Part5:   Protein  non-­‐folding     Základy  molekulární  biofyziky     (in  English)   Proteins  need  to  fold  into  their  3D  structure     to  have  biological  acCvity   Structure-­‐func*on  paradigm  in  biochemistry   NOT  ALL  proteins  need  to  fold  into  their  3D  structure     to  have  biological  acCvity   …  the  picture  is  not  so  simple   INTRINSICALLY  DISORDERED  PROTEINS   Defini&on:   INTRINSICALLY  DISORDERED  PROTEINS  (IDP)   Synonyms:   Intrinsically  unfolded  proteins,    naCvely  unfolded  proteins,     intrinsically  unstructured  proteins     IDP  proteins  characterized  by  lack  of  stable  ter*ary  structure.       DISORDERED  PROTEIN  REGIONS   Loops  between  2º  elements   Linkers  between  folded  domains   Tails  of  the  folded  proteins   Defini&on:   FuncConal  protein  regions  longer  than  30  AA  characterized  by  lack   of  stable  secondary  &  ter*ary  structure.     Folding   Misfolding   Non-­‐folding   The  modern  understanding  of  the  fate  of  a  polypep*de  chain     inside  a  cell.   Amyloid-­‐like  fibrils   New model of structure-function relationships Protein   quartet   MG   RC   ordered   (Uversky)   PMG   Func*on  can  arise  from  any  of  these  conforma*ons     or  transi*ons  between  them.     PMG  pre-­‐molten  globule   MG      molten  globule   RC          random  coil   Disorder  in  complete  genomes  [%]     Dunker et al. (2000) Genome Inf. 11, 161 L  >  30   L  >  40   L  >  50   Bacilus  sub&lis   (Bacteria)   Drosophila  melanogaster   (Eukaryota)   disordered   ordered   30%   15%   8%   63%   51%   41%   Structural disorder: evolutionary success 20   40   60      0   IDR  (<40)  protein,  %   Domain  of  life   Bacteria   Archea   Eukaryota   Vuce*c  et  al.  (2002)  Proteins  52,  573   Iakoucheva  et  al.  (2002)  J.  Mol.  Biol.  323,  573   regulatory   signaling   biosynthe*c   metabolic   0 20 40 60 80 protein  (%)   30<                40<                50<                60  <   length  of  disordered  region   Disorder prevails in regulatory proteins Disorder correlates with complex size Hegyi  et  al.  (2007)  BMC  Struct.  Biol.  7,  65   0 20 40 60 80 100 0 20 40 60 80 occurrence predicted disorder single 11-100 2-4 5-10 Larger  protein  complexes  have  more  disorder   IDP regions are abundant in disease related proteins. Uversky et al., BMC Genomics, 2008   Physico-chemical properties of intrinsically unfolded proteins Stereo-­‐chemical  proper*es  of  IUP   IUPs  are  dynamic,  but  their  structures  are  NOT  random     Dij = − γiγ jµ0 4π 2 r3 (3cos2 θ −1 2 Experimental  evidence:    non-­‐zero  residual  dipolar  couplings   Physico-­‐chemical  proper*es  of  IUP    IUPs  have  a  low  “density”    connected  with  large  radius  of  gyra*on.   100  AA   500  AA   10 2 10 3 10 4 10 5 10 6 10 7 Number of residues Hydrodynamicvolume,Å 3 Native MG PMG U (RC) IUPPMG IUPRC Uversky (2002) Prot. Sci. 11, Overall  structure  of  IUPs     Sequence  signatures  of  disorder   Dunker  et  al.  (2001)  J.  Mol.  Graph.  Model.  19,  26   • low  content  of  bulky,  hydrophobic  amino  acids     • high  propor*on  of  polar  and  charged  amino  acids.     • ofen  low  complexity  sequences,  i.e.  overrepresenta*on  of  a  few  residues.     Sequence  signatures  of  disorder   Dunker  et  al.  (2001)  J.  Mol.  Graph.  Model.  19,  26   order-promoting disorder-promoting IUPs: AA charge & hydrophobicity Uversky  (2002)  Eur.  J.  Biochem.  269,  2   Folded     Unfolded     Mean  hydrophobicity   Mean  net  charge   Folded     Unfolded     AA charge & hydrophobicity (Uversky plot) IUPs: “turned out” response to heat Folded   unfolded   IUPs: counter ions might promote folding IUPs: membrane field can promote 2º structure Uversky  (2009)  Protein  J.   IUPs properties overview: • IUP  stereochemistry  is  sensiCve  to  environmental  factors   • Large  hydrodynamic  radius     • High  content  of  polar  AA   • High  solvent  accessibility   • Low  content  of  hydrophobic,  bulky  AA   • Resistant  to  heat   • Dynamic           Biophysical tools for identification of IUPs methods  that  are  sensi*ve  to  molecular  size,  density  or  hydrodynamic  drag:   size  exclusion  chromatography,  analyCcal  ultracentrifugaCon,     small  angle  X-­‐ray  sca[ering  (SAXS),  or  NMR  measurements  of  the  diffusion  constant.       methods  able  to  detect  a  lack  of  secondary  structure:     far-­‐UV  (170-­‐250  nm)  circular  dichroism,  infrared  spectroscopy,  NMR  spectroscopy     methods  able  to  probe  solvent  accessibility:     Limited  proteolysis  proteases,  hydrogen-­‐deuterium  exchange  (MS  and  NMR)       The  primary  method  ….   to  obtain  informaCon  on  disordered  regions  of  a  protein  is  NMR  spectroscopy.   (QuanCtaCve  descripCon:  residual  structure  &  dynamics)     …others  (SDS  PAGE,  AFM,  DLS,  …)   Coupled  folding  and  binding The  ability  of  disordered  proteins  to  bind,  and  thus  to  exert  a  func*on,     shows  that  stability  is  not  a  required  condi*on.   Folding  upon  binding   Restric&ng  mobility  upon  binding   IUPs   Entropic  chains   Transient  binding   Permanent  binding   Recogni*on   Display  sites   Chaperons   Effectors   Assemblers   Scavengers   Func*ons  of  intrinsically  unfolded  proteins:   Directly  funcCon  due  to   disorder  as  linker,  bristle,   spring   Sites  of  PTM   Assist  folding   of  protein  or   RNA   Modulate  acCvity   of  partner  molecule   Assemble  complexes     Store  or  neutralize   small  ligands   IUPs:  Entropic  chains   e.g.  entropic  clock   IUPs:  Scavengers   Ca2+ + PO4 3- Ca3(PO4)2 Casein  –  prevenCng  Ca2+  precipitaCon   IUPs:  Assemblers/Scafolds   Assemble  complexes  –  IUP  brings  binding  partners  together   Specificity  without  strong  binding     (binding  promiscuity,  increased  speed  of  interac*on)   Func*onal  advantages  of  IUPs   Possible  mechanisms  of  IDP  func*on  in  signal   transduc*on Ordered  proteins   IDP   Shape  shifing Database  of  Protein  Disorder  &  IDP  predictors h[p://www.disprot.org/