Last Tuesday and Beyond

• Common 2° structural elements: influenced by 1° structure

– alpha helices– beta strands – beta turns

• Structure vs. function– Fibrous (collagen, silk)– Globular

• Determination of 3-D structure– 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• Domains

– 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” structuresCommon, stable motifs in which 2o structural elements

come together

• loop• corner• barrels• Greek key• Jellyroll• -meander• unit

Greek key

-meander Jellyroll

Rules for protein folds

1. Burial of H-phobic– Requires 2 layers of 2°

structure2. -helix and -sheets

– Different ‘layers’ of structure because H-bonding

3. Adjacent peptide segments in structure, sometimes adjacent in sequence

4. Connections cannot cross/form knots

5. -conformation most stable with right twist

Simplifying 3D structure

• Classify according to 2° components

• All • All • (alternate)• & (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– 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• Denaturants1. Heat2. pH (strong acids/bases)3. Organic solvents4. Salts (urea, guanidine HCl)5. Detergents6. Reducing agents7. Heavy metal ions8. 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– 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• Sequences prone to

or

2. Mid-range interaction• eg. two helices

come together

3. Longer range interactions• Two loops interact

Denatured state

Native state

“Molten globule” model

• Molten globule model– Peptide collapses into compact state

• Hphobic on inside, Hphilic on outside• ‘molten globule’

– Protein folds from this

• Describe with a free energy funnel– Thousands of unfolded conformations

• Highly unstable– Some collapse, some start forming 2o

structure• Semistable intermediates

– At bottom, single/few native structures with a small set of conformation