Amino Acids and Stereochemistry

• C? has 4 different groups attached
• side chain (unique to each amino acid)
• amino group
• carboxyl group
• proton

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Amino Acids and Stereochemistry (contd)

• Can arrange these in two different ways around a
  tetrahedral carbon

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Classification of Common Amino Acids

• Side chains unique, differ in polarity
• Hydrophilic
• Uncharged
• relatively soluble in water
• Charged
• Acidic
• Basic
• Hydrophobic
• relatively insoluble
• tend to self associate

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Hydrophobic

• Alanine Ala A
• -CH3
• Valine Val V
• -CH(CH3)2
• Leucine Leu L
• -CH2CH(CH3)2

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Hydrophobic (contd)

• Isoleucine Ile I
• CH(CH3)(CH2CH3)
• Methionine Met M
• CH2SCH2CH3
• Phenylalanine Phe F
• -CH2(C6H5)

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Hydrophobic

• Tryptophan Trp W

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Hydrophilic and Uncharged

• Serine Ser S
• CH2OH
• Threonine Thr T
• CH(OH)(CH3)

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Hydrophilic and Uncharged (contd)

• Tyrosine Tyr Y

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Hydrophilic and Uncharged (contd)

• Glycine Gly G
• H
• Asparagine Asn N
• CH2CONH2
• Glutamine Gln Q
• CH2CH2CONH2
• Cysteine Cys C
• CH2SH

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Hydrophilic and Uncharged (contd)

• Pro line Pro P

Note cis (C? same side as CO) is preferred for
Pro
Rigid ring - can induce kinks in polypeptide chain
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Hydrophilic and Charged

• Aspartic acid Asp D
• CH2COOH
• Glutamic acid Glu E
• CH2CH2COOH
• Histidine His H

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Hydrophilic and Charged (contd)

• Lysine Lys K
• CH2CH2CH2CH2NH2
• Arginine Arg R

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Nonprotein Amino Acids

• EX ?-aminobutyric acid
• accumulates under stress conditions

GABA chemical agent for transmission of nerve
impulses
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4 Elements of Protein Structure

• Primary
• gt 200 kJ/mol
• Secondary
• 10 kJ/mol
• Tertiary
• lt 5 kJ/mol
• Quaternary - oligomeric (gt 1 chain) only

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Protein Structure

• Primary
• Order of amino acids in linear chain
• N - terminus (start, by convention)
• C-terminus (end)
• Secondary
• Portions of linear chain fold into regular
  conformations
• ?-helix
• ?-sheet
• Other structures ?- or reverse turns, omega
  loops Random coil

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Protein Structure (contd)

• Tertiary structure
• Overall shape
• Quaternary structure
• In proteins with several peptide chains
• EXAMPLE hemoglobin

1A3O from Homo Sapiens
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Primary Structure - Homology

• Cyt c amino acid residues from several species

homology
From Fig. 3.1 in Moore, G.R. Pettigrew, G.W.
Cytochromes c. Evolutionary, Structural and
Physicochemical Aspects. Berlin
Springer-Verlag, 1990.
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Secondary Structure - ?-Helix

• Cylindrical shape
• Stabilized by H-bonds between CO of residue i
  and amide proton of residue i3
• Typically 10 -15 amino acid residues and 3-4
  turns
• Each turn
• contains 3.6 amino acid residues
• covers distance of 5.41 Å

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Secondary Structure - ?-Helix (contd)

• Amino acids with extended side chains
• Hydrophilic and hydrophobic amino acids on
  opposite faces of cylinder

1IRL from Homo sapiens Mott, H. R., Baines,
B. S., Hall, R. M., Cooke, R. M., Driscoll, P.C.,
Weir, M. P., Campbell, I. D. The solution
structure of the F42A mutant of human interleukin
2. J Mol Biol 247 pp. 979 (1995).
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Secondary Structure - ?-Sheet

• Sheet-like structure
• Consisting of 2-6 strands (3-10 amino acids each)
  stabilized by H-bonds
• Parallel
• Antiparallel (more common)
• Contain amino acids branched at ?-C
• e.g., isoleucine, threonine, valine

