Protein Structure and Function

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Lecture 13

• Protein Structure and Function

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Proteins -- Of the first rank..

• Proteins are single, unbranched chains of amino
  acid with diverse functions
• catalysis
• transport and storage
• motion
• mechanical support
• regulation
• host/pathogen interaction

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From the M. tuberculosis genome.
Of 3924 ORFs virulence, detoxification,
adaptation 3 lipid metabolism
6 information pathways 5 cell wall and
cell processes 14 intermediary
metabolism and respiration
23 regulatory proteins 5 unknown
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Amino acids
Avg. MW 113 Least common Cys, Met, Trp (5 of
total) Most common Leu, Ser, Gly, Glu (32 of
total) Handy rule YFP MW AAs X 110
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Hydrophilic amino acids
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Hydrophobic and special amino acids
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The peptide bond
Peptide bonds are rigid and planar.
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Four levels of structural organization

• Primary
• Secondary
• Tertiary
• Quaternary

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Four levels of structural organization

• Primary the linear sequence of amino acids.
• Secondary the localized organization of parts of
  a polypeptide chain (e.g., the ? helix or ?
  sheet).
• Tertiary the overall, three-dimensional
  arrangement of the polypeptide chain.
• Quaternary the association of two or more
  polypeptides into a multi-subunit complex.

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Secondary structure the ? helix
Solved by Pauling and Corey. Very compact. Very
stable. Very rigid. Very common. Cant contain
proline.
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The ? helix is everywhere
When we consider that the fibrous proteins of
the epidermis, the keratinous tissues, the chief
muscle protein and now the fibrinogen of the
blood all spring from the same peculiar shape of
molecule, and are therefore probably all
adaptations of a single root idea, we seem to
glimpse one of the great coordinating facts in
the lineage of biological molecules. -- Bailey,
Astbury and Rudall, Nature, 1943
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The beta sheet
Solved by Pauling, Corey. Very extended. Can be
parallel, anti-parallel. Can form
barrels. b-turn 180 in 4 AAs.
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Primary and secondary structure in hemagglutinin
(HA)
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Tertiary and quaternary structure in hemagglutinin
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Motifs regular combinations of secondary
structures
Two or more helices interact. Requires
amphipathic helices.
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Membrane proteins in a lipid bilayer
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Integral membrane proteins
- interact extensively with hydrocarbon chains of
membrane lipids - span lipid bilayer (single pass
vs multipass ? helix) - can be displaced from
bilayer through use of detergents sodium
dodecyl sulfate (charged, proteins
denatured) triton (uncharged, proteins maintain
some activity)
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Membrane protein topologies
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Peripheral membrane proteins
- Attached to lipid bilayer covalently fatty
acid chains prenyl groups oligosaccharide (GPI
anchors) - noncovalent interactions with other
membrane proteins, many times integral proteins -
can be displaced by changes in ionic strength or
pH
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Lipid anchors
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Membrane proteins are dynamic
- diffuse laterally or rotationally within the
plane of the membrane (esp. lipid anchors) - some
protein confinement can occur within lipid
bilayer polarized cells - apical, basal and
lateral domains protein tethers - cytoskeletal
or extracellular matrix protein aggregates -
restrict movement
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Protein Structure Analysis

• Technique of choice depends on your needs.

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Protein Structure Analysis

• Limited proteolysis
• Circular dichroism (CD)
• X-ray crystallography
• NMR
• Technique of choice depends on your needs.

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Circular dichroism (CD)

• Measures differences in the absorption of
  polarized light due to structural asymmetry.
• Ordered structure results in a spectrum that can
  have both positive and negative signals.
• Reveals presence of secondary structure.
• Compare WT and mutant proteins.

http//www.ap-lab.com/circular_dichroism.htm
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Limitations of circular dichroism
Requires fairly large (mg) quantities of
protein. Cannot determine entire 3D structure.
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X-ray crystallography

• Beams of x-rays passed through a crystal of
  purified protein.
• Can be done with
• protein
• protein/DNA
• protein/RNA
• protein/small molecule

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X-ray crystallography

• X-ray wavelengths are short - resolve atoms
• Diffraction pattern - complex, based on atoms
• Modification of protein with heavy metal -
• shifts pattern, allows interpretation

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X-ray Crystallography

• Issues
• Need gram quantities of protein.
• NEED CRYSTAL.
• Need heavy metal version.
• Flexible regions dont form structure.
• Membrane proteins very difficult.

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Diffraction pattern
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NMR Nuclear Magnetic Resonance

• Concentrated protein solution
• Magnetic field
• Effect of radio frequencies on the resonance of
  different atoms is measured.

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NMR Nuclear Magnetic Resonance

• Behavior of any atom is influenced by neighboring
  atoms -
• more closely spaced residues are more perturbed
  than distant residues
• can calculate distances based on perturbation

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An idealized NMR spectrum
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NMR spectrum of a protein
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NMR nuclear magnetic resonance

• Dont need crystals
• Limited to proteins less than 20 kD
• OK for domains from larger proteins.