Characterizing Palindromic Strand Exchange in Alpha-crystallin Oligomerization

Characterizing  palindromic  strand  exchange in  alpha-­crystallin  oligomerization Introduction Materials  and  methods Alpha-­crystallin,  a  member  of  the  small  heat  shock  protein  (sHSP)  class,   is  expressed  in  eye  lens,  muscle,  and  brain  tissue  where  it  contributes  to   homeostasis  by  chaperoning  and  thus  preventing  the  pathogenic  aggrega-­ tion  of  damaged  or  unfolded  proteins.  As  a  major  cytoplasmic  component   in  the  eye  lens,  alpha-­crystallin  is  further  essential  for  producing  a  high  in-­ dex  of  refraction.  Despite  its  high  concentrations,  alpha-­crystallin  does  not   itself  aggregate  or  crystallize,  a  property  facilitated  by  its  populating  dy-­ namic,  polydisperse  assemblies.  Oligomerization  in  this  manner  is  a  com-­ mon  feature  of  the  sHSPs  and  is  likely  connected  to  their  chaperone  func-­ tion.  Structural  studies1  suggest  that  one  mechanism  of  polydispersity  may   be  the  ability  of  a  palindromic  sequence  centered  on  the  sHSP  IXI  motif   LQWKHDOSKD%FU\VWDOOLQ Į% LVRIRUPFWHUPLQXVWRELQGELGLUHFWLRQDOO\WR other  monomers.  Bidirectional  strand  exchanges  would  result  in  heteroge-­ neous  oligomeric  structures  while  maintaining  near  identical  residue  inter-­ actions. Binding  orientation  between  a  peptide  model  of  the  c-­terminal  strand  ex-­ FKDQJHVHTXHQFHDQGWKHĮ%PRQRPHULVPHDVXUHGXVLQJVLQJOHSDLU )5(7:HKDYHPXWDWHGĮ%WRLQWURGXFHDF\VWHLQHUHVLGXHIRUVLWHVSHFLI-­ ic  labeling.  The  protein  and  peptide  are  labeled  so  that  binding  in  opposing   GLUHFWLRQVZLOOEHLQGLFDWHGE\GLIIHUHQWREVHUYHG)5(7HI¿FLHQFLHV , P, , P , , P Chaperone  function  is  assessed  using  an  insulin  model  system.  Insulin  is   induced  to  aggregate  and  formation  of  aggregates,  in  the  presence  or  ab-­ VHQFHRIĮ%LVPRQLWRUHGRYHUWLPH Preliminary  results (PLVVLRQVSHFWUDVKRZODEHOHGFRPSRQHQWV Į%GRQRU , acceptor UHODWLYH intensity peptide-­acceptor donor ...VSGPERTIPITREEKPAVAAPKK  (c-­term) Figure  1:  &U\VWDOVWUXFWXUHRIWUXQFDWHGĮ%FRQVWUXFWVKRZLQJFWHUPLQDO VWUDQGH[FKDQJHEHWZHHQWZRPRQRPHUV1.  At  bottom  is  the  palindromic  c-­ter-­ PLQDOVHTXHQFHZLWKKLJKOLJKWHG,;,PRWLI Objective  and  approach Objective: ‡Characterize  the  role  of  palindrom-­ LFVWUDQGH[FKDQJHLQĮ%ROLJRPHUL-­ zation  and  chaperone  function Approach: ‡Measure  binding  of  designed  palin-­ GURPLFSHSWLGHVHTXHQFHVRIĮ%LQ solution ‡Use  single-­pair  FRET  to  distinguish   binding  orientation ‡Design  and  characterize  sequences   with  orientation  bias ‡0XWDWHĮ%ZLWKQHZVWUDQGH[-­ change  sequences  to  determine  link   to  oligomerization  and  function Figure  3:  (PLVVLRQVSHFWUDRIIUHHGRQRUIUHHDFFHSWRUGRQRUODEHOHGĮ%DQG acceptor-­labeled  strand  exchange  peptide.  The  system  components  are  estab-­ lished  and  suitable  for  FRET  studies. Į%F\VWHLQHPXWDQWUHWDLQVFKDSHURQHDFWLYLW\ QRĮ% Design  novel  strand   exchange  sequences   0XWDWHĮ%ZLWK characterized  sequences $VVHVVHIIHFWVRQĮ% chaperone  activity Figure  2:  Experimental  approach. number  of   HYHQWV LQWHUDFWLRQRIĮ% and  peptide )5(7HI¿FLHQF\ Figure  5:  Į%SURWHLQDQGWKHVWUDQGH[FKDQJHSHSWLGHELQGLQVROXWLRQDVLQGL-­ FDWHGE\WKHGHWHFWLRQRIKLJK)5(7HI¿FLHQF\YDOXHV2EVHUYHGGLVWULEXWLRQVDUH EURDGHUWKDQH[SHFWHGDQGVRGRQRWUHYHDOVSHFL¿FELQGLQJRULHQWDWLRQV0HWK-­ RGVZLOOEHWHVWHGDQGUH¿QHGIRUJUHDWHUVHQVLWLYLW\ Conclusions  and  future  work Our  results  show  that  the  experimental  system  components  are  established   and  are  amenable  to  clarifying  the  importance  of  a  proposed  mechanism  of   alpha-­crystallin  polydispersity  and  chaperone  activity.   Future  work: ‡5H¿QH)5(7PHWKRGVIRUJUHDWHUVHQVLWLYLW\WRHQDEOHGLIIHUHQWLDWLRQRI binding  orientation ‡  Employ  molecular  dynamics  to  aid  in  the  design  of  additional  strand  ex-­ change  sequences ZDYHOHQJWK QP Molecular  dynamics  simulations Measure  binding  directionality with  peptide  model   Į%ELQGVVWUDQGH[FKDQJHSHSWLGH Acknowledgements We  thank  Raysa  Cabrejo  and  Karen  Cheng  for  related  work  that  was   adapted  for  use  in  this  project  and  Jonathan  Gough  for  molecular  dynam-­ ics  help.  Funding  was  provided  by  the  Amherst  College  Department  of   Chemistry. ZLOGW\SH  Į%   UHODWLYH aggregation Further  information F\VPXWDQWĮ%   minutes Figure  4:  ,QVXOLQ  DJJUHJDWLRQDVVD\FRPSDULVRQRIZLOGW\SHĮ%DQGF\VWHLQPX-­ WDQWĮ%7KHPXWDQWUHWDLQVFKDSHURQHDFWLYLW\FRQILUPLQJLWVUHOHYDQFHIRUIXUWKHU structural  and  functional  studies. Literature  cited: 1.  Laganowsky  and  Eisenberg  (2010),  Protein  Science  19:1031-­43.