N-term

C-term

The Folding of WW Domain The Folding of WW Domain FBP28 FBP28

(Formin binding protein):(Formin binding protein):

Xavier PerioleXavier Periole

WW domainsWW domains

• Two highly conserved Two highly conserved tryptophans tryptophans

• ~ 35-45 aa. ~ 35-45 aa.

• Non-catalytic signaling Non-catalytic signaling proteins (protein-protein proteins (protein-protein recognition) recognition)

• >200 non-redundant >200 non-redundant proteins (2003)proteins (2003)

• Bind proline-rich sequences. Bind proline-rich sequences.

• 6 classes (sequence 6 classes (sequence preference).preference).

WW domains:WW domains:

• Fast and reversible folding Fast and reversible folding • Highly conserved structureHighly conserved structure• Rapid formation of amyloid fibres (Rapid formation of amyloid fibres (Protein Conformational DisorderProtein Conformational Disorder))

• Collaboration with Guy Lippens (NMR-Lille)Collaboration with Guy Lippens (NMR-Lille)

WW Domain FBP28 (Formin binding protein):WW Domain FBP28 (Formin binding protein):

• Fastest folding of the family (10s of Fastest folding of the family (10s of s)s)• The fold is conserved upon mutationsThe fold is conserved upon mutations

• Temp, mutations and sequence truncation modulate a three- to two-Temp, mutations and sequence truncation modulate a three- to two-state folding pathwaystate folding pathway

• The N- (res 1-5) and C-termini (res 34-37) are not required: 28 residuesThe N- (res 1-5) and C-termini (res 34-37) are not required: 28 residues

WW Domain FBP28WW Domain FBP28

Xavier PerioleXavier Periole

Very stable and highly conservedVery stable and highly conserved

slightly curved three-stranded antiparallel slightly curved three-stranded antiparallel ß-sheetß-sheet

N-term

C-term

Xavier PerioleXavier Periole

WW Domain FBP28WW Domain FBP28

• 38 Replicas 38 Replicas • T: from 282 to 498KT: from 282 to 498K• in waterin water• GROMOS96 FF GROMOS96 FF

38*200ns=7.638*200ns=7.6ss

Xavier PerioleXavier Periole

WW Domain FBP28WW Domain FBP28

RMSD from the X-ray structureRMSD from the X-ray structure

T=280KT=280K

T=500KT=500K

Xavier PerioleXavier Periole

WW Domain FBP28WW Domain FBP28

X-rayX-ray rmsdrmsd0.22 nm0.22 nm

rmsdrmsd0.14 nm0.14 nm

Structures with minimum deviation from the X-rayStructures with minimum deviation from the X-ray

Xavier PerioleXavier Periole

WW Domain FBP28WW Domain FBP28

RMSD from the X-ray structureRMSD from the X-ray structure

Xavier PerioleXavier Periole

WW Domain FBP28WW Domain FBP28

rmsd vs. Rrmsd vs. Rgyrgyr

Xavier PerioleXavier Periole

WW Domain FBP28WW Domain FBP28

Comparison with the Native StateComparison with the Native State

rmsd vs. Sec. Struct.rmsd vs. Sec. Struct.

Xavier PerioleXavier Periole

WW Domain FBP28WW Domain FBP28

rmsd vs. native contactsrmsd vs. native contacts

Xavier PerioleXavier Periole

WW Domain FBP28WW Domain FBP28

The Real The Real CaseCase

Build the Transition StateBuild the Transition State

1.1. Characterize the limits native/unfolded statesCharacterize the limits native/unfolded states

• Build the intermediary stateBuild the intermediary state

• Unfolding SimulationUnfolding Simulation

2.2. Use Use -values as restraints in REMD-values as restraints in REMD

Xavier PerioleXavier Periole

The Real The Real CaseCase

REMD from the native state: Unfolding simulationsREMD from the native state: Unfolding simulations

Xavier PerioleXavier Periole

The Real The Real CaseCase

-values as restraints in MD:-values as restraints in MD:

A=1A=0Mutation does not

affect the TSEMutation doesaffect the TSE

mutDN

mutDTmut GG /

a) b)rmsd: 4.4 1.3rmsd: 4.4 1.3ÅÅ

rmsd (rmsd (ÅÅ))

Xavier PerioleXavier Periole