Using two recently-Using two recently-developed molecular developed molecular

dynamics protocols for dynamics protocols for protein foldingprotein folding

Timothy H. ClickTimothy H. ClickDepartment of Chemistry and Department of Chemistry and

BiochemistryBiochemistryUniversity of OklahomaUniversity of Oklahoma

Norman, OklahomaNorman, Oklahoma

2

OutlineOutline

Introduction to MD protocolsIntroduction to MD protocols Previous workPrevious work Simulations of tryptophan zipper 2Simulations of tryptophan zipper 2 Simulation of Simulation of StreptococcalStreptococcal protein G B1 protein G B1

domain (residues 41-56)domain (residues 41-56) ConclusionsConclusions Future directionsFuture directions AcknowledgementsAcknowledgements

3

Protein geometry Protein geometry optimizationoptimization

Dill, K.A.; Chan, H.S. Nat. Struct. Biol., 1997, 4, 10-19.

4

11stst MD protocol — DIVE MD protocol — DIVE

Disrupted Velocity (DIVE) search Disrupted Velocity (DIVE) search protocolprotocol Velocity reassignment of coordinate historiesVelocity reassignment of coordinate histories

Magnitude rescaling — energy perturbationMagnitude rescaling — energy perturbation Direction changesDirection changes Reassignment every n steps (defined by user)Reassignment every n steps (defined by user)

Heating and cooling cyclesHeating and cooling cycles Conformations sampled near absolute zeroConformations sampled near absolute zero Overall, protocol disrupts equilibriumOverall, protocol disrupts equilibrium Energy barriers overcome or circumventedEnergy barriers overcome or circumvented Several potential energy minima sampledSeveral potential energy minima sampled

5

How conformations are How conformations are selectedselected

Disrupted Velocity for trpzip2

-500

-495

-490

-485

-480

-475

-470

-465

-460

-455

-450

0 500 1000 1500 2000 2500 3000 3500 4000

Time (ps)

Po

ten

tia

l e

ne

rgy

(k

ca

l/m

ol)

Disrupted velocity for trpzip2

-500.00

-495.00

-490.00

-485.00

-480.00

-475.00

-470.00

-465.00

-460.00

0 500 1000 1500 2000 2500 3000 3500 4000

Time (ps)

Po

ten

tial

en

erg

y (k

cal/

mo

l)

βlowto

DIP

βlow2to

DIP

β

βlow

6

22ndnd MD protocol — DIP MD protocol — DIP

Divergent Path (DIP) search strategyDivergent Path (DIP) search strategy Coordinate histories at same constant Coordinate histories at same constant

temperaturetemperature Simulations involve multiple coordinate historiesSimulations involve multiple coordinate histories Individual coordinate histories randomly Individual coordinate histories randomly

assigned initial velocitiesassigned initial velocities Velocities can be altered allowing for different Velocities can be altered allowing for different

conditionsconditions Constant temperatures maintained by rescaling Constant temperatures maintained by rescaling

velocity magnitudesvelocity magnitudes Broader sampling of potential energy surface Broader sampling of potential energy surface

allowedallowed

7

DIP simulationDIP simulation

8

DIP simulation (cont’d)DIP simulation (cont’d)<E>= -624.22 ± 8.27

kcal/mol<RMSD> = 1.6 ± 0.5 Å

<E>= -632.42 ± 8.44

kcal/mol<RMSD> = 1.8 ± 0.2 Å

<E>= -587.16 ± 9.50

kcal/mol<RMSD> = 13.2 ± 0.5 Å

<E>= -633.94 ± 7.87

kcal/mol<RMSD> = 1.5 ± 0.2 Å

<E>= -592.02 ± 8.38

kcal/mol<RMSD> = 12.5 ± 0.5 Å

<E>= -600.29 ± 13.05

kcal/mol<RMSD> = 11.3 ± 0.7 Å

nmr

9

Protocol proceduresProtocol procedures

Modified Amber force field (Okur,A.; Modified Amber force field (Okur,A.; Strockbine, B.; Hornak, V.; Simmerling, C., Strockbine, B.; Hornak, V.; Simmerling, C., J. J. Comput. Chem.Comput. Chem., , 20032003, 21), 21)

Constraints on atoms covalently bonded to Constraints on atoms covalently bonded to hydrogenhydrogen

