A Protein Folding Nucleus in the Gas

PhaseJessica Thomas and David Pratt

University of Pittsburgh

Michel Mons Franccedilois Piuzzi Eric Gloaguen and Benjamin

TardivelLaboratoire Francis Perrin

Motivation

Poupon and Mornon1 Folding Nuclei segments of proteins that fold first

Molinari and Coworkers2 Identified folding nuclei in β-lactoglobulin

What intermolecular forces are found in a folding nucleus (And what does this tell us about protein folding) Samples = segments of WYSLA from β-lactoglobulin

Acetyl- and ndashNH2 caps simulate longer chains

1 A Poupon and J-P Mornon FEBS Letters (1999) 452 283

2 L Ragona et al Biochemistry (2002) 41 2786

Experimental

Sample Preparation Laser ablation

Spectroscopic Methods UV ionization with mass spec

UVIR hole burning

S0

S1

IR

UV

Laser Ablation

Reduces decomposition of large biomolecules

Laser 2nd harmonic of NdYAG

Graphite matrix

UVIR Lab

MiniYAG

UV laser

Ablation

OPO YAG LaserIR laser

Prism creates second UV path through chamber

Excimer Laser Dye Laser

Chambers Jet and MS

Detection

Acetyl-Tryptophan(W)-Tyrosine(Y)-Amide Two UV chromophores

Results WYp

WYp Ionization

Trp gives broad UV absorption spectrum with sharp features

Use UV spectrum to identify electronic transitions for UVIR hole-burning

Wavenumbers (cm-1)

Inte

nsit

y (a

rbit

rary

uni

ts)

C7 C7C10 C5C5

WYp UVIR

Five N-H bonds one O-H bond Possible intramolecular interactions

Within backbone C5 C7 C10 Terminus NH2 sym and antisym Involving rings N-Hπ O-Hπ Other free N-H free O-H

Interactions cause shifts in IR spectrum

C7 or C10

IR Spectra of WYp

C5 or N-Hmiddotmiddotmiddotπ (one band)

C5 andor N-Hmiddotmiddotmiddotπ

(Two bands)

N-H of indole or antisym

C10 Bound O-H

C7

Conformer A

Conformers A + B

N-H Stretch Region O-H Stretch Region

Preliminary assignments based on previous results

Chin et al JACS (2005) 127 707

Chin et al JACS (2005) 127 11900

C7 C7

Initial Assignment of WYp

C10

Conformer A

Conformer B

(2) C7 C10

N-Hπ (2) N-Hπ

Free N-H of indole

Free N-H of indole

Bound O-H Bound O-H

Conformer A

Conformer B

Experimental amp Calculated Spectra

Conformer A

Conformers A amp B

Calculated 2Double C7M05-2X6-31gScaled by 09373

Calculated 1Straight ChainM05-2X6-31g (dp)Scaled by 09373

Calculations in progress

Results WYSp

Acetyl-Tryptophan(W)-Tyrosine(Y)-Serine(S)-Amide Same two UV chromophores

WYSp UVIR

Experimental

Red shifting due to H-bonds

Calculated 1M05-2X6-31g

Scaled by 09373NH2 sym NH2 antisym

O-H of Tyr

O-H of Ser

N-H of indole

(3) Backbone N-H

Calculations in progress

Conclusions

Presence of serine changes backbone configuration

Larger molecules tend to form fewer conformers Tryptamine = 7 WYp = 2 WYSp = 1

Peptide acts as a folding nucleus due to H-bonds

Ongoing and Future Work

Adapt ablation for Pittsburgh labs vibrationally and rotationally resolved spectroscopy

WYSLp Identified parent molecule via mass spec Signal did not last long amp high ablation power needed Significant fragmentation

Related Molecules [[[Tyr-Ser]-Leu]-Ala] (narrower spectrum without Trp)

Acknowledgements

Sophie Herbeaux (undergraduate Universiteacute Paris-Sud) for assistance in the lab

CEA (France) University of Pittsburgh and NSF for financial support of the project and my visit to the Laboratoire Francis Perrin

