Centre  of  the  University  and  School  of  Milan  for  Bioscience  Educa3on

www.cusmibio.unimi.it

INVISIBLE  FORMS:  PROTEINS  IN  3-­‐D  

16-­‐19  April  2011  Copenhagen  Denmark

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INVISIBLE  FORMS:  PROTEINS  IN  3-­‐D  

Biological  macromolecules  measure  only   a  few  nanometres  and  cannot  be  observed  even  with  the  strongest  light  microscope.  One  of  the  major  technologies  available  to  make  protein  structures  ‘visible’  is  X-­‐ray  crystallography.The  3D  coordinates  of  each  single  atom  are  collected  in   a  file  and  can  be  viewed  using  dedicated  soFware.

GeHng  regular  crystals  is  the  first  step  of  3D  protein  structure  determina3on.  X-­‐ray  irradia3on  of  crystals  allows  magnifica3on  at  atomic  resolu3on  and  determina3on  of  the  3D  structure  of  the  molecule.  The  spa3al  structure  of  a  protein  is  very   important  for   its  func3on.   These  structures  are  collected   in  specialized  databases  and  can  be  viewed   using   dedicated   soFware   to   compare   structures  of   normal   and   mutated  proteins,  design  new  vaccines  and  therapeu3c  molecules.This   workshop   provides   an   interdisciplinary   exercise   for   biology,   chemistry   and  bioinforma3cs.  

Main  techniques  for  3D  structure  determinaNon  of  proteins

Accurate   3D   macromolecular   structures   are   obtained   by   two   main   techniques:  Nuclear   MagneNc   Resonance   (NMR)   and   X-­‐ray   crystallography   (83%).   When   a  protein   with   unknown   structure   shows  a  good   level  of   sequence   iden3ty   with   a  protein  with  known  structure,   a  rough  structure  prevision  can  also  be  obtained  by  compara3ve  modelling.  This  kind  of  analysis,  although  less  accurate,  can  also  provide  important   insights  on  the  structure/func3on  of  a  protein,  but   at  present   templates  are  available  for  only  20%  of  protein  domains.    

Whatever  method   is  used  to  determine  the  structure  of  a  macromolecule,   the  3D  coordinates  of  each  single  atom  are  collected  in  a  file,  deposited  in  the  PDB  (Protein  Data  Bank)  database  and  can  be  viewed  using  dedicated  soFware.  

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Determinazione della

struttura in 3D

Crystal  characteriza3on

Crystal  diffrac3on  spectrum

Interpreta3on  of  electron  density

3D  structure

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Nuclear  MagneNc  Resonance  is  a  type  of  spectrometry  in  which  proteins  are  hit  with  radio  waves  while  they're  in  a  strong  magne3c  field.  Since  atomic  nuclei  selec3vely  absorb  electromagne3c   radia3ons,  the  peaks  you  see  represent   individual  atoms  in  the  protein  -­‐  actually   the  interac3ons  between  specific  atoms.  The  posi3ons  of  the  peaks  and  their  sizes  are  used  to  determine  the  distances  between  the  atoms,  and  to  put   together   a  model   of   the  molecule   that   fits   those   constraints  using   complex  soFware.  One  of  the  major  limits  of  NMR  is  that  only   structures  of  rela3vely   small  proteins  (no  more  than  200  aminoacids)  can  be  resolved.

