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Last Tuesday and Beyond
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Common 2° structural elements: influenced by 1°
structure
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alpha helices
beta strands
beta turns
Structure vs. function
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Fibrous (collagen, silk)
Globular
Determination of 3-D structure
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X-ray crystallography
NMR
Organization of protein structure
• Subdomains within proteins
• Motifs: common ways 2° structural
elements interact
• The process of protein folding and
unfolding
3-D substructure
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Domains
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Compact, globular units found in
large proteins
– Folds independently
– Retain structure even when
separated from rest of protein
– Genetic manipulation:
» Can be added, removed,
swapped…(often) with
impunity
– Result from gene fusion
during evolution
– Often bring together different
functions
» Regulatory & catalytic
Myosin
“Supersecondary” structures
Common, stable motifs in which 2o structural elements
come together
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b-a-b loop
a-a corner
b-barrels
Greek key
Jellyroll
b-meander
a-a unit
Greek key
b-meander
Jellyroll
Rules for protein folds
1. Burial of H-phobic
– Requires 2 layers of 2°
structure
2. a-helix and b-sheets
– Different ‘layers’ of
structure because Hbonding
3. Adjacent peptide segments
in structure, sometimes
adjacent in sequence
4. Connections cannot
cross/form knots
5. b-conformation most stable
with right twist
Simplifying 3D structure
• Classify according to 2°
components
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All a
All b
a/b (alternate)
a & b (segregated)
• Many different folds
• But fewer than 1000 may exist in all
proteins
• 3o structure conserved
3-D structure examples
Family and superfamily
Multisubunit proteins
• Separate subunits
– Sometimes different domains and
functions
• Catalysis/regulation
• Structural/catalysis
• Multistep catalysis
• Hemoglobin
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4 polypeptide chains
4 prosthetic (heme) groups
2 a chains (141 AA)
2 b chains (146 AA)
Arranged as symmetric pairs
• a/b subunit
• Tetramer or dimer of a/b protomers
Limitations on protein size
– Theoretically unlimited, but not so in practice
– Genetic coding capacity of nucleic acids
– Accuracy of protein biosynthesis
• More efficient to make many copies of small than one
large protein
• >~100000: multiple subunits (more than one polypeptide
chain, more than one gene)
– Reduce probability for error
• 1/10000 amino acid
Protein folding
• Ribonuclease
– ‘Denatured’ with urea
– Disulfides broken with a
‘reducing agent’ (BME)
• inactive protein
– Urea and BME removed
• Active, refolded protein
• Protein has ‘renatured’
– Sequence confers 3-D
structure  activity
Protein denaturation
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Denaturants
Heat
pH (strong acids/bases)
Organic solvents
Salts (urea, guanidine HCl)
Detergents
Reducing agents
Heavy metal ions
Mechanical stress
• Only ‘weak’ and S-S interactions are
broken
– No peptide covalent bonds
• Detect by spectroscopy, eg.
fluorescence of aromatic residues
Protein folding: strand → native
• Cannot be completely random
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100 residues: 10100 possible conformations
Theoretically 1077 years for protein to fold
Actual time scale: milliseconds to seconds
“Levinthal’s paradox”
How?
• Driven by physics/chemistry: Anfinsen’s experiment
One model: ‘hierarchical’
1. ‘Local’ structures
fold
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Denatured state
Sequences prone to
a or b
2. Mid-range
interaction
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eg. two a helices
come together
3. Longer range
interactions
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Two loops interact
Native state
“Molten globule” model
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Molten globule model
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Peptide collapses into compact state
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Hphobic on inside, Hphilic on outside
‘molten globule’
Protein folds from this
Describe with a free energy funnel
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Thousands of unfolded conformations
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Some collapse, some start forming 2o
structure
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Highly unstable
Semistable intermediates
At bottom, single/few native structures
with a small set of conformation