Protein Basics Maureen Hillenmeyer 020402

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Protein BasicsMaureen Hillenmeyer02-04-02

• Protein function
• Protein structure
• Primary
• Amino acids
• Linkage
• Protein conformation framework
• Dihedral angles
• Ramachandran plots
• Sequence similarity and variation

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Protein Function in Cell

• Enzymes
• Catalyze biological reactions
• Structural role
• Cell wall
• Cell membrane
• Cytoplasm

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Protein Structure
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Protein Structure
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Model Molecule Hemoglobin
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Hemoglobin Background

• Protein in red blood cells

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Red Blood Cell (Erythrocyte)
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Hemoglobin Background

• Protein in red blood cells
• Composed of four subunits, each containing a heme
  group a ring-like structure with a central iron
  atom that binds oxygen

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Heme Groups in Hemoglobin
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Hemoglobin Background

• Protein in red blood cells
• Composed of four subunits, each containing a heme
  group a ring-like structure with a central iron
  atom that binds oxygen
• Picks up oxygen in lungs, releases it in
  peripheral tissues (e.g. muscles)

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Hemoglobin Quaternary Structure
Two alpha subunits and two beta subunits (141 AA
per alpha, 146 AA per beta)
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Hemoglobin Tertiary Structure
One beta subunit (8 alpha helices)
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Hemoglobin Secondary Structure
alpha helix
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Structure Stabilizing Interactions

• Noncovalent
• Van der Waals forces (transient, weak electrical
  attraction of one atom for another)
• Hydrophobic (clustering of nonpolar groups)
• Hydrogen bonding

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Hydrogen Bonding

• Involves three atoms
• Donor electronegative atom (D)
• (Nitrogen or Oxygen in proteins)
• Hydrogen bound to donor (H)
• Acceptor electronegative atom (A) in close
  proximity

D H
A
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D-H Interaction

• Polarization due to electron withdrawal from the
  hydrogen to D giving D partial negative charge
  and the H a partial positive charge
• Proximity of the Acceptor A causes further charge
  separation

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D-H Interaction

• Polarization due to electron withdrawal from the
  hydrogen to D giving D partial negative charge
  and the H a partial positive charge
• Proximity of the Acceptor A causes further charge
  separation
• Result
• Closer approach of A to H
• Higher interaction energy than a simple van der
  Waals interaction

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Hydrogen Bonding And Secondary Structure
beta-sheet
alpha-helix
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Structure Stabilizing Interactions

• Noncovalent
• Van der Waals forces (transient, weak electrical
  attraction of one atom for another)
• Hydrophobic (clustering of nonpolar groups)
• Hydrogen bonding
• Covalent
• Disulfide bonds

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Disulfide Bonds

• Side chain of cysteine contains highly reactive
  thiol group
• Two thiol groups form a disulfide bond

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Disulfide Bridge
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Disulfide Bonds

• Side chain of cysteine contains highly reactive
  thiol group
• Two thiol groups form a disulfide bond
• Contribute to the stability of the folded state
  by linking distant parts of the polypeptide
  chain 

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Disulfide Bridge Linking Distant Amino Acids
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Hemoglobin Primary Structure
NH2-Val-His-Leu-Thr-Pro-Glu-Glu- Lys-Ser-Ala-Val-T
hr-Ala-Leu-Trp- Gly-Lys-Val-Asn-Val-Asp-Glu-Val- G
ly-Gly-Glu-..
beta subunit amino acid sequence
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Protein Structure - Primary

• Protein chain of amino acids joined by peptide
  bonds

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Protein Structure - Primary

• Protein chain of amino acids joined by peptide
  bonds
• Amino Acid
• Central carbon (Ca) attached to
• Hydrogen (H)
• Amino group (-NH2)
• Carboxyl group (-COOH)
• Side chain (R)

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General Amino Acid Structure
H
COOH
H2N
Ca
R
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General Amino Acid Structure At pH 7.0
H
COO-
H3N
Ca
R
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General Amino Acid Structure
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Amino Acids

• Chiral

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Chirality Glyceraldehyde
L-glyderaldehyde
D-glyderaldehyde
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Amino Acids

• Chiral
• 20 naturally occuring distinguishing side chain

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20 Naturally-occurring Amino Acids
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Amino Acids

• Chiral
• 20 naturally occuring distinguishing side chain
• Classification
• Non-polar (hydrophobic)
• Charged polar
• Uncharged polar

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Alanine Nonpolar
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Serine Uncharged Polar
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Aspartic AcidCharged Polar
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GlycineNonpolar (special case)
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Peptide Bond