 X-­‐ray  crystallography  is  a  method  of  determining  the  arrangement  of  atoms  within  a    crystal  in  which  a  beam  of   X-­‐rays  strikes  a  crystal  and  diffracts  into  many   specific  direc3ons.   From   the   angles   and   intensi3es   of   these   diffracted   beams,   a  crystallographer  can  produce  a  three-­‐dimensional  picture  of  the  density  of  electrons  within  the  crystal.  From  this  electron  density,  the  mean  posi3ons  of  the  atoms  in  the  crystal   can   be   determined,   as   well   as   their   chemical   bonds   and   various   other  informa3on.To  perform  X-­‐ray   crystallography,  it   is  necessary   to  grow  crystals  with  edges  around  0.1-­‐0.3  mm.  Finding  the  op3mal  condi3ons  of  crystal  growth  is  not  an  easy  task.  Nevertheless  crystals  of  complex  and  large  macromolecules  have  been  obtained.     In  1954   Max   Perutz's   group   developed   X-­‐ray   cristallography   methods   to   solve   the  molecular  structure  of  globular  proteins  and  define  the  structure  of  haemoglobin.  In  1953  Watson  and  Crick  published  their   model  of   the  DNA   double  helix    based  on  crystallography   data  provided  by   Rosalind  Franklin.  More  recently,   the  2009   Nobel  Prize  for  chemistry  was  assigned  for   the  defini3on  of  the  3D  structure  of  a  complex  organelle  such  a  ribosome.  Obtaining  the  crystal  of  the  first  macromolecular  complex  took  almost  30  years!  

Which  are  the  main  applications  of  biocrystallography?

study  protein  structure  and  functions  relationship

study  protein-­‐ligand  interactions

drug  design

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Fig.  1  The  vapour  diffusion  method  is  the  most   frequently  used  technique  to  grow  protein  crystals.  A.  A  small  amount  of  a  crystallisa3on  solu3on  is  put  into  the  chamber  reservoir.    A  drop  of  protein  solu3on  and  a  drop  of  crystallisa3on  solu3on  are  placed  onto    the  siHng  drop  post.  B.  The  chamber  is  sealed  to  start  the  crystallisa3on  process.

Among  the  various  methods  of  growing  protein  crystals,  the  most  common  is  Vapour  Diffusion  (Hanging  Drop  Method).A   drop   of   protein   solu3on   is   suspended   over   a   reservoir   containing   buffer   and  crystalliza3on  solu3on.  Water  diffuses  from  the  drop  to  the  solu3on  leaving  the  drop  with  op3mal  crystal  growth  condi3ons  (Fig.  1).  

The  protein  we  will  crystallize  in  this  lab  ac3vity  is  chicken  lysozyme  (129  aminoacids,  from  hen  egg  white).Lysozyme   is   a   potent   bactericide   agent.   It   is   a   small   enzyme   that   aiacks   the  protec3ve  cell  walls  of  bacteria.  Bacteria  build  a  tough  skin  of  carbohydrate  chains,  interlocked  by   short   pep3de  strands,   that   braces  their   delicate  membrane  against  the   cell's   high   osmo3c   pressure.   Lysozyme   breaks   these   carbohydrate   chains,  destroying  the  structural  integrity  of  the  cell  wall.  The  bacteria  burst  under  their  own  internal   pressure.   Substrates   of   lysozyme  i nc lude   pep3dog l ycan   ( F i g . 2 ) ,   t he  polysaccharide  component   of   the  cell  walls  of   certain  bacteria;   lysozyme  hydrolyzes  the  ß(1-­‐4)   glycosidic   bond   between   residues  of  N-­‐acetylmuramic   acid   (NAM)   and   N-­‐acetylglucosamine  (NAG),   the  two  monomer  sugars  of  pep3doglycan  chains.    Lysozyme  is  one  of  the  natural  immunity  weapons  against  infec3ons.   It   is   abundant   in   all   vertebrate  secre3ons  to  resist  infec3on  of  body  exposed  surfaces;   in   humans   lysozyme   is  present   in  tears  and  mucus.    Unfortunately,   lysozyme  is  a   large   molecule   that   is   not   par3cularly  useful  as  a  drug.

