What Can Fluorescence Tell You about Biomolecular Structure and Function Part I

What can fluorescence tell you about biomolecular structure and function? Part I Chem 46B February  1,  2011 Outline   • What  are  the  ways  in  which  light  physically   interacts  with  materials?  (i.e.  no  photochemistry)   • What  is  fluorescence?   • What  can  fluorescence  tell  you  about   biomolecular  structure?   • Example  I:  Steady  State  fluorescence  –  metal   binding  to  transferrin   – Biological  moOvaOon   – Fluorescence  Experiments  (intensity  quenching,   steady  state  polarizOon)   What  are  the  ways  in  which  light  physically  interacts  with   materials?  (i.e.  no  photochemistry)   Emission of Light YES After heating? Incandescence NO Luminescence After using mechanical forces? YES YES After excitation with sound? Sonoluminescence Triboluminescence After excitation with light? Immediately? YES fluorescence YES NO Inanimate? NO phosphorescence YES chemiluminescence NO bioluminescence What  is  fluorescence?   3 S1   1 2 2 ν4 ν3 ν2 ν1 S1   4 So   So   Fluorescent  Emission  in  Atoms   S1   Fluorescent  Emission  in  Molecules   5 T1   6 So   Phosphorescent  Emission  from  Molecules   What  you  learn  depends  upon  your   experimental  methodology  (bulk)   • Steady  state     – ExcitaOon  and  emission   • fingerprints     – Energies  and  IntensiOes   • Compare  w  similar  molecules     – Intensity  quenching  from  ligand   • Kd,  Km,  Ki   – Solvent  shiWs  of  spectra   • Calculate  energy  differences   – Emission  PolarizaOon   • Bound/Unbound  ligands   • Averaged  rotaOonal    Omes   – FRET   • Average  donor  acceptor   distances   • Time/Frequency  resolved     – LifeOmes   • Buried/exposed   • Unique  environments   – LifeOme  distribuOons     • Dynamics   • Local  moOons   – Time  resolved  FRET   • Accurate  donor/acceptor  distance   • Temp  dependence  to  FRET  yields   info  on  dynamics   – Time  resolved  PolarizaOon   • LimiOng  polarizaOon,  rotaOonal   correlaOon  Omes,  diffusion  Omes.   What  you  learn  depends  upon  your   experimental  methodology  (single  molecule)   • Steady  state     – ExcitaOon  and  emission   • fingerprints     – Energies  and  IntensiOes   • Compare  w  similar  molecules     – Intensity  quenching  from  ligand   • Kd,  Km,  Ki   – Solvent  shiWs  of  spectra   • Calculate  energy  differences   – Emission  PolarizaOon   • Bound/Unbound  ligands   • Averaged  rotaOonal    Omes   – FRET   • Average  donor  acceptor   distances   • Time/Frequency  resolved     – LifeOmes   • Buried/exposed   • Unique  environments   – LifeOme  distribuOons     • Dynamics   • Local  moOons   – Time  resolved  FRET   • Accurate  donor/acceptor  distance   • Temp  dependence  to  FRET  yields   info  on  dynamics   – Time  resolved  PolarizaOon   • LimiOng  polarizaOon,  rotaOonal   correlaOon  Omes,  diffusion  Omes.   What  you  learn  depends  upon  your   experimental  methodology  (bulk)   • Steady  state     – Excita3on  and  emission   • fingerprints     – Energies  and  IntensiOes   • Compare  w  similar  molecules     – Intensity  quenching  from  ligand   • Kd,  Km,  Ki   – Solvent  shiWs  of  spectra   • Calculate  energy  differences   – Emission  PolarizaOon   • Bound/Unbound  ligands   • Averaged  rotaOonal    Omes   – FRET   • Average  donor  acceptor  distances   • Time/Frequency  resolved     – LifeOmes   • Buried/exposed   • Unique  environments   – LifeOme  distribuOons     • Dynamics   • Local  moOons   – Time  resolved  FRET   • Accurate  donor/acceptor  distance   • Temp  dependence  to  FRET  yields   info  on  dynamics   – Time  resolved  PolarizaOon   • LimiOng  polarizaOon,  rotaOonal   correlaOon  Omes,  diffusion  Omes.   Using  fluorescence  spectroscopy  to  probe   binding  of  ruthenium  compounds  to  human   serum  transferrin   • Funding:  John  Abele  Faculty  Development  Fund   • Collaborators   – – – – Renzo  Cini  (U.  Siena,  Italy)   Lauren  Benson  ’08  (now  chem  teacher)   Zandra  Walton  ’09     Phoebe  Arbogast  ’10   • 1981,  my  first  paper  measured  energy  transfer  in  transferrin   • What  was  known  about  transferrin   then?   • a  non-­‐heme  iron  protein  that  exists   in  human  blood  to  transport  Fe   throughout  body   • Fe(III)  binding  site  is  shown     • Fe(III)  Kd  <  10-­‐27  M…pH  7.4   • Metal  is  bound  at  two  idenOcal  sites   on  two  different  domains   • SynergisOc  anion,  typically   carbonate  necessary   • Other  trivalent  metals  also  bind  (i.e.   Mn(III),  Tb(III))     • What  do  we  know  today?     • Two  structurally  similar  lobes  (binding   site  previous  slide)   ConformaOonal  change  occurs  upon   metal  binding     induces  a  binding  site  cleW  closure   • occurs  with  two  domains  rotaOng   • 63o  with  respect  to  each  other  about   hinge   MetalloTf  shape  is  more  compact   • ConformaOonal  change  about  Trp  in   • the  hinge  region  between  two   domains   Green  circles  are  fluorescent   • reporter  molecules  Trp  (W)   • • Serum  Tf    in  vivo  is  typically   about  30%  saturated  with   iron  (pH  =  7.4)   • Only  Fe-­‐Tf  binds  to   receptor  on  the  cell  surface   • Fe-­‐Tf  enters  the  cell  in  a   vesicle     • Inside  vesicle  (low  pH~5.5)   Fe(III)  is  released   • apoTf  and  receptor  are   transported  back  to  cell   surface,  where  they   dissociate Transferrin  and  its  receptor   Fluorescence  properOes  of  Transferrin     •    Research  Plan  is  to  use   fluorescence  to  follow  metal   binding    of  Ru(III)  compounds  (why   Ru(III)?  –  more  in  a  moment)   •  Use  the  protein  Trp  fluorescence   (6  Trp)   •  How  do  6  Trp  contribute  to  bulk   Tf  fluorescence?   •  One  lobe  of  Fe-­‐Tf  shown  here-­‐   only  3  Trp   Single  W  dele3on  mutants  created  in  Tf     •  He  et  al  made  W  mutants  to  probe   their  fluorescence  contribuOons   •  W8  in  hydrophobic  box,  surrounded  by   three  F,  a  K  residue  is  nearby,   fluorescence  totally  quenched,   independent  of  metal  binding   •  W128  fluorescence  blue  shiWed  and   quenched  by  metal  binding   •  W264  fluorescence  independent  of   metal  binding,  solvent  accessible  and   primary  contributor  to  fluorescence       • Other  metals  can  bind  to   transferrin   • Ruthenium  is  similar  to  iron   (group  VIII  of  the  transiOon   metals)   • Ruthenium  can  be   transported  to  Ossues  as  a   ruthenium-­‐transferrin   complex   • x-­‐ray  crystallography  shows   that  Ru-­‐In2  binds  to  Tf  at   metal  binding  site     • Binding  occurs  through  the   imidazole  ring  of  the  His   residue,  displacement  of  Cl-­‐   • ChemotherapeuOc  [RuIn2Cl4]-­‐   retains  its  acOvity  against   colon  cancer  cells  when   bound  to  transferrin   [RuInd2Cl4]-­‐   Ru-Indazole shown to bind to apoTf •  Structure  of  trans-­‐indazole   •  tetrachlorobis(indazole   thuthenate(III)     •  Kratz  et  al  1994   •  Used  against  colon  cancer  trial   drug  called  KP1019   •How  does  it  bind?   What  you  learn  depends  upon  your   experimental  methodology   • Steady  state     – ExcitaOon  and  emission   • fingerprints     – Energies  and  IntensiOes   • Compare  w  similar  molecules     – Intensity  quenching  from  ligand   • Kd,  Km,  Ki   – Solvent  shiWs  of  spectra   • Calculate  energy  differences   – Emission  PolarizaOon   • Bound/Unbound  ligands   • Averaged  rotaOonal    Omes   – FRET   • Average  donor  acceptor   distances   • Time/Frequency  resolved     – LifeOmes   • Buried/exposed   • Unique  environments   – LifeOme  distribuOons     • Dynamics   • Local  moOons   – Time  resolved  FRET   • Accurate  donor/acceptor  distance   • Temp  dependence  to  FRET  yields   info  on  dynamics   – Time  resolved  PolarizaOon   • LimiOng  polarizaOon,  rotaOonal   correlaOon  Omes,  diffusion  Omes.   Fluorescence  PolarizaOon   Fluorescent  Life3me  =  τfl • If  τfl  <<  θrot    (buried  W)   side  group  doesn’t  rotate   during  excited  state   life3me  and  emiMed  light   will  be  polarized   • If  τfl  >>  θrot  (freely   rota3ng  W)  side  group   rotates  during  excited   state  life3me  and  emiMed   light  will  be  depolarized      Rota3onal  correla3on  3me  =  θrot     Steady  state  bulk  Trp  polarizaOon  as  a   reporter  for  metal  binding   • Fe(III)-­‐Tf  shows  a  very  low   polarizaOon  of  W  indicaOng   free  rotaOon  from  dominant   fluorescent  W248  consistent   Fe  binding  closes  metal   binding  cleW  but  opens  Tf   overall  conformaOon     Fe-­‐Tf   