Tentative Assignment of WYSp

N-H of indole Bound

N-H backbone 3 Free

N-H backbone 2 N-Hπ w indole

N-H backbone 1 Free

O-H of Ser Doubly H-bonded

NH2 antisym -

O-H of Tyr H-bonded

Ablated WYSp Mass Spec

WYSp

WYSp ndash 30 amu

Oil in chamber

Smaller fragments

IonizationFluorescence Lab

Chambers Jet and MS

Dye Laser YAG Laser

PMT camera amp Monochrometer

Excimer LaserDye Laser

Mini YAG

Electronics

Computers amp Oscilloscope

Ablation

UV laser Chambers and Detection

Timing

Display and Storage

UVUV Hole Burning

3rd harmonic pumps coumarin 540A

General Valve nozzle 300 μm

~10-6 mbar

3 ndash 6 bar Ar

Motivation

Poupon and Mornon1 Folding Nuclei segments of proteins that fold first

Molinari and Coworkers2 Identified folding nuclei in β-lactoglobulin

What intermolecular forces are found in a folding nucleus (And what does this tell us about protein folding) Samples = segments of WYSLA from β-lactoglobulin

Acetyl- and ndashNH2 caps simulate longer chains

1 A Poupon and J-P Mornon FEBS Letters (1999) 452 283

2 L Ragona et al Biochemistry (2002) 41 2786

Experimental

Sample Preparation Laser ablation

Spectroscopic Methods UV ionization with mass spec

UVIR hole burning

S0

S1

IR

UV

Laser Ablation

Reduces decomposition of large biomolecules

Laser 2nd harmonic of NdYAG

Graphite matrix

UVIR Lab

MiniYAG

UV laser

Ablation

OPO YAG LaserIR laser

Prism creates second UV path through chamber

Excimer Laser Dye Laser

Chambers Jet and MS

Detection

Acetyl-Tryptophan(W)-Tyrosine(Y)-Amide Two UV chromophores

Results WYp

WYp Ionization

Trp gives broad UV absorption spectrum with sharp features

Use UV spectrum to identify electronic transitions for UVIR hole-burning

Wavenumbers (cm-1)

Inte

nsit

y (a

rbit

rary

uni

ts)

C7 C7C10 C5C5

WYp UVIR

Five N-H bonds one O-H bond Possible intramolecular interactions

Within backbone C5 C7 C10 Terminus NH2 sym and antisym Involving rings N-Hπ O-Hπ Other free N-H free O-H

Interactions cause shifts in IR spectrum

C7 or C10

IR Spectra of WYp

C5 or N-Hmiddotmiddotmiddotπ (one band)

C5 andor N-Hmiddotmiddotmiddotπ

(Two bands)

N-H of indole or antisym

C10 Bound O-H

C7

Conformer A

Conformers A + B

N-H Stretch Region O-H Stretch Region

Preliminary assignments based on previous results

Chin et al JACS (2005) 127 707

Chin et al JACS (2005) 127 11900

C7 C7

Initial Assignment of WYp

C10

Conformer A

Conformer B

(2) C7 C10

N-Hπ (2) N-Hπ

Free N-H of indole

Free N-H of indole

Bound O-H Bound O-H

Conformer A

Conformer B

Experimental amp Calculated Spectra

Conformer A

Conformers A amp B

Calculated 2Double C7M05-2X6-31gScaled by 09373

Calculated 1Straight ChainM05-2X6-31g (dp)Scaled by 09373

Calculations in progress

Results WYSp

Acetyl-Tryptophan(W)-Tyrosine(Y)-Serine(S)-Amide Same two UV chromophores

WYSp UVIR

Experimental

Red shifting due to H-bonds

Calculated 1M05-2X6-31g

Scaled by 09373NH2 sym NH2 antisym

O-H of Tyr

O-H of Ser

N-H of indole

(3) Backbone N-H

Calculations in progress

Conclusions

Presence of serine changes backbone configuration

Larger molecules tend to form fewer conformers Tryptamine = 7 WYp = 2 WYSp = 1

Peptide acts as a folding nucleus due to H-bonds

Ongoing and Future Work

Adapt ablation for Pittsburgh labs vibrationally and rotationally resolved spectroscopy

WYSLp Identified parent molecule via mass spec Signal did not last long amp high ablation power needed Significant fragmentation

Related Molecules [[[Tyr-Ser]-Leu]-Ala] (narrower spectrum without Trp)

Acknowledgements

Sophie Herbeaux (undergraduate Universiteacute Paris-Sud) for assistance in the lab