PROTOCOL

Equipment,  materials  and  chemicals

•crystallisa3on  plates  

•sealing  tape  (5  cm)  

•1  ml  and  1  μl  micropipeie

•A  microscope  to  observe  the  crystals

•Storage  space  at  20  °C

•Hen  egg  white  lysozyme    (a  single  chain  of  129  aa  residues)  40  mg/ml  in  H2O

•crystalliza3on  solu3on  NaCl  3.4  M  (20%  w/vol)

•Na-­‐acetate  buffer  pH  4  1M

•bidis3lled  H2O

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Fig.  2  Peptidoglycan  structure

A   small   amount   of   a   crystallisa3on   solu3on   is   added   to   the   reservoir   of   the  crystallisa3on   chamber.   A   drop   of   protein   solu3on   and   a   drop   of   crystallisa3on  solu3on  (1:1  vol)  are  pipeied  onto  the  siHng  drop  post,  located  in  the  centre  of  the  chamber.

well 1 well 2Final  crystallisaNon  condiNons:

well  1:  NaCl  8%,  Acetate  Buffer  pH4,  50  mM

well  2:  NaCl  9%,  Acetate  Buffer  pH4,  50  mM

NaCl  (200  μl)

Buffer  pH  4.0  (25  μl)

H2O  (275  μl)  

NaCl  (225  μl)

Buffer  pH  4.0  (25  μl)

H2O  (250  μl)

Final  crystallisaNon  condiNons:

well  1:  NaCl  8%,  Acetate  Buffer  pH4,  50  mM

well  2:  NaCl  9%,  Acetate  Buffer  pH4,  50  mM

In  details:Pipeie   0.5   ml   of   crystallisa3on   solu3on   (prepared   by   mixing   precipita3on  solu3on  NaCl  20%  +  Na-­‐Acetate  Buffer  1M  +  H2O)  in  2  wells  of  the  crystallisa3on  plate  (according  to  the  volumes  of  each  solu3on  given  in  the  table  above).Pipeie  2  μl  of  iysozyme  solu3on  (  40mg/ml)    into  the  crystallisa3on  cup  on  the  siHng  drop  post  in  each  well.  Pipeie  2   μl  of   the  reservoir   solu3on  into  the  crystallisa3on  cup  on  the  siHng  drop  post  in  each  well.  Mix  gently.  The  final  volume  of  the  drop  is  4  μl.Immediately  seal  the  chamber  to  avoid  evapora3on  and  to  guarantee  the  correct  vapour  diffusion  equilibrium  in  the  chamber.  Leave  the  plates  at  21  °C.  WARNING:  crystal  forma3on  requires  absolute  s3llnessCrystal  growth  can  be  monitored  directly  under   the  microscope  aFer  1-­‐2  hours.  The  plates  may   be  stored  un3l  the  next   day   for   final  analysis.  AFer   about  1-­‐2  weeks,  crystals  will  have  grown  to  their  final  size.  (A  sealed  plate  will  keep  up  to  a  year,  some3mes  even  longer).By   comparing   the   results   from   the   2   chambers,   determine   the   op3mal  condi3ons  for  crystallisa3on.

Since  the  concentra3on  of  salt  ions  is  higher  in  the  crystallisa3on  solu3on  than  in  the  mixture  on  the  siHng  drop  post,  solvent  molecules  will  move  from  the  protein  drop  to   the   reservoir   by   vapour   diffusion   in   the   gas   phase.   During   this   process,   the  solubility   of   the  protein   in   the   drop   decreases.   The  protein   solu3on   in   the  drop  eventually   becomes  supersaturated,  which   is  a  thermodynamically   unstable   state.  This  causes  some  of  the  protein  in  the  drop  either  to  form  crystal  nuclei  that  finally  grow   into   larger   protein   crystals,   or   to   precipitate  as  amorphous  protein  which  is  useless  for  X-­‐ray   analysis.  Crystallisa3on  and  precipita3on  are  compe3ng  processes,  so  it  is  extremely  important  to  find  the  op3mal  condi3ons  favouring  crystallisa3on.

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FROM  CRYSTALS  TO  3D  STRUCTURES

The  steps  necessary   to   analyze  the  crystals  and   reconstruct   the  3D  structure  of   a  protein  are  depicted  in  Fig  3  and  4.      