Implicit solventImplicit solvent 2 fs time step2 fs time step 4,000,000 steps4,000,000 steps Velocity disruption every 20,000 steps (DIVE)Velocity disruption every 20,000 steps (DIVE) T = 300 ± 20 K (DIP)T = 300 ± 20 K (DIP) 6 independent coordinate histories/simulation6 independent coordinate histories/simulation

10

Previous work with Previous work with αα--heliceshelices

Zunnan HuangZunnan Huang 13-residue polyalanine13-residue polyalanine Trp-cage (Trp-cage (αα-helix and 3-helix and 31010-helix-helix

Huang and Zhanyong GuoHuang and Zhanyong Guo Peptide FPeptide F

Timothy H. ClickTimothy H. Click C-peptide of ribonuclease A (residues 1-13)C-peptide of ribonuclease A (residues 1-13)

11

Tryptophan zipper 2 Tryptophan zipper 2 (trpzip2)(trpzip2)

De novo 12-residue polypeptideDe novo 12-residue polypeptide Sequence (SSequence (S11WTWENGKWTWKWTWENGKWTWK1212-NH2)-NH2) PDB code 1LE1 (20 NMR models)PDB code 1LE1 (20 NMR models) Stable β-sheet in aqueous solution by cross-Stable β-sheet in aqueous solution by cross-

stranded pairs of four tryptophansstranded pairs of four tryptophans Simulations completed by other groupsSimulations completed by other groups

1 Cochran, A.G.; Skelton, N.J.; Starovasnik, M.A. P. Natl. Acad. Sci. USA, 2001, 98, 5578-5583.

12

Trpzip2 DIVE ResultsTrpzip2 DIVE Results

extlowE = -496.59 kcal/mol

RMSD 6.9 Ẳ

βlowE = -494.05 kcal/mol

RMSD 5.0 Ẳ

αlowE = -498.22 kcal/mol

RMSD 6.1 Ẳ

β*E = -489.44 kcal/mol

RMSD 0.9 Ẳ

extlow2E = -499.23 kcal/mol

RMSD 7.0 Ẳ

βlow2E = -497.06 kcal/mol

RMSD 5.7 Ẳ

αlow2E = -498.17 kcal/mol

RMSD 6.1 Ẳ

13

Trpzip2 DIP ResultsTrpzip2 DIP Results

ext<E> = -360.82 ± 7.66

kcal/mol<RMSD> 6.5 ± 0.8 Ẳ

β<E> = -381.65 ± 4.55

kcal/mol<RMSD> 0.9 ± 0.1 Ẳ

α<E> = -369.79 ± 9.01

kcal/mol<RMSD> 6.7 ± 0.4 Ẳ

extlow<E> = -375.47 ± 6.28

kcal/mol<RMSD> 7.5 ± 0.1 Ẳ

βlow<E> = -374.14 ± 7.18

kcal/mol<RMSD> 7.0 ± 0.3 Ẳ

αlow<E> = -368.77 ± 7.38

kcal/mol<RMSD> 6.1 ± 0.1 Ẳ

extlow2<E> = -381.44 ± 5.74

kcal/mol<RMSD> 7.4 ± 0.1 Ẳ

βlow2<E> = -378.75 ± 6.04

kcal/mol<RMSD> 7.0 ± 0.3 Ẳ

αlow2<E> = -368.93 ± 7.54

kcal/mol<RMSD> 6.2 ± 0.3 Ẳ

β*<E> = -381.65 ± 5.04

kcal/mol<RMSD> 0.8 ± 0.1 Ẳ

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Trpzip2 Trpzip2 SummarySummary

PES rough at low temperaturesPES rough at low temperatures ββ-hairpin challenging secondary structure-hairpin challenging secondary structure ββ-hairpin as relative global PE -hairpin as relative global PE

conformationconformation αα-helices metastable conformation-helices metastable conformation

15

B1 domain of B1 domain of StreptococcalStreptococcal protein Gprotein G

Natural Natural ββ-hairpin stable in aqueous -hairpin stable in aqueous solution.solution. Sequence (GSequence (G4141EWTYDDATKTFTVTEEWTYDDATKTFTVTE5656)) PDB 2GB1 (x-ray crystal structure)PDB 2GB1 (x-ray crystal structure) Stabilization factorsStabilization factors

Hydrophobic coreHydrophobic core Terminal salt bridgeTerminal salt bridge

Several simulationsSeveral simulations

22 Gronenborn, A. M.; Filpula, D. R.; Essig, N. Z.; Achari, A.; Gronenborn, A. M.; Filpula, D. R.; Essig, N. Z.; Achari, A.; Whitlow, M.; Wingfield, P. T.; Clore, G. M. Science, Whitlow, M.; Wingfield, P. T.; Clore, G. M. Science, 19911991, , 253, 657-661.253, 657-661.