CEA (France) University of Pittsburgh and NSF for financial support of the project and my visit to the Laboratoire Francis Perrin

Tentative Assignment of WYSp

N-H of indole Bound

N-H backbone 3 Free

N-H backbone 2 N-Hπ w indole

N-H backbone 1 Free

O-H of Ser Doubly H-bonded

NH2 antisym -

O-H of Tyr H-bonded

Ablated WYSp Mass Spec

WYSp

WYSp ndash 30 amu

Oil in chamber

Smaller fragments

IonizationFluorescence Lab

Chambers Jet and MS

Dye Laser YAG Laser

PMT camera amp Monochrometer

Excimer LaserDye Laser

Mini YAG

Electronics

Computers amp Oscilloscope

Ablation

UV laser Chambers and Detection

Timing

Display and Storage

UVUV Hole Burning

3rd harmonic pumps coumarin 540A

General Valve nozzle 300 μm

~10-6 mbar

3 ndash 6 bar Ar

Experimental

Sample Preparation Laser ablation

Spectroscopic Methods UV ionization with mass spec

UVIR hole burning

S0

S1

IR

UV

Laser Ablation

Reduces decomposition of large biomolecules

Laser 2nd harmonic of NdYAG

Graphite matrix

UVIR Lab

MiniYAG

UV laser

Ablation

OPO YAG LaserIR laser

Prism creates second UV path through chamber

Excimer Laser Dye Laser

Chambers Jet and MS

Detection

Acetyl-Tryptophan(W)-Tyrosine(Y)-Amide Two UV chromophores

Results WYp

WYp Ionization

Trp gives broad UV absorption spectrum with sharp features

Use UV spectrum to identify electronic transitions for UVIR hole-burning

Wavenumbers (cm-1)

Inte

nsit

y (a

rbit

rary

uni

ts)

C7 C7C10 C5C5

WYp UVIR

Five N-H bonds one O-H bond Possible intramolecular interactions

Within backbone C5 C7 C10 Terminus NH2 sym and antisym Involving rings N-Hπ O-Hπ Other free N-H free O-H

Interactions cause shifts in IR spectrum

C7 or C10

IR Spectra of WYp

C5 or N-Hmiddotmiddotmiddotπ (one band)

C5 andor N-Hmiddotmiddotmiddotπ

(Two bands)

N-H of indole or antisym

C10 Bound O-H

C7

Conformer A

Conformers A + B

N-H Stretch Region O-H Stretch Region

Preliminary assignments based on previous results

Chin et al JACS (2005) 127 707

Chin et al JACS (2005) 127 11900

C7 C7

Initial Assignment of WYp

C10

Conformer A

Conformer B

(2) C7 C10

N-Hπ (2) N-Hπ

Free N-H of indole

Free N-H of indole

Bound O-H Bound O-H

Conformer A

Conformer B

Experimental amp Calculated Spectra

Conformer A

Conformers A amp B

Calculated 2Double C7M05-2X6-31gScaled by 09373

Calculated 1Straight ChainM05-2X6-31g (dp)Scaled by 09373

Calculations in progress

Results WYSp

Acetyl-Tryptophan(W)-Tyrosine(Y)-Serine(S)-Amide Same two UV chromophores

WYSp UVIR

Experimental

Red shifting due to H-bonds

Calculated 1M05-2X6-31g

Scaled by 09373NH2 sym NH2 antisym

O-H of Tyr

O-H of Ser

N-H of indole

(3) Backbone N-H

Calculations in progress

Conclusions

Presence of serine changes backbone configuration

Larger molecules tend to form fewer conformers Tryptamine = 7 WYp = 2 WYSp = 1

Peptide acts as a folding nucleus due to H-bonds

Ongoing and Future Work

Adapt ablation for Pittsburgh labs vibrationally and rotationally resolved spectroscopy

WYSLp Identified parent molecule via mass spec Signal did not last long amp high ablation power needed Significant fragmentation

Related Molecules [[[Tyr-Ser]-Leu]-Ala] (narrower spectrum without Trp)

Acknowledgements

Sophie Herbeaux (undergraduate Universiteacute Paris-Sud) for assistance in the lab

CEA (France) University of Pittsburgh and NSF for financial support of the project and my visit to the Laboratoire Francis Perrin

