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” structures Common, stable motifs in which 2 o 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 ° structure  -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  -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 3 o 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 Denaturants 1.Heat 2.pH (strong acids/bases) 3.Organic solvents 4.Salts (urea, guanidine HCl) 5.Detergents 6.Reducing agents 7.Heavy metal ions 8.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: possible conformations –Theoretically 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 2 o structure Semistable intermediates –At bottom, single/few native structures with a small set of conformation