All  files  containing  molecular  coordinates  of   the  molecules  whose  3D  structure  has  been  determined  are  collected  (in  different  formats)  in  the  PDB  (“Protein  Database)  hip://www.rcsb.org/pdb/home/home.do   and   can   be   viewed   with   dedicated  soFwares.  We  will  use  YASARA  soFware.

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Fig.3.   X-­‐ray   diffrac3on.   The   x-­‐rays   are  diffracted  in  a  predictable  paiern  based  on  the  regular   crystal   laHce   structure   formed  by   the   protein.   The   diffracted   X-­‐rays  produce   a   paiern   of   spots   on   a  photographic  plate.

Fig.4  From  the  angles  and  intensi3es  of  the  diffracted  beams,  crystallographers  produce  a  three-­‐dimensional  picture  of  the  electron  density  within  the  crystal  and  reconstruct  the  polypeptide  aminoacid  chain.

SCENARIO

You  will  be  given  3   protein  par3al  sequences:  your   task  is  to  find  to  which  protein  

they   belong  to  and  which  of   them  can  be  responsible  for   the  severe  anaphylac3c  reac3on    in  the  woman.  

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In  this  scenario,  the  molecular  analysis  is  cri3cal  to  the  iden3fica3on  of   toxic   substances   which   may   have   caused   harm   to   a   person’s  health.A   young   woman   is   admiied   to   the   emergency   room   of   a   local  hospital  with   severe   symptoms  of   anaphylac3c   reac3on.   She  was  enjoying  a  dinner  at  an  italian  restaurant  with  a  friend  when  the  first  symptoms  occurred.   She  refers  to  be  allergic   to   egg   proteins  and  soya  and  therefore  to  have  chosen  food  which  should  not  contain  any  of  the  substances  she  knows  to  be  allergic  to.Her  friend,  a  lawyer,  wants  to  suit  the  restaurant  for  food  fraud.

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>Protein 1CCDKPLLEKS HCIAEVEKDA IPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRR HPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKL KHLVDEPQNL IKQNCDQFEK LGEYGFQNAL IVRYTRKVPQ VSTPTLVEVS

>Protein 2DQAMEDIKQM EAESISSSEE IVPNSVEQKH IQKEDVPSER YLGYLEQLLR LKKYKVPQLE IVPNSAEERL HSMKEGIHAQ QKEPMIGVNQ ELAYFYPELF RQFYQLDAYP SGAWYYVPLG TQYTDAPSFS DIPNPIGSEN SEKTTMPLW

>Protein 3KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSS

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To  find  the  protein  to  which  each  par3al  sequence   belongs   to   open   hip://mrs.cmbi.ru.nl/mrs-­‐web/,   the   MRS  homepage,  a search engine for biological and medical databanks.  

Click  the  link  Blast,  on  the  top  page  grey  bar.Paste   the   aa  sequence  of   protein   1   in  

the  white  box.    Don’t  forget  the  >  symbol  in   front   of   the   protein   name   and  sequence,   to   conform   to   the   correct  FASTA  format.  Check   that   the   op3on   Filter   sequence  

(low  complexity)  is  selected.    ClicK  Run  Blast,  top  right  in  the  page.

The  new   page  shows  a  table   giving   the   number   of   hits,   i.e.   similar   to   the  query  sequence  and   the   degree   of   similarity   of   each   alignment;   Blast   has   iden3fied   48  proteins  whose  sequence  has  a  certain  degree  of  similarity  to  your  query.  Click  the  first   row  in  the  table  to  obtain   the  complete  hit   list.  The  first   hit  has  the  highest   BitScore  and   the   lowest   E-­‐value,   i.e.   the  match  has  the  highest   sta3s3cal  significance.  The  first  hit  corresponds  to  albu_bovin,  i.e.  to  bovine  serum  albumin.  