Tentative Assignment of WYSp

N-H of indole Bound

N-H backbone 3 Free

N-H backbone 2 N-Hπ w indole

N-H backbone 1 Free

O-H of Ser Doubly H-bonded

NH2 antisym -

O-H of Tyr H-bonded

Ablated WYSp Mass Spec

WYSp

WYSp ndash 30 amu

Oil in chamber

Smaller fragments

IonizationFluorescence Lab

Chambers Jet and MS

Dye Laser YAG Laser

PMT camera amp Monochrometer

Excimer LaserDye Laser

Mini YAG

Electronics

Computers amp Oscilloscope

Ablation

UV laser Chambers and Detection

Timing

Display and Storage

UVUV Hole Burning

3rd harmonic pumps coumarin 540A

General Valve nozzle 300 μm

~10-6 mbar

3 ndash 6 bar Ar

Laser Ablation

Reduces decomposition of large biomolecules

Laser 2nd harmonic of NdYAG

Graphite matrix

UVIR Lab

MiniYAG

UV laser

Ablation

OPO YAG LaserIR laser

Prism creates second UV path through chamber

Excimer Laser Dye Laser

Chambers Jet and MS

Detection

Acetyl-Tryptophan(W)-Tyrosine(Y)-Amide Two UV chromophores

Results WYp

WYp Ionization

Trp gives broad UV absorption spectrum with sharp features

Use UV spectrum to identify electronic transitions for UVIR hole-burning

Wavenumbers (cm-1)

Inte

nsit

y (a

rbit

rary

uni

ts)

C7 C7C10 C5C5

WYp UVIR

Five N-H bonds one O-H bond Possible intramolecular interactions

Within backbone C5 C7 C10 Terminus NH2 sym and antisym Involving rings N-Hπ O-Hπ Other free N-H free O-H

Interactions cause shifts in IR spectrum

C7 or C10

IR Spectra of WYp

C5 or N-Hmiddotmiddotmiddotπ (one band)

C5 andor N-Hmiddotmiddotmiddotπ

(Two bands)

N-H of indole or antisym

C10 Bound O-H

C7

Conformer A

Conformers A + B

N-H Stretch Region O-H Stretch Region

Preliminary assignments based on previous results

Chin et al JACS (2005) 127 707

Chin et al JACS (2005) 127 11900

C7 C7

Initial Assignment of WYp

C10

Conformer A

Conformer B

(2) C7 C10

N-Hπ (2) N-Hπ

Free N-H of indole

Free N-H of indole

Bound O-H Bound O-H

Conformer A

Conformer B

Experimental amp Calculated Spectra

Conformer A

Conformers A amp B

Calculated 2Double C7M05-2X6-31gScaled by 09373

Calculated 1Straight ChainM05-2X6-31g (dp)Scaled by 09373

Calculations in progress

Results WYSp

Acetyl-Tryptophan(W)-Tyrosine(Y)-Serine(S)-Amide Same two UV chromophores

WYSp UVIR

Experimental

Red shifting due to H-bonds

Calculated 1M05-2X6-31g

Scaled by 09373NH2 sym NH2 antisym

O-H of Tyr

O-H of Ser

N-H of indole

(3) Backbone N-H

Calculations in progress

Conclusions

Presence of serine changes backbone configuration

Larger molecules tend to form fewer conformers Tryptamine = 7 WYp = 2 WYSp = 1

Peptide acts as a folding nucleus due to H-bonds

Ongoing and Future Work

Adapt ablation for Pittsburgh labs vibrationally and rotationally resolved spectroscopy

WYSLp Identified parent molecule via mass spec Signal did not last long amp high ablation power needed Significant fragmentation

Related Molecules [[[Tyr-Ser]-Leu]-Ala] (narrower spectrum without Trp)

Acknowledgements

Sophie Herbeaux (undergraduate Universiteacute Paris-Sud) for assistance in the lab

CEA (France) University of Pittsburgh and NSF for financial support of the project and my visit to the Laboratoire Francis Perrin