16

Protein G DIVE resultsProtein G DIVE results

extlowE = -784.40 kcal/mol

RMSD 7.4 Ẳ

βlowE = -774.96 kcal/mol

RMSD 6.8 Ẳ

αlowE = -783.53 kcal/mol

RMSD 8.4 Ẳ

β*E = -770.54 kcal/mol

RMSD 0.9 Ẳ

extlow2E = -785.85 kcal/mol

RMSD 7.3 Ẳ

βlow2E = -781.75 kcal/mol

RMSD 6.4 Ẳ

αlow2E = -785.64 kcal/mol

RMSD 8.4 Ẳ

17

Protein G DIP resultsProtein G DIP results

ext<E> = -612.63 ± 9.31

kcal/mol<RMSD> 10.6 ± 0.6 Ẳ

β<E> = -638.42 ± 5.84

kcal/mol<RMSD> 1.6 ± 0.4 Ẳ

α<E> = -646.18 ± 6.90

kcal/mol<RMSD> 8.9 ± 0.2 Ẳ

extlow<E> = -640.60 ± 7.13

kcal/mol<RMSD> 9.1 ± 0.3 Ẳ

βlow<E> = -651.05 ± 6.34

kcal/mol<RMSD> 6.9 ± 1.2 Ẳ

αlow<E> = -646.14 ± 6.01

kcal/mol<RMSD> 9.0 ± 0.3 Ẳ

extlow2<E> = -633.74 ± 7.13

kcal/mol<RMSD> 9.0 ± 0.7 Ẳ

βlow2<E> = -643.91 ± 7.86

kcal/mol<RMSD> 9.0 ± 0.3 Ẳ

αlow2<E> = -650.14 ± 6.33

kcal/mol<RMSD> 9.0 ± 0.2 Ẳ

β*<E> = -644.28 ± 6.08

kcal/mol<RMSD> 1.6 ± 0.2 Ẳ

18

Protein G summaryProtein G summary

ββ-hairpin stable at 300 K-hairpin stable at 300 K Helical conformation lower in energyHelical conformation lower in energy

Better energy compensationBetter energy compensation33

Agreement with other simulationAgreement with other simulation44

Various factors may overstabilize Various factors may overstabilize helices (e.g., implicit solvent, salt helices (e.g., implicit solvent, salt bridges)bridges)

33 Muñoz, V.; Thompson, P. A.; Hofrichter, J.; Eaton, W. A. Muñoz, V.; Thompson, P. A.; Hofrichter, J.; Eaton, W. A. NatureNature, , 19971997, 390, 196-199., 390, 196-199.44 Krivov, S. V.; Karplus, M. P. Natl. Acad. Sci., USA, Krivov, S. V.; Karplus, M. P. Natl. Acad. Sci., USA, 20042004, , 101, 14766-14770.101, 14766-14770.

19

ConclusionsConclusions

DIVE and DIP locate several PE minimaDIVE and DIP locate several PE minima PES mapped by DIVEPES mapped by DIVE PES of conformations at desired PES of conformations at desired

temperature with DIPtemperature with DIP Conformations in good, if not excellent, Conformations in good, if not excellent,

agreement with experimental agreement with experimental structures using DIP and DIVEstructures using DIP and DIVE

20

Future directionsFuture directions

Continue validation of MD protocols with Continue validation of MD protocols with larger larger ββ-sheet-sheet

Further test MD protocols with tertiary Further test MD protocols with tertiary structurestructure

Predict structure of small proteinPredict structure of small protein

21

AcknowledgementsAcknowledgements

Ralph A. WheelerRalph A. Wheeler Zunnan Huang and Adam HixsonZunnan Huang and Adam Hixson National Research Service Award 5 F31 National Research Service Award 5 F31

GM067560-03 to THC from the GM067560-03 to THC from the NIH/NIGMSNIH/NIGMS

Oklahoma Center for the Advancement of Oklahoma Center for the Advancement of Science and Technology (OCAST) HR01-Science and Technology (OCAST) HR01-148148

Oklahoma Supercomputing Center for Oklahoma Supercomputing Center for Education and Research (OSCER)Education and Research (OSCER)

NSF/NRAC supercomputer time MCA96-NSF/NRAC supercomputer time MCA96-N019N019