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Secondary Structure - ?-sheet (contd)

• Silk fibroin (poly Ala-Gly) (theoretical)
• High ?-sheet content
• 400,000 Da

1SLK theoretical Fossey, S. A., Nemethy, G.,
Gibson, K. D., Scheraga, H. A. Conformational
energy studies of beta-sheets of model silk
fibroin peptides. I. Sheets of poly(Ala-Gly)
chains. Biopolymers 31 pp. 1529 (1991).
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Stabilizing Intramolecular Interactions

• H-bonds
• 3 kcal/mol
• Electrostatic interactions - between charged
  amino acid side chains
• salt bridges
• Disulfide bonds
• 2 S-H S-S 2H
• Hydrophobic interactions
• 3-5 kcal/mol (entropic)

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Proteins
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Proteins - Functionally Diverse
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Proteins - Diverse

• E. coli - 1000 different proteins
• Total number of proteins gt 1010

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Chemical Stability

• Proteolysis
• digestion of peptide bonds by proteases
• Hydrolysis
• Enzymatic
• Chemical
• Oxidation
• serine
• cysteine
• Aggregation
• Adsorption

• Deamidation
• asparagine
• Phosphorylation and glycation
• ?-elimination
• Isopeptide formation
• Racemization
• Maillard chemistry
• reaction of amines with reducing sugars

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Conformational Stability Affected by

• pH
• Hydrophobic aggregation
• Pressure
• Shear (mixing, flow, UF)
• Temperature
• Sorption at interfaces
• Metal binding
• Solvent effects

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Deterioration of Proteins

• Thermal Inactivation
• Cold inactivation
• Reversible
• Heat inactivation (gt 600C)
• Irreversible
• Loss of activity
• Microbiological reaction
• Sterile buffer, solutions, glassware, gloves

• Adsorption
• Plasticware rather than glassware
• Clean glassware
• Shear
• Mix solutions gently
• Vortex mixer or sonication vs. inversion

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Deterioration of Proteins - Thermal Inactivation

• Hydrolysis
• avoid acidic/alkaline pH
• Deamidation
• Sulfhydryl oxidation
• reducing agents, e.g., dithiothreitol (DTT) or
  ?-mercaptoethanol
• Disulfide rearrangement
• avoid pH gt 10
• t1/2 (S-S) 10 h at room temperature

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Storage Stabilization of Proteins

• Chemical additives
• 50 glycerol (polyhydroxyl cpds - PHCs)
• 50 methanol (fp depression)
• Salting
• 3M (NH4)2SO4
• lt 0.15M (isotonic in living organisms)
• Freeze/thaw

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Storage Stabilization of Proteins

• Lyophilization
• Chemical modification
• Crosslinking (gluteraldehyde)
• x-ray crystallography

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Storage Stabilization of Proteins

• Dialysis
• Remove low MW impurities
• Ultrafiltration (UF)

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Proteins and pH

• Since proteins are made of amino acids,
• at any pH above the pKa, molecule will be
  negatively charged (R-COO-)
• at any pH below the pKa, the molecule will be
  positively charged (R-NH3)
• at any pH more than 1.0 unit away from pKa,
  moiety can be considered to be essentially
  completely ionized

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At Physiological pH...

• Every amino acid is a zwitterion
• Asp and Glu negatively charged
• Cys and Tyr protonated
• Arg and Lys positively charged

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pI

• pH at which protein has no net charge
• Proteins have low solubility near pI

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Basic Protein

• pI at high pH
• R-NH3 R-NH2 H
• at neutral pH, protein is positively charged
• HPLC use cation exchange

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Acidic Protein

• pI at low pH
• R-COOH RCOO- H
• at neutral pH, protein is negatively charged
• HPLC use anion exchange

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Protein Solubility

• Varies widely
• Depends on hydrophilicity/hydrophobicity of
  surface amino acid residue side chains
• Varying pH may affect
• Proteins less soluble near pI
• Animal proteins pI near 5.5-6.0
• Plant/bacterial proteins pI near 4.5-5.0

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Protein Solubility (contd)