Tentative Assignment of WYSp

N-H of indole Bound

N-H backbone 3 Free

N-H backbone 2 N-Hπ w indole

N-H backbone 1 Free

O-H of Ser Doubly H-bonded

NH2 antisym -

O-H of Tyr H-bonded

Ablated WYSp Mass Spec

WYSp

WYSp ndash 30 amu

Oil in chamber

Smaller fragments

IonizationFluorescence Lab

Chambers Jet and MS

Dye Laser YAG Laser

PMT camera amp Monochrometer

Excimer LaserDye Laser

Mini YAG

Electronics

Computers amp Oscilloscope

Ablation

UV laser Chambers and Detection

Timing

Display and Storage

UVUV Hole Burning

3rd harmonic pumps coumarin 540A

General Valve nozzle 300 μm

~10-6 mbar

3 ndash 6 bar Ar

UVIR Lab

MiniYAG

UV laser

Ablation

OPO YAG LaserIR laser

Prism creates second UV path through chamber

Excimer Laser Dye Laser

Chambers Jet and MS

Detection

Acetyl-Tryptophan(W)-Tyrosine(Y)-Amide Two UV chromophores

Results WYp

WYp Ionization

Trp gives broad UV absorption spectrum with sharp features

Use UV spectrum to identify electronic transitions for UVIR hole-burning

Wavenumbers (cm-1)

Inte

nsit

y (a

rbit

rary

uni

ts)

C7 C7C10 C5C5

WYp UVIR

Five N-H bonds one O-H bond Possible intramolecular interactions

Within backbone C5 C7 C10 Terminus NH2 sym and antisym Involving rings N-Hπ O-Hπ Other free N-H free O-H

Interactions cause shifts in IR spectrum

C7 or C10

IR Spectra of WYp

C5 or N-Hmiddotmiddotmiddotπ (one band)

C5 andor N-Hmiddotmiddotmiddotπ

(Two bands)

N-H of indole or antisym

C10 Bound O-H

C7

Conformer A

Conformers A + B

N-H Stretch Region O-H Stretch Region

Preliminary assignments based on previous results

Chin et al JACS (2005) 127 707

Chin et al JACS (2005) 127 11900

C7 C7

Initial Assignment of WYp

C10

Conformer A

Conformer B

(2) C7 C10

N-Hπ (2) N-Hπ

Free N-H of indole

Free N-H of indole

Bound O-H Bound O-H

Conformer A

Conformer B

Experimental amp Calculated Spectra

Conformer A

Conformers A amp B

Calculated 2Double C7M05-2X6-31gScaled by 09373

Calculated 1Straight ChainM05-2X6-31g (dp)Scaled by 09373

Calculations in progress

Results WYSp

Acetyl-Tryptophan(W)-Tyrosine(Y)-Serine(S)-Amide Same two UV chromophores

WYSp UVIR

Experimental

Red shifting due to H-bonds

Calculated 1M05-2X6-31g

Scaled by 09373NH2 sym NH2 antisym

O-H of Tyr

O-H of Ser

N-H of indole

(3) Backbone N-H

Calculations in progress

Conclusions

Presence of serine changes backbone configuration

Larger molecules tend to form fewer conformers Tryptamine = 7 WYp = 2 WYSp = 1

Peptide acts as a folding nucleus due to H-bonds

Ongoing and Future Work

Adapt ablation for Pittsburgh labs vibrationally and rotationally resolved spectroscopy

WYSLp Identified parent molecule via mass spec Signal did not last long amp high ablation power needed Significant fragmentation

Related Molecules [[[Tyr-Ser]-Leu]-Ala] (narrower spectrum without Trp)

Acknowledgements

Sophie Herbeaux (undergraduate Universiteacute Paris-Sud) for assistance in the lab

CEA (France) University of Pittsburgh and NSF for financial support of the project and my visit to the Laboratoire Francis Perrin

Tentative Assignment of WYSp

N-H of indole Bound

N-H backbone 3 Free

N-H backbone 2 N-Hπ w indole

N-H backbone 1 Free

O-H of Ser Doubly H-bonded

NH2 antisym -

O-H of Tyr H-bonded

Ablated WYSp Mass Spec

WYSp

WYSp ndash 30 amu

Oil in chamber

Smaller fragments

IonizationFluorescence Lab

Chambers Jet and MS

Dye Laser YAG Laser

PMT camera amp Monochrometer

Excimer LaserDye Laser

Mini YAG

Electronics

Computers amp Oscilloscope

Ablation

UV laser Chambers and Detection

Timing

Display and Storage

UVUV Hole Burning

3rd harmonic pumps coumarin 540A

General Valve nozzle 300 μm

~10-6 mbar

3 ndash 6 bar Ar

Acetyl-Tryptophan(W)-Tyrosine(Y)-Amide Two UV chromophores

Results WYp

WYp Ionization

Trp gives broad UV absorption spectrum with sharp features

Use UV spectrum to identify electronic transitions for UVIR hole-burning

Wavenumbers (cm-1)