Look  carefully  at  the  hit  list.  For  each  protein  different  codes  and  values  are  reported:•-­‐   the   ID   column   with   the   iden3fica3on   code   of   the   protein   sequence   in   the  

database  (the  protein  name  followed  by  the  name  of  the  organism);•-­‐   the  Coverage  column  with  the  color   code  degree  of  correspondence  between  

the  query   sequence  and  the  homologous  sequence  in  the  database;  a  red  line  means  that   the  alignment  of   the  query   and  albu_bovin  is  complete;   blue  and  light  blue  lines  mean  a  par3al  correspondence;

•-­‐   the  Descrip3on  column  with  a  short  descrip3on  of  the  hit  protein;•-­‐   the   Hsps,   Bitscore   and   E-­‐value   columns   with   the   values   giving   the  

sta3s3cal  significance  of  each  match.

Click  the  code  albu_bovin.  In  the  new  page  you  will  find  many  data  about  the  protein,  its  func3on  and  its  effects  on  human  health.    

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BLAST   (Basic   Local   Alignment   Search  Tool)   is   a   collection   of   searching  programs   for   biological   sequence  databases.   The   program   uses   the   BLAST  algorithm   to   compare   protein   or   DNA  sequence   queries   to   protein   or   DNA  sequence   databases.   The   Basic   Local  Alignment   Search   Tool   (BLAST)   Dinds  regions   of   local   similarity   between  sequences.   The   program   compares  nucleotide   or   protein   sequences   to  sequence   databases   and   calculates   the  statistical   signiDicance  of  matches.  BLAST  can   be   used   to   infer   functional   and  evolutionary   relationships   between  sequences   as   well   as   help   identify  members  of  gene  families.

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The  page  is  ar3culated  in  different  sec3ons.  Consult  the  sec3ons  Entry   informa3on,  Name  and  origin,  Comments,  Features  key  and  Sequence  informa3on  to  answer  the  following  ques3ons  about  the  protein  albu_bovin.

Return  to  the  hit   list  page.  Click  the  red  line  corrresponding  to  albu_bovin.  You  will  find  that  the  150  aminoacids  of  your  query  completely  correspond  (100%  Iden3ty)  to  150   residues  of   albu_bovin;   click   the   red   line  again   to   find  a   sec3on  where   the  alignment  of    protein1  and  albu_bovin  is  shown.The  sequence  of  protein  1   is  iden3fied  by   the  leier  Q  (query)  and  the  sequence  of  albu_bovin  is  iden3fied  by  the  leier  S  (sequence).      Between  these  two  sequences  is  the    sequence  of  the  shared  aminoacids.  Numbers  on  the  leF  and  right  correspond  to  aminoacid  posi3ons.  Note  that  the  150  residues  of  the  query  sequence  correspond  to  residues  301-­‐  450  of  bovin  albumin.    

Following  the  same  procedure,  iden3fy  protein  2   and  protein  3,   find   the  requested   info   and  answer  the  6  ques3ons  in  the  box.  Draw   your   conclusions  about   the  case  of   the  allergic  woman.  Among   the   3   proteins,   only   lysozyme,  abundant  in  hen  egg  white,  can  be  responsible  for  the  allergic  reac3on  shown  by  the  woman.    

Which  entrée  in  the  Menu  chosen  by  the  woman  may  contain  egg  white  lysozyme?    

Egg  white  lysozyme  is  largely  used  in  cheese  produc3on  for  its  ly3c  effect  on  bacteria  cell  wall.   Lysozyme  is  efficient   in   countering   “late  blowing”  during   the  ripening  of  cheese,  such  as  parmesan,  provolone  and  the  like.   “Late  blowing”   is  caused  by   the  outgrowth   of   clostridial  spores   (origina3ng   mainly   from   the   use   of   silage   in   the  feeding  of  dairy  caile)  present  in  raw  milk.

