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McGill University
BIOC 212
Jason Young

A)  Protein  Folding   1.  Elements  of  protein  structure     Polypeptides   • Amino  acid  residues  covalently  link  into  polypeptides   • Peptide  bonds  are  polar  and  uncharged   • Rotation  of  the  backbone  occurs  around  the  central  carbon  of  a  residue,  peptide  bonds  cannot   rotate   • Both  the  side  chain  and  the  peptide  backbone  can  form  non-­‐covalent  bonds  with  amino  acids  and   other  biological  molecules   • 100-­‐800  amino  acids  long,  weigh  12-­‐90  kDa   • Non-­‐covalent  bonds  between  the  residues  stabilize  polypeptides   o Hydrophobic  interactions  (drives  protein  folding,  protein-­‐membrane,  transport  across   membrane)   o Hydrogen  bonds  (peptide  backbone,  polar  side  chains)   o Van  der  Waals  interactions  (transient,  between  all  atoms)   o Ionic  bonds  (charged  atoms,  can’t  be  formed  with  backbone)   • Covalent  disulfide  bonds  between  cysteine  residues  reinforce  structure  of  extracellular  proteins  or   proteins  inside  secretory  organelles  (need  redox  ∴  NOT  for  cytosolic  proteins)   Protein  folding   • 2°  structure  is  stabilized  by  local  hydrogen  bond  between  the  backbone.  Side  chains  point   outwards  and  aren’t  involved  in  stabilizing  structure.   o α-­‐helix  (backbone  is  curving   o β-­‐sheet  (backbone  is  stretched  to  form  extended  stran   • 3°  structure  is  stabilized  (driven)  by  hydrophobic  interactions   between  2°  structures  (native  state  =  most  stable  conformation)   o Polar  side  chains  exposed,  hydrophobic  side  chains   hidden  inside   o Long-­‐range  folding  from  interactions  between  residues   that  are  scattered  far  apart  in  1°  sequence   • 4°  structure  is  stabilized  by  hydrophobic  interactions  between  the  subunits  (dimer=2  subunits,   oligomer=many  subunits)   • Proteins  with  similar  1°  sequences  fold  similarly  ¦  similar  native  states  ¦  similar  functions   Other  features   • 1°  sequence  determines  functional  surface  interactions  with  other  molecules.     o The  range  of  molecules  recognized/bound  can  be  narrow  or  broad.   o Enzymes  increase  the  rate  of  covalent  chemical  reactions   o Allosteric  proteins  change  conformation  depending  on  substrate  binding   • Domains:  independently  folded  units  within  a  protein   o Some  domains  need  a  ligand  (cofactor,  protein  subunit  etc.)  to  be  stable   o Some  fold  stably  without  a  ligand,  but  bind  it  reversibly  as  a  part  of  their  function   o Modular  domains  are  relatively  independent  so  their  function  can  be  inserted  into  many   different  proteins.     § They  form  non-­‐covalent  interactions  with  specific  signals  on  other  macromolecules   to  allow  regulation  of  function   • Protein  families   o Proteins/domains  with  similar  sequences  and  structures  usually  have  related  functional   mechanisms   o Homology  (similarity)  indicates  conservation  through  evolution   o Structural  similarity  is  still  different  from  functional  similarity  (they  can  look  totally   different  and  still  do  the  same  thing).   2.  Molecular  chaperones     Protein  folding  process   • Thermodynamically  favored,  spontaneous,  assisted  by  different  biological  mechanisms,  1°   sequence  determines  specific  contact  between  residues  aka  determines  folded  structure.   o Established  by  Christian  Anfinsen  in  1972   • Proceeds  from  unfolded  (takes  up  most  space)  ¦  folding  intermediates  ¦  molten  globule  ⇌  native   state  (tightly  packed)   o Folding  intermediates   § Incomplete  3°  structure  (emerge  linearly  from  ribosome  after  synthesis,  ligand  not   available,  denatured)   § Flexible,  loosely  packed   § Not  all  hydrophobic  side  chains  are  buried  (risk  of  aggregation)   • Quality  control  mechanisms  deal  with  folding  intermediates  that  are  off-­‐pathway  (non-­‐ productive):   o Chaperones  assist  in  correctly  folding  proteins   o Proteasomes  degrade  unfolded  proteins  that  can’t  be  fixed   Chaperones   • Assist  in  folding  of  specific  intermediates  that  need  help     • Not  part  of  native  state   • Recognize  and  bind  to  exposed  hydrophobic  regions  to  prevent  aggregation   • Highly  expressed  under  stress  conditions  (i.e.  