• Joins amino acids

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Peptide Bond Formation
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Peptide Chain
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Peptide Bond

• Joins amino acids
• 40 double bond character
• Caused by resonance

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Peptide bond

• Joins amino acids
• 40 double bond character
• Caused by resonance
• Results in shorter bond length

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Peptide Bond Lengths
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Peptide bond

• Joins amino acids
• 40 double bond character
• Caused by resonance
• Results in shorter bond length
• Double bond disallows rotation

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Protein Conformation Framework

• Bond rotation determines protein folding, 3D
  structure

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Bond Rotation Determines Protein Folding
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Protein Conformation Framework

• Bond rotation determines protein folding, 3D
  structure
• Torsion angle (dihedral angle) t
• Measures orientation of four linked atoms in a
  molecule A, B, C, D

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Protein Conformation Framework

• Bond rotation determines protein folding, 3D
  structure
• Torsion angle (dihedral angle) t
• Measures orientation of four linked atoms in a
  molecule A, B, C, D
• tABCD defined as the angle between the normal to
  the plane of atoms A-B-C and normal to the plane
  of atoms B-C-D

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Ethane Rotation
A
A
D
D
B
B
C
C
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Protein Conformation Framework

• Bond rotation determines protein folding, 3D
  structure
• Torsion angle (dihedral angle) t
• Measures orientation of four linked atoms in a
  molecule A, B, C, D
• tABCD defined as the angle between the normal to
  the plane of atoms A-B-C and normal to the plane
  of atoms B-C-D
• Three repeating torsion angles along protein
  backbone ?, f, ?

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Backbone Torsion Angles
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Backbone Torsion Angles

• Dihedral angle ? rotation about the peptide
  bond, namely Ca1-C-N- Ca2

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Backbone Torsion Angles
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Backbone Torsion Angles

• Dihedral angle ? rotation about the peptide
  bond, namely Ca1-C-N- Ca2
• Dihedral angle f rotation about the bond
  between N and Ca

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Backbone Torsion Angles
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Backbone Torsion Angles

• Dihedral angle ? rotation about the peptide
  bond, namely Ca1-C-N- Ca2
• Dihedral angle f rotation about the bond
  between N and Ca
• Dihedral angle ? rotation about the bond
  between Ca and the carbonyl carbon

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Backbone Torsion Angles
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Backbone Torsion Angles

• ? angle tends to be planar (0º - cis, or 180 º -
  trans) due to delocalization of carbonyl p
  electrons and nitrogen lone pair

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Backbone Torsion Angles

• ? angle tends to be planar (0º - cis, or 180 º -
  trans) due to delocalization of carbonyl pi
  electrons and nitrogen lone pair
• f and ? are flexible, therefore rotation occurs
  here

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Backbone Torsion Angles
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Backbone Torsion Angles

• ? angle tends to be planar (0º - cis, or 180 º -
  trans) due to delocalization of carbonyl pi
  electrons and nitrogen lone pair
• f and ? are flexible, therefore rotation occurs
  here
• However, f and ? of a given amino acid residue
  are limited due to steric hindrance

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Steric Hindrance

• Interference to rotation caused by spatial
  arrangement of atoms within molecule
• Atoms cannot overlap
• Atom size defined by van der Waals radii
• Electron clouds repel each other

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Backbone Torsion Angles

• ? angle tends to be planar (0º - cis, or 180 º -
  trans) due to delocalization of carbonyl pi
  electrons and nitrogen lone pair
• f and ? are flexible, therefore rotation occurs
  here
• However, f and ? of a given amino acid residue
  are limited due to steric hindrance
• Only 10 of the f, ? combinations are generally
  observed for proteins
• First noticed by G.N. Ramachandran

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G.N. Ramachandran

• Used computer models of small polypeptides to
  systematically vary f and ? with the objective of
  finding stable conformations
• For each conformation, the structure was examined
  for close contacts between atoms
• Atoms were treated as hard spheres with
  dimensions corresponding to their van der Waals
  radii
• Therefore, f and ? angles which cause spheres to
  collide correspond to sterically disallowed
  conformations of the polypeptide backbone

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Ramachandran Plot

• Plot of f vs. ?
• The computed angles which are sterically allowed
  fall on certain regions of plot

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Computed Ramachandran Plot
White sterically disallowed conformations
(atoms come closer than sum of van der Waals
radii) Blue sterically allowed conformations
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Ramachandran Plot

• Plot of f vs. ?
• Computed sterically allowed angles fall on
  certain regions of plot
• Experimentally determined angles fall on same
  regions

