Thermodynamic  and  Kine/c  Characteriza/on   of  Folding  Mechanisms  of  CheY  Circular   Permutants   Tyler  Ogden   Brooks  Group   Hampshire  College  

Sequence-­‐dependent  folding  behavior   Free  Energy  Landscape   Theory  

•  Folding  characterized  by  free   energy  minima   •  Analyze  at  Tf  to  see  basins  of   unfolded  and  na/ve  state  

CheY  protein  

•  (αβ)5   •  How  do  cuts  between  secondary   structures  affect  folding?     •  Inform  protein  engineering   Hills  and  Brooks.  JMB  (2008)  382,  485-­‐495  

Three  Circular  Permutant  Systems   CpB2:  Cut  between  residues   35-­‐36   • End  of  β2  in  N-­‐terminal   CpB3:  cut  between  residues   62-­‐63   • In  turn  between  β3α3;   splits  C-­‐terminal  and  N-­‐ terminal   CpB4:  cut  between  residues   77-­‐78   • In  turn  between  α3β4  in  C-­‐ terminal  

The  Go-­‐Model   •  Residues  as  Cα  ‘beads’   •  Na/ve  contacts:   –  Side-­‐chain  side-­‐chain  interac/ons   –  Backbone  H-­‐bonds   •  Lennard-­‐Jones  poten/al  for  i  -­‐  j  >  2  

σij 12 σij 10 σij 6 Vij = �ij [13( ) − 18( ) + 4( ) ] rij rij rij •  Non-­‐na/ve  repulsion  

σij 12 Vij = �ij [13( ) ] rij •  This  simple  model  has  been  successfully  applied  to  inves/gate   folding  mechanisms  of  several  proteins  in  the  past   Karanicolas  and  Brooks.  Prot  Sci  (2002)  11:2351  

Simula/on  Details   •  Thermodynamic    -­‐  Replica  Exchange  

–  10,000  exchange  cycles  (exchange  every  20,000  steps)   –  4  temperature  windows  (.87  Tf,  .97  Tf,  1.08  Tf,  1.20  Tf)     –  Umbrella  bias:  8  rgyr  biases  spanning  1.0  Rg0    to  2.0  Rg0   –  Using  gorex.pl  from  the  MRMTSB  toolset     –  Unbiased  data  using  WHAM   0 g

•  Kine/c    

–  48  independent  unbiased  MD  simula/ons  at  .87  Tf  for  2  x   108  /me  steps   –  Unfolded  state  obtained  from  hea/ng  to  1.5  Tf   –  Time  step  is  22fs     –  Langevin  Dynamics  

Folding  of  CheY1  

1Hills  and  Brooks.  JMB  (2008)  382,  485-­‐495  

 

•  N-­‐terminus  nuclea/on   •  Frustra/on  in  α2β3α3β4   tetrad   •  C-­‐terminus  folding  is   accompanied  by  loosening  of   en/re  structure   •  Non-­‐obligate  intermediate   –  Structured  N-­‐terminus   –  Unstructured  C-­‐terminus   •  Agrees  with  experimental   results2   2Kathuria,  Day,  Wallace  &  Majhews.  JMB    

(2008)  382,  467-­‐484  

Qtotal  

Change  in  Tf   Na/ve  State  Stability   • Tf  at  peak   Tf  CpB2  =  280  K       Tf    CpB3  =  285  K   Tf  CpB4  =  287  K  

CpB2-­‐Energy  Landscape  and   Folding   1 1

0.6

0.8 0.6





0.8

C-term N-term Helices 2&3 Helix 3&4 Strands 3&4 Strands 4&5

0.4

0.4

0.2 0 0

N-term CpB2 C-term CpB2 N-term WT C-term WT

0.2

0.2

0.4 0.6 Qtotal

0.8

1

0 0

1000 500 1500 Time (arbitrary units)

2000

CpB4-­‐Energy  Landscape  and   Folding  

CpB3  –  Energy  Landscape  and   Folding  (Major  Error  in  Kine/cs)  

Conclusions   •  Simula/ons  predict  cut-­‐dependent  folding  for   a  constant  amino  acid  sequence   •  Cuts  change  stability  of  na/ve  state   •  Loca/on  of  intermediate  on  folding  pathway   may  vary  (CpB3)   •  Suggests  possibility  of  tuning  protein  folding   pathways    

Acknowledgements   •  •  •  •  •  • 

Karunesh  Arora  (UMICH)   Charlie  (UMICH)   David  Braun  (UMICH)   Ron  Hills  (UNE)   John  Karanicolas  (KU)   Bob  Majhews    (UMASS  Medical  School)