HSP  family),  but  also  essential  under  normal   conditions   • There  is  cooperation  between  types  of  chaperones  (in  cytosol  and  ER)   o ATP-­‐independent  chaperones:  prevent  aggregation  by  binding  and  covering  exposed   hydrophobic  regions  of  intermediates,  can  also  catalyze  some  folding  steps   § Calreticulin  &  calnexin,  PPIase   § PPIases  increase  proline  rotation  to  speed  up  folding  (proline’s  extra  covalent  bond   limits  its  rate  of  folding)   o ATP-­‐dependent  chaperones:  ATPase  cycle  regulates  binding  and  release  of  substrate  to   actively  promote  folding   § Chaperonins,  HSP70,  HSP90   • Chaperones  have  co-­‐evolved  with  their  substrates  (chaperonin  families  are  not  universal,   members  are  not  found  in  all  organisms  and  organelles)     HSP70  family   • 70kDa  monomers  with  two  domains   • Multiple  functions  other  than  protein  folding   • Human  Hsc70  is  always  produced,  Hsp70  is  inducible   • States:   o Folded:  ATPase  domain  holding  peptide  binding  domain   open,  like  hands  holding  pliers  open   § ATP-­‐bound:  no  substrate  binding   o Separate:   § ADP-­‐bound:  peptide  binding  domain  is  shut  tight  on  substrate   § Nucleotide  free   • Substrate  binding   o Recognizes  short,  7  amino  acid  sequences  (multiple  binding  sites  per  polypeptide)     o Occurs  as  the  polypeptide  is  being  synthesized  (first  chaperone  to  act  on  a  protein)   o Large  range  of  unfolded  proteins  can  be  bound   o Substrate  can’t  be  too  coiled,  only  extended  portions  will  fit   • Co-­‐chaperone  (regulates  chaperone  function)   o DNAJ  (necessary)   § J  domain:     • Modular  domain,  ATPase  activity   • Binds  transiently  to  Hsp70-­‐ATP     • Activates  Hsp70  to  hydrolize  ATP   § Substrate  binding  domain:  (Type  1&2)   • Act  as  ATP-­‐independent  chaperones   § Types  of  DNAJ   • Type  1:     o Domains:  J  domain,  conserved  substrate  binding  domain,  dimerization   o Bind  short  hydrophobic  sequences     o Homodimers:  2  x  50kDa  subunits   • Type  2:  divergent  substrate  binding  domains   • Type  3:  no  substrate  binding,  activate  specialized  HSP70  functions,  NO   FOLDING   o NEF   § Replace  ADP  with  ATP  to  open  up  Hsp70’s  ATPase  domain  and  releases  its  substrate   • Process:  multiple  fast  cycles  of  substrate  binding  and  releasing   1. DNAJ  binds  substrate     2. J  domain  stimulates  ATP  hydrolysis  by  HSP70.  Substrate  is  transferred  and  bound  by   HSP70  in  ADP  state.   3. NEFs  cause  ATP  re-­‐binding  and  release  of  substrate       3.  Multi-­‐chaperone  systems     Chaperonin  family   • Large  complexes  with  multiple  subunits  (840kDa)   o GroEL  (two  rings  that  join  at  their  ATPase  domains,  apical  domains  face  out)  +  GroES  (cap)   • GroEL  in  e.coli,  TRiC  in  humans:  specialized  for  folding  certain  proteins,  acts  after  Hsp70  in  late   stages  of  folding  because  it  can  only  fit  partially  folded/compact  proteins  in  its  cavity   • States:   o Apical  domain  down:  (when  no  nucleotide  bound)   § Hydrophobic  surface  is  exposed,  provides  multiple  substrate  binding  sites   o Apical  domain  up:  (when  ATP  or  ADP  is  bound)   § Hydrophobic  surface  binds  to  GroES  cap   § Substrate  can  be  encapsulated  and  folded  in  cavity  formed  by  polar  insides  of  GroEL   • Cycle:   GroEL Cycle • binds substrate with hydrophobic domains (no nucleotide top ring) • ATP binding (top ring) encloses substrate inside a polar cavity under GroES cap – substrate is not bound but is free inside cavity – confinement promotes folding protein'folding'occurs'during'hydrolysis,'no'conformational'change'of'GroEL'then$it's$ready$to$open$cap$and$release$ • Substrate is enclosed while ATP is hydrolyzed to ADP substrate • no nucleotide (top ring) and ATP (bottom ring) release G
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