While   the  use  of   lysozyme  as  cheese  preserva3ve  is  allowed   in   the  produc3on  of  many   cheese   types,   its   use   (and   that   of   ensilaged   forages)   is   forbidden   in   the  

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1.  Protein  name2.  Species  of  origin3.  Func3on4.  Lenght  in  aminoacids5.  Is  this  protein  present  in  the  food  ingested  by  the  woman?6.  Could  this  protein  be  responsible  for  the  allergic  reac3on  suffered  by  the  woman?  Explain  your  answer

disciplinary   of   produc3on   of   PDO   (Protected   DesignaNon   of   Origin)   Parmigiano-­‐Reggiano.   Lysozyme   (not   exceeding   300   mg/kg)   can   instead   be   added   to   Grana  Padano,   a  cheese   similar   to   Parmigiano   Reggiano   and   oFen   used   as  a  Reggiano  surrogate,   but   whose   organolec3c   quali3es   (and   price)   are   far   below   those   of  Reggiano.  Strong  suspect  exists  that  the  Carpaccio  served  to  the  woman,  differently  from  what  declared  on  the  Restaurant  Menu,  was  accompanied  by  slivers  of  Grana  Padano  (and  not  of  the  more  expensive  Parmigiano  Reggiano).

The  woman  and  her  lawyer  have  good  chance  of  winning  the  case  for   fraud  brought  to  the  restaurant.

WORKING  WITH  YASARA

Yasara  is  a  graphics  program  used  to  visualise  and  manipulate  protein  models.  It  was  developed  by   Elmar   Krieger,  mainly   at   the  CMB.   If  Yasara  is  not   installed  on  your  computer  yet,  go  to  the    the  soFware  sec3on  hip://www.bioinforma3csatschool.eu/soFware.html  to  see  how  to  do  that.AFer   the   prac3cum,   you   will   have   a  chance  to   look   at   several   other   interes3ng  proteins.  You  can  also  use  the  program  to  make  nice  pictures  for  reports  and  science  projects.  

Exercise  1:Start  Yasar.   In  Yasara,  you  can  load  a  polypep3de  using  the  menu  on  the  top  leF  of  the  window:  File  >  Load  >  Complete  scene.  Choose  the  file  introduc3on.sce  and  click  OK.  

You  can  now   see  the  structure  of   the  pep3de  with  sequence  Asp-­‐His-­‐Arg-­‐Gly-­‐Gly-­‐Met-­‐Lys-­‐Tyr   in  the  so-­‐called  ball  and  s3ck  representa3on.  Individual  atoms  are  shown  as  balls,  connected  by   s3cks  represen3ng  the  atomic  bonds.  Note:  the  hydrogen  atoms  are  not  represented   because   that   would   make   the   model  much  more   complicated.   Below,   you  can  see  a  2D  representa3on  of  the  same  pep3de.  You  will  no3ce  a  "cloud"   around   the  pep3de.   This   represents   the  Van   der  Waals  surface.   This  surface   shows  how  much  space  the  atoms  actually   occupy:   there  is  barely  any  empty  space  between  adjacent  atoms.  

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If  you  move  your  mouse  cursor  along  the  boiom  of  the  Yasara  screen,  the  sequence  bar  appears.  To  keep  this  bar  visible,  click  on  the  blue  pushpin  at  the  leF  end  of  the  bar.  

When  you  click  on  a  residue  in  the  sequence  bar,   the  Cα-­‐atom  of  this  residue  will  flash.   If  you  push  Ctrl  while  you  click  on  the  residue,  the  protein  will  rotate  and  zoom  so  that  you  can   clearly   see   the   Cα-­‐atom.   You   can  manipulate  the  protein  by  holding  the  mouse  buions   and   moving   your   mouse.   Try   the  following:  •LeF:  Rotate•Middle:  Translate•Right:  Zoom

The  atoms  are  coloured  by   atom  type.   If   you   click  on   an  atom,   extra  informa3on  about  this  atom  will  appear  on  the  leF  side  of  the  window.  

Exercise  2:Look  up  which  types  of  atoms  (elements)  can  be  found  in  proteins,  using  this  list  of  amino  acids.  Look  at  different  atoms  in  Yasara  and  find  out  which  elements  are  coloured  red,  dark  blue,  green,  and  light  blue.