apoTf   • Removal  of  Fe  changes  Tf   polarizaOon  to  much  higher   value,  suggesOng  burying  of     W248  in  apoTf     Ru(III)  Indazole  with  apoTf     •  [Ru(III)(In)2Cl4]-­‐1  binding  to  apoTf   •  intensity  quenches   •  anisotropy  drops   •  binding  not  as  strong  as  Fe(III)   •  does  not  depend  on  CO3-­‐2   Transferrin  fluorescence  +  ruthenium   compounds   Tf  fluoresces  at  320  nm  when  excited  at   295  nm,  anisotropy  0.139     +  [Ru(im)]+3    ,  slight  intensity  quench,   anisotropy  unchanged  (0.143)   +[Ru(in1)]+3,    slight  intensity  increase,   anisotropy  decreases  to  0.033   +Ru(in2)+3  intensity,  down,  then  up   anisotropy  decreases  to  0.062   Anisotropy  of  Terbium-­‐Tf  +  Ru(III)   complexes   Protein  Anisotropy  295x/320m   •  Tb(III)  binding  does  NOT  cause  drop  anisotropy     •  Anisotropy  quenched  with  Fe(III)  and  Ru(III)(In)2   •Anisotropy  NOT  quenched  with  Ru(III)(Im)2   •  Ru(III)(In)2  and  Tb(III)  bind  simultaneously  to  Tf   Cini  Ru(III)  compounds   • Phosphines  show  selecOve   cytotoxic  and  anOcancer   properOes   • Thiopyrimidines  possess   anOviral  and  photochemical   properOes  that  may  prove   useful  in  the  design  of  new   photodynamic  cancer   therapies       • Thiopurines  are  currently  used   •Ru-­‐847   as  anOleukemic  and  anOviral   Cis-­‐[Ru(PPh3)2(TPYM)2]       agents       PPh3=  triphenylphosphine,     TPYM=  2-­‐thio-­‐1,3,  pyrimidine       MW=  847.9     What  is  the  chemo-­‐strategy?   FIRST,  a  tangent:  Siena,  Italy  has   one  of  the  oldest,  craziest  horse   races  in  the  world   • Each  July,  over  60,000  people   gather  to  see  ten  horses  and   riders  each  represenOng  a   parOcular  neighborhood  in  Siena   run  this  dirt  track.   • Riders  don’t  use  saddles  or  have   sOrrups   • Horses  don’t  need  their  riders  to   win.     • What  is  the  chemo  strategy?   • Upsevng  your   rival  rider  from  its   horse  is  legal.   • Goal  is  to  have  a   Oght  grip  on  your   horse  at  the  start,   and  an  ability  to   dismount  quickly   What  is  the  chemo-­‐strategy?   • This  wooden  horse  stands   at  the  entrance  to  a  church   in  Siena,  Italy   • We  can  imagine  the   transferrin,  like  the  horse,   might  be  able  to  carry  a  Ru (III)  molecule  (the  rider)  to  a   cancer  cell   • Once  at  the  cell,  the   transferrin  releases  the  Ru (III)  which  gets  reduced  to   Ru(II)  kills  the  cancer  cell.       Ru(III)-­‐847  with  apoTf     •  Ru(III)-­‐847  binding  to  apoTf   •  intensity  quenches  (no  shiW)   •  weak  binding,  not  specific   •  anisotropy  does  not  decrease   More  Cini  Ru(III)  compounds   •  Ru-­‐866     Trans,  trans,  trans-­‐[RuCl2(PPh3)2(THZ)2]       THZ=  thiazole-­‐1,3       MW=  866.8    g /mole   Ru-­‐1314 Ru(PPh3)2(HTPR)2]Cl2 HTPR=6-thiopurine-riboside MW = 1314. g/mole Ru(III)  1314  with  apoTf     •  Ru(III)-­‐1314  binding  to  apoTf   •  intensity  quenches   •  anisotropy  does  not  change   •  weak  non-­‐specific  binding     Cellular  uptake  of  Ru(III)  into  cancer  cells   •Ru(III)  complexes  (KP1019)  are   beqer  transported  into  cells   when  transferrin  contains  Fe(III)   •Suggests  Tf  is  able  to  carry   BOTH  Fe(III)  and  Ru(III)   DNA*Berberine  +  Ru(III)   Berberine  is  a  fluorescent  DNA   dye  that  reports  on  DNA   structure   DNA*  Berberine  emission  max   525  nm/365  nm  excite,   anisotropy    0.147   +  [Ru(bpy)3]+2    shows  slight   berberine  quenching,   anisotropy  0.134   +  Ru(Im)2  shows  slight   berberine  quenching,   anisotropy  0.132   +  Ru(In)2    shows  no  berberine   quenching,  anisotropy  0.153   How  the  Brain  Rusts:   Researchers  at  the  University  of   Warwick  and  the  Indian  InsOtute   of  Technology  Kanpur  have   discovered  that  the  mechanism   that  we  rely  on  to  transport  iron   safely  through  our  blood  stream   can,  in  certain  circumstances,   collapse  into  a  state  which  grows   long  worm-­‐like  "fibrils"  banded  by   lines  of  iron  rust.  This  process   could  provide  the  first  insight  into   how  iron  gets  deposited  in  the   brain  to  cause  some  forms  of   Parkinson’s  &  Alzheimer’s  and   HunOngton’s  diseases.