Inte

nsit

y (a

rbit

rary

uni

ts)

C7 C7C10 C5C5

WYp UVIR

Five N-H bonds one O-H bond Possible intramolecular interactions

Within backbone C5 C7 C10 Terminus NH2 sym and antisym Involving rings N-Hπ O-Hπ Other free N-H free O-H

Interactions cause shifts in IR spectrum

C7 or C10

IR Spectra of WYp

C5 or N-Hmiddotmiddotmiddotπ (one band)

C5 andor N-Hmiddotmiddotmiddotπ

(Two bands)

N-H of indole or antisym

C10 Bound O-H

C7

Conformer A

Conformers A + B

N-H Stretch Region O-H Stretch Region

Preliminary assignments based on previous results

Chin et al JACS (2005) 127 707

Chin et al JACS (2005) 127 11900

C7 C7

Initial Assignment of WYp

C10

Conformer A

Conformer B

(2) C7 C10

N-Hπ (2) N-Hπ

Free N-H of indole

Free N-H of indole

Bound O-H Bound O-H

Conformer A

Conformer B

Experimental amp Calculated Spectra

Conformer A

Conformers A amp B

Calculated 2Double C7M05-2X6-31gScaled by 09373

Calculated 1Straight ChainM05-2X6-31g (dp)Scaled by 09373

Calculations in progress

Results WYSp

Acetyl-Tryptophan(W)-Tyrosine(Y)-Serine(S)-Amide Same two UV chromophores

WYSp UVIR

Experimental

Red shifting due to H-bonds

Calculated 1M05-2X6-31g

Scaled by 09373NH2 sym NH2 antisym

O-H of Tyr

O-H of Ser

N-H of indole

(3) Backbone N-H

Calculations in progress

Conclusions

Presence of serine changes backbone configuration

Larger molecules tend to form fewer conformers Tryptamine = 7 WYp = 2 WYSp = 1

Peptide acts as a folding nucleus due to H-bonds

Ongoing and Future Work

Adapt ablation for Pittsburgh labs vibrationally and rotationally resolved spectroscopy

WYSLp Identified parent molecule via mass spec Signal did not last long amp high ablation power needed Significant fragmentation

Related Molecules [[[Tyr-Ser]-Leu]-Ala] (narrower spectrum without Trp)

Acknowledgements

Sophie Herbeaux (undergraduate Universiteacute Paris-Sud) for assistance in the lab

CEA (France) University of Pittsburgh and NSF for financial support of the project and my visit to the Laboratoire Francis Perrin

Tentative Assignment of WYSp

N-H of indole Bound

N-H backbone 3 Free

N-H backbone 2 N-Hπ w indole

N-H backbone 1 Free

O-H of Ser Doubly H-bonded

NH2 antisym -

O-H of Tyr H-bonded

Ablated WYSp Mass Spec

WYSp

WYSp ndash 30 amu

Oil in chamber

Smaller fragments

IonizationFluorescence Lab

Chambers Jet and MS

Dye Laser YAG Laser

PMT camera amp Monochrometer

Excimer LaserDye Laser

Mini YAG

Electronics

Computers amp Oscilloscope

Ablation

UV laser Chambers and Detection

Timing

Display and Storage

UVUV Hole Burning

3rd harmonic pumps coumarin 540A

General Valve nozzle 300 μm

~10-6 mbar

3 ndash 6 bar Ar

WYp Ionization

Trp gives broad UV absorption spectrum with sharp features

Use UV spectrum to identify electronic transitions for UVIR hole-burning

Wavenumbers (cm-1)

Inte

nsit

y (a

rbit

rary

uni

ts)

C7 C7C10 C5C5

WYp UVIR

Five N-H bonds one O-H bond Possible intramolecular interactions

Within backbone C5 C7 C10 Terminus NH2 sym and antisym Involving rings N-Hπ O-Hπ Other free N-H free O-H