• Can enhance by addition of detergent or solvent
• Form micelle
• Add sodium dodecyl sulfate (SDS) or Triton-X
  (non-ionic)
• Dissolve or precipitate by addition of ethanol or
  acetone
• Disadvantages must remove may denature

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Enzymes

• Proteins that catalyze biological reactions
• E S -gt E P
• Categorized according to S
• Proteases - break down proteins
• Amylases - break down starch into simple sugars
• Lipases - split fats
• Catalases - break down H2O2

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Advantages

• Enantioselective (FDA)
• Regioselective
• Chemoselective
• Cheap
• React under mild conditions (T, P)
• Green alternatives
• Minimal waste cleanup
• Recyclable

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Disadvantages

• Purity
• Inactivation
• Solvent compatibility
• proteolysis
• T, P
• Downstream processing
• Low volume productivity

Issues for Chemists
Chemical Engineering Issues
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Forms Used

• Soluble enzyme
• Cost
• Stability
• Immobilized enzyme
• Kinetic limitations
• Cross-linked enzyme
• Whole cells

Desireable but requires high purity
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Application of UV to Proteins

• 3 aromatic amino acids (?-? chromophores)
• tyrosine
• tryptophan
• phenylalanine

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Amino Acids
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Molar Absorptivity for Selected Amino Acids
Taken from Franks, F. Protein Biotechnology
Humana Totowa, 1993.
NB pKa tyrosine 9.5
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Cofactors Can Be Useful Diagnostic Features

• EX heme group

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Cofactors

• Nicotinamide adenine dinucleotide (NADH)
• flavin adenine dinucleotide (FADH)
• Heme group (Fe in protoporphyrin IX)

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Application of UV to Proteins

• Cofactors
• Heme (?400105 M-1 cm-1)
• NADH (?3406.2 x 106 M-1 cm-1 ?26014 x 106 M-1
  cm-1)
• NAD (?26018 x 106 M-1 cm-1)

Taken from Campbell, I.D. Dwek, R.A. Biological
Spectroscopy Benjamin Cummings Menlo Park,
1984.
Highly delocalized ?-systems
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Cyt c

• Function Redox protein involved in cell
  apoptosis and respiration
• Structure heme protein
• FW 12,384 (horse)
• 6-c low spin
• axial ligands H18, M80
• 2 internal conserved H2Os

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UV-vis of 10 µM Cytochrome c

• Three sets of bands
• Soret
• Q-bands
• 695-nm band
• Intensity and energy redox-dependent

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Spectroscopic Methods of Protein Analysis
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Circular Dichroism (CD)

• A spectroscopic probe of protein secondary
  structure
• Based on differential absorption of lcp/rcp light
  by chromophores

Taken from Franks, F. Protein Biotechnology
Humana Totowa, 1993.
57
Fluorescence

• Natural fluorophores (few)
• Aromatic amino acids (guess which ones!)
• Cofactors
• FADH
• NADH

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Natural Fluorophores
Note ? is the quantum yield think of this as
analogous to ? in UV-vis spectroscopy
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Selected Fluorescent Probes
Note extensively delocalized ?-systems
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Useful Resources

• (1) Bell, J. E. Bell, E. T. Proteins and
  Enzymes Prentice-Hall Englewood Cliffs, 1988.
• (2) Campbell, I. D. Dwek, R. A. Biological
  Spectroscopy The Benjamin/Cummings Publishing
  Company Menlo Park, 1984.
• (3) Cooper, T. G. The Tools of Biochemistry
  John Wiley Sons New York, 1977.
• (4) Franks, F., Ed. Protein Biotechnology.
  Isolation, Characterization, and Stabilization
  Humana Press Totowa, 1993.
• (5) Seidman, L. A. Moore, C. J. Basic
  Laboratory Methods for Biotechnology. Textbook
  and Laboratory Reference Prentice-Hall Upper
  Saddle River, 2000.
• (6) Stein, S., Ed. Fundamentals of Protein
  Biotechnology Marcel Dekker New York, 1990.

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Useful Resources (contd)

• http//www.worthpublishers.com/lehninger3d/