Proteins

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Proteins

• Most of the biotech drugs are proteins or
  peptides
• Amino acids are the building blocks of proteins
• The genetic code specifies 20 different amino
  acids

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Amino acids are grouped according to their side
chains
Hydrophobic (nonpolar) Charged (acidic or
basic) Polar (uncharged)
Side chains often determine secondary structure
and protein function
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Basic Protein Structures
Primary structure linear amino acid
sequence Secondary structure shape of initial
folded protein alpha helix, beta sheet,
turns Tertiary structure completely folded
protein - monomer Quaternary structure subunit
assembly monomer, dimer, tetramer, etc.
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Protein Structures
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Primary Structure
The linear amino acid sequence of amino acids
along a polypeptide chain
Leu Ser Val Thr
Leu Ser Val Thr Ser Ala
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Peptide Bonds
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Secondary Structure
Formation of secondary structure along a
polypeptide chain. Occurs mainly as alpha helices
and beta sheets.
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Alpha Helix
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Beta Sheet
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Beta-Alpha-Beta Motif
Several secondary structures will form a motif
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Tertiary structure
Several motifs combine to form globular
structures called domains 50-350 amino
acids Modular unit from which proteins are made
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Quaternary structure
When a protein consists of more than one
polypeptide domain (e.g., dimer)
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Forces in Protein Structures
Hydrophobic interactions - hydrophobic residues
are generally buried within globular structure
hydrophilic residues reside on
surface Hydrogen bonds - hydrogen atom shared
between 2 electronegative atoms (generally
oxygen and nitrogen) Electrostatic forces -
occurs between two charged molecules (same
charge repulsion) Van der Waals - weak
interactions between all atoms due to net
attractive interactions between dipoles
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Hydrophobicity Plot of a Membrane Protein
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Hydration
Water molecules bound to protein both internally
and externally Can form hydrogen bonds with
protein atoms Can occupy active site in presence
or absence of substrate Can help to stabilize
protein structure Serves as solvent on protein
surface
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Protein Folding and Modifications

• Protein folding
• Proteins must be properly folded for function
• Unfolded proteins are insoluble and unstable
• Post-translation modifications
• May be required for protein function -case by
  case basis
• e.g., phosphorylation, glycosylation

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Protein Folding
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Disulfide Bond Formation
Important for proper folding of some
proteins Disulfide bonds (cysteines) must be
properly oxidixed for industrial applications
the use of reducing agent such as DTT may be
necessary
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Disulfide Bond Formation
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Folding Process - unfolded to globular protein
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Protein Folding Gone Bad
Expression in E. coli may lead to inclusion
bodies improperly folded or unfolded
proteins Can use detergents to stabilize and
allow protein to fold basically unfold and then
allow protein to fold In humans..can lead to
disease state
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Protein Re-Folding
Refolding proteins - occurs through
intermediates - slow removal of denaturants
(urea or guanidinium) via dialysis dilution
in buffer (no denaturants) Only useful in the
lab.
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Alzheimers - Improper Folding
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Alzheimers Plaques and Tangles
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Alzheimer Amyloid Production
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Refolding -gt Sticky Proteins -gt Aggregates
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Glycosylation
Most extensive post-translational modification
made by eukaryotic cells. Addition of
carbohydrates (mannose, glucose, galactose) to a
reactive side chain of an amino acid.
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Why do proteins need to be glycosylated?
Involved in protein interactions Helps to
solubilize and stabilize proteins Effects protein
biosynthesis and secretion Immune protection For
many glycoproteins Full glycosylation full
biological activity
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Glycosylation
Recombinant proteins expressed in eukaryotic
cells will be glycosylated. E. coli does NOT
glycosylate proteins.
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Glycosylation
N-linked glycan Amide N of Asn O-linked
glycan OH group Ser, Thr
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N-linked Glycosylation
Contains N-acetylglucosamine linked to amide
group of Asn N-linked motif Asn-X-Ser/Thr
where X represents any amino acid Common
pentasaccharide core (trimannosyl core)
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N-linked Glycosylation
N-linked complex glycan
Trimannosyl core
Asn
Asn
Mannose
N-acetylneuraminic acid
Galactose
N-acetylglucosamine
Fucose
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O-linked Glycosylation

• Contains N-acetylgalactosamine linked to
• hydroxyl group of Ser or Thr

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O-linked Glycosylation
Ser
N-acetylneuraminic acid
N-acetylgalactosamine
Galactose
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Filgrastim MW core protein 18.6 kDa
G-CSF MW glycoprotein approx. 20 kDa
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Recombinant G-CSF
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Erythropoietin (EPO) a clinically useful
glycoprotein
Primary regulator of erythropoiesis Gold
Standard for treating anemia associated with
renal failure 30 kDa protein ? 40 of proteins
mass is composed of carbohydrates
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Carbohydrate Structure of EPO Varies
Isoform (No. of Sialic Acid residues)
3 N-linked chains
1 O-linked chains
14 13 12 11 10 9
Br. J. Cancer (2001) 84 (Supplement 1), 3-10
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In vivo Efficacy of EPO Isoforms
Br. J. Cancer (2001) 84 (Supplement 1), 3-10