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Experimental Ramachandran Plot
f, ? distribution in 42 high-resolution protein
structures (x-ray crystallography)
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Ramachandran Plot And Secondary Structure

• Repeating values of f and ? along the chain
  result in regular structure
• For example, repeating values of f -57 and ?
  -47 give a right-handed helical fold (the
  alpha-helix)

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The structure of cytochrome C shows many segments
of helix and the Ramachandran plot shows a tight
grouping of f, ? angles near -50,-50
cytochrome C Ramachandran plot
alpha-helix
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Similarly, repetitive values in the region of f
-110 to 140 and ? 110 to 135 give beta
sheets. The structure of plastocyanin is
composed mostly of beta sheets the Ramachandran
plot shows values in the 110, 130 region
plastocyanin Ramachandran plot
beta-sheet
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Ramachandran PlotAnd Secondary Structure

• White sterically disallowed conformations
• Red sterically allowed regions if strict
  (greater) radii are used (namely right-handed
  alpha helix and beta sheet)
• Yellow sterically allowed if shorter radii are
  used (i.e. atoms allowed closer together brings
  out left-handed helix)

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Sample Ramachandran Plot
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Alanine Ramachandran Plot
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Arginine Ramachandran Plot
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Glutamine Ramachandran Plot
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Glycine Ramachandran Plot
Note more allowed regions due to less steric
hindrance - Turns
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Proline Ramachandran Plot
Note less allowed regions due to structure
rigidity
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f, ? and Secondary Structure
Name f ? Structure
------------------- -------
------- --------------------------------- alpha-L
57 47 left-handed alpha
helix 3-10 Helix -49 -26
right-handed. p helix -57 -80
right-handed. Type II helices -79 150
left-handed helices
formed by polyglycine
and
polyproline. Collagen -51 153
right-handed coil formed
of three left handed

helicies.  
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Sequence Similarity

• Sequence similarity implies structural,
  functional, and evolutionary commonality

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Homologous ProteinsEnterotoxin and Cholera toxin
Enterotoxin
Cholera toxin
80 homology
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Sequence Similarity

• Sequence similarity implies structural,
  functional, and evolutionary commonality
• Low sequence similarity implies little structural
  similarity

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Nonhomologous ProteinsCytochrome and Barstar
Cytochrome
Barstar
Less than 20 homology
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Sequence Similarity

• Sequence similarity implies structural,
  functional, and evolutionary commonality
• Low sequence similarity implies little structural
  similarity
• Small mutations generally well-tolerated by
  native structure with exceptions!

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Sequence Similarity Exception

• Sickle-cell anemia resulting from one residue
  change in hemoglobin protein
• Replace highly polar (hydrophilic) glutamate with
  nonpolar (hydrophobic) valine

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Sickle-cell mutation in hemoglobin sequence
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Normal Trait

• Hemoglobin molecules exist as single, isolated
  units in RBC, whether oxygen bound or not
• Cells maintain basic disc shape, whether
  transporting oxygen or not

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Sickle-cell Trait

• Oxy-hemoglobin is isolated, but de-oxyhemoglobin
  sticks together in polymers, distorting RBC
• Some cells take on sickle shape

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Sickle-cell
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RBC Distortion

• Hydrophobic valine replaces hydrophilic glutamate
• Causes hemoglobin molecules to repel water and be
  attracted to one another
• Leads to the formation of long hemoglobin
  filaments

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Hemoglobin Polymerization
Normal
Mutant
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RBC Distortion

• Hydrophobic valine replaces hydrophilic glutamate
• Causes hemoglobin molecules to repel water and be
  attracted to one another
• Leads to the formation of long hemoglobin
  filaments
• Filaments distort the shape of red blood cells
  (analogy icicle in a water balloon)
• Rigid structure of sickle cells blocks
  capillaries and prevents red blood cells from
  delivering oxygen

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Capillary Blockage
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Sickle-cell Trait

• Oxy-hemoglobin is isolated, but de-oxyhemoglobin
  sticks together in polymers, distorting RBC
• Some cells take on sickle shape
• When hemoglobin again binds oxygen, again becomes
  isolated
• Cyclic alteration damages hemoglobin and
  ultimately RBC itself

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Protein The Machinery of Life
NH2-Val-His-Leu-Thr-Pro-Glu-Glu- Lys-Ser-Ala-Val-T
hr-Ala-Leu-Trp- Gly-Lys-Val-Asn-Val-Asp-Glu-Val- G
ly-Gly-Glu-..

• Life is the mode of existence of proteins, and
  this mode of existence essentially consists in
  the constant self-renewal of the chemical
  constituents of these substances.
• Friedrich Engles, 1878