Interactions cause shifts in IR spectrum

C7 or C10

IR Spectra of WYp

C5 or N-Hmiddotmiddotmiddotπ (one band)

C5 andor N-Hmiddotmiddotmiddotπ

(Two bands)

N-H of indole or antisym

C10 Bound O-H

C7

Conformer A

Conformers A + B

N-H Stretch Region O-H Stretch Region

Preliminary assignments based on previous results

Chin et al JACS (2005) 127 707

Chin et al JACS (2005) 127 11900

C7 C7

Initial Assignment of WYp

C10

Conformer A

Conformer B

(2) C7 C10

N-Hπ (2) N-Hπ

Free N-H of indole

Free N-H of indole

Bound O-H Bound O-H

Conformer A

Conformer B

Experimental amp Calculated Spectra

Conformer A

Conformers A amp B

Calculated 2Double C7M05-2X6-31gScaled by 09373

Calculated 1Straight ChainM05-2X6-31g (dp)Scaled by 09373

Calculations in progress

Results WYSp

Acetyl-Tryptophan(W)-Tyrosine(Y)-Serine(S)-Amide Same two UV chromophores

WYSp UVIR

Experimental

Red shifting due to H-bonds

Calculated 1M05-2X6-31g

Scaled by 09373NH2 sym NH2 antisym

O-H of Tyr

O-H of Ser

N-H of indole

(3) Backbone N-H

Calculations in progress

Conclusions

Presence of serine changes backbone configuration

Larger molecules tend to form fewer conformers Tryptamine = 7 WYp = 2 WYSp = 1

Peptide acts as a folding nucleus due to H-bonds

Ongoing and Future Work

Adapt ablation for Pittsburgh labs vibrationally and rotationally resolved spectroscopy

WYSLp Identified parent molecule via mass spec Signal did not last long amp high ablation power needed Significant fragmentation

Related Molecules [[[Tyr-Ser]-Leu]-Ala] (narrower spectrum without Trp)

Acknowledgements

Sophie Herbeaux (undergraduate Universiteacute Paris-Sud) for assistance in the lab

CEA (France) University of Pittsburgh and NSF for financial support of the project and my visit to the Laboratoire Francis Perrin

Tentative Assignment of WYSp

N-H of indole Bound

N-H backbone 3 Free

N-H backbone 2 N-Hπ w indole

N-H backbone 1 Free

O-H of Ser Doubly H-bonded

NH2 antisym -

O-H of Tyr H-bonded

Ablated WYSp Mass Spec

WYSp

WYSp ndash 30 amu

Oil in chamber

Smaller fragments

IonizationFluorescence Lab

Chambers Jet and MS

Dye Laser YAG Laser

PMT camera amp Monochrometer

Excimer LaserDye Laser

Mini YAG

Electronics

Computers amp Oscilloscope

Ablation

UV laser Chambers and Detection

Timing

Display and Storage

UVUV Hole Burning

3rd harmonic pumps coumarin 540A

General Valve nozzle 300 μm

~10-6 mbar

3 ndash 6 bar Ar

C7 C7C10 C5C5

WYp UVIR

Five N-H bonds one O-H bond Possible intramolecular interactions

Within backbone C5 C7 C10 Terminus NH2 sym and antisym Involving rings N-Hπ O-Hπ Other free N-H free O-H

Interactions cause shifts in IR spectrum

C7 or C10

IR Spectra of WYp

C5 or N-Hmiddotmiddotmiddotπ (one band)

C5 andor N-Hmiddotmiddotmiddotπ

(Two bands)