Exercise  3:In  the  representa3on   that   you  see,   covalent   bonds  are  shown  as  short   s3cks  of   a  specific  colour.  Explain  the  difference  between  the  yellow  and  white  bonds  using  the    list  of  amino  acid  structures.  The  3D   structure  shows  three  of   the  four   different   types  of   interac3ons  that   are  important  for  protein  folding.  These  different  interac3ons  are  represented  by  green,  blue  and  orange  s3cks.  

Exercise  4:Which  interac3on  belongs  to  each  colour?  Locate  these  interac3ons  in  the  2D  representa3on    of  the  pep3de.  

Exercise  5:Reset  Yasara  by   clicking  File  >  New  and  then  Yes.  Now,   load  the  file     1JYV.pdb  (you  can  download  lysozyme’s  structure  in  .pdb  format,  at  the  site:    hip://www.rcsb.org/pdb/results/results.do?ou�ormat=&qrid=932BF699&tabtoshow=Current   )    via  File  >  Load  >  PDB  file  .

You   now   see   lysozyme   in  Ball   representa3on:   you   can   see  all   atoms  (except   the  hydrogens)   as   large   balls.   This   is  not   a   very   clear   representa3on.   Fortunately,   a  number   of   alterna3ve   representa3ons   exist.   You   can   browse   through   them   by  pushing  the  F1  through  F8  keys  on  your  keyboard:

F1:  Ball  representa3on  (individual  atoms)F2:  Ball-­‐and-­‐s3ck  representa3on  (individual  atoms  +  bonds)

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F3:  S3ck  representa3on  (the  atoms  look  like  s3cks)  F4:  Cα-­‐trace  (only  the  Cα  are  represented,  connected  with  s3cks)F5:  Backbone-­‐trace  (showing  only  the  backbone,  no  side  chains)F6:  Cartoon  representa3on  (secondary  structure-­‐elements)F7:  Alterna3ve  cartoon  representa3onF8:  Add/Remove  the  side  chains  of  the  residues  (the  "R"  groups)  in  any  other  representa3on

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Exercise  6:

Using   F5,  F6   and  F7   you  can  easily   recognise  secondary   structure  elements.  Which  secondary  structures  match  the  red  and  blue  parts  or  the  protein?  

Exercise  7:  

Find  the  following  five  structure-­‐elements:  a  double  bonded  oxygen  atom,  a  pep3de  bond,  a  his3dine  side  chain,  an  alpha  helix  and  a  disulfide  bond.  Use  the  appropriate  representa3on  of  the  protein  for  each  element  and  write  those  down.

The  acNve  site

We  will  now  look  for  the  ac3ve  site  of  the  protein.  That  is  where  the  most  important  amino   acids   are   located   and   where   the   actual   chemical   reac3on   takes   place.  !

A  characteris3c  feature  of  the  ac3ve  site  of  enzymes  is  that  they  (almost)  always  lye  in  a  cavity  or  cleF  on  the  surface  of  the  protein.  Therefore,  a  quick-­‐and-­‐dirty  way  to  find  the  ac3ve  site  is  to  look  for  the  largest  cavity  on  the  surface  of  the  protein.  

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Exercise  8:Why  is  the  ac3ve  site  usually  located  in  a  cavity?  

Exercise  9:Find  the  ac3ve  site  by  rota3ng  the  protein  and  zooming.  Choose  the  representa3on  that  you  think  is  most  appropriate.  

The   Cus-­‐Mi-­‐Bio   staff,   composed   of   both   University   Professors   and   High   School  teachers,  are  the  scien3fic  editors  and  authors  of  the  contents  of  this  Handbook.  Workshop  LeadersCinzia  Grazioli  and  Cris3na  GriHHigh  school  teacher  fully  working    at  CusMiBio,  via  Celoria  20  Milan,  Italy

ScienNfic  supervisorGiovanna  VialeProfessor  of  Biology  and  GeneDcs,  Dept.  of  Biology  and  GeneDcs  for  Medical  Sciences,  University  of  Milan,  via  VioJ  3/5,  Milan,  Italy

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