N-H of indole or antisym

C10 Bound O-H

C7

Conformer A

Conformers A + B

N-H Stretch Region O-H Stretch Region

Preliminary assignments based on previous results

Chin et al JACS (2005) 127 707

Chin et al JACS (2005) 127 11900

C7 C7

Initial Assignment of WYp

C10

Conformer A

Conformer B

(2) C7 C10

N-Hπ (2) N-Hπ

Free N-H of indole

Free N-H of indole

Bound O-H Bound O-H

Conformer A

Conformer B

Experimental amp Calculated Spectra

Conformer A

Conformers A amp B

Calculated 2Double C7M05-2X6-31gScaled by 09373

Calculated 1Straight ChainM05-2X6-31g (dp)Scaled by 09373

Calculations in progress

Results WYSp

Acetyl-Tryptophan(W)-Tyrosine(Y)-Serine(S)-Amide Same two UV chromophores

WYSp UVIR

Experimental

Red shifting due to H-bonds

Calculated 1M05-2X6-31g

Scaled by 09373NH2 sym NH2 antisym

O-H of Tyr

O-H of Ser

N-H of indole

(3) Backbone N-H

Calculations in progress

Conclusions

Presence of serine changes backbone configuration

Larger molecules tend to form fewer conformers Tryptamine = 7 WYp = 2 WYSp = 1

Peptide acts as a folding nucleus due to H-bonds

Ongoing and Future Work

Adapt ablation for Pittsburgh labs vibrationally and rotationally resolved spectroscopy

WYSLp Identified parent molecule via mass spec Signal did not last long amp high ablation power needed Significant fragmentation

Related Molecules [[[Tyr-Ser]-Leu]-Ala] (narrower spectrum without Trp)

Acknowledgements

Sophie Herbeaux (undergraduate Universiteacute Paris-Sud) for assistance in the lab

CEA (France) University of Pittsburgh and NSF for financial support of the project and my visit to the Laboratoire Francis Perrin

Tentative Assignment of WYSp

N-H of indole Bound

N-H backbone 3 Free

N-H backbone 2 N-Hπ w indole

N-H backbone 1 Free

O-H of Ser Doubly H-bonded

NH2 antisym -

O-H of Tyr H-bonded

Ablated WYSp Mass Spec

WYSp

WYSp ndash 30 amu

Oil in chamber

Smaller fragments

IonizationFluorescence Lab

Chambers Jet and MS

Dye Laser YAG Laser

PMT camera amp Monochrometer

Excimer LaserDye Laser

Mini YAG

Electronics

Computers amp Oscilloscope

Ablation

UV laser Chambers and Detection

Timing

Display and Storage

UVUV Hole Burning

3rd harmonic pumps coumarin 540A

General Valve nozzle 300 μm

~10-6 mbar

3 ndash 6 bar Ar

C7 or C10

IR Spectra of WYp

C5 or N-Hmiddotmiddotmiddotπ (one band)

C5 andor N-Hmiddotmiddotmiddotπ

(Two bands)

N-H of indole or antisym

C10 Bound O-H

C7

Conformer A

Conformers A + B

N-H Stretch Region O-H Stretch Region

Preliminary assignments based on previous results

Chin et al JACS (2005) 127 707

Chin et al JACS (2005) 127 11900

C7 C7

Initial Assignment of WYp

C10

Conformer A

Conformer B

(2) C7 C10

N-Hπ (2) N-Hπ

Free N-H of indole

Free N-H of indole

Bound O-H Bound O-H

Conformer A

Conformer B

Experimental amp Calculated Spectra

Conformer A

Conformers A amp B

Calculated 2Double C7M05-2X6-31gScaled by 09373

Calculated 1Straight ChainM05-2X6-31g (dp)Scaled by 09373

Calculations in progress

Results WYSp

Acetyl-Tryptophan(W)-Tyrosine(Y)-Serine(S)-Amide Same two UV chromophores

WYSp UVIR

Experimental

Red shifting due to H-bonds

Calculated 1M05-2X6-31g

Scaled by 09373NH2 sym NH2 antisym

O-H of Tyr

O-H of Ser

N-H of indole

(3) Backbone N-H

Calculations in progress

Conclusions

Presence of serine changes backbone configuration

Larger molecules tend to form fewer conformers Tryptamine = 7 WYp = 2 WYSp = 1

Peptide acts as a folding nucleus due to H-bonds

Ongoing and Future Work

Adapt ablation for Pittsburgh labs vibrationally and rotationally resolved spectroscopy

WYSLp Identified parent molecule via mass spec Signal did not last long amp high ablation power needed Significant fragmentation

Related Molecules [[[Tyr-Ser]-Leu]-Ala] (narrower spectrum without Trp)

Acknowledgements

Sophie Herbeaux (undergraduate Universiteacute Paris-Sud) for assistance in the lab

CEA (France) University of Pittsburgh and NSF for financial support of the project and my visit to the Laboratoire Francis Perrin

Tentative Assignment of WYSp

N-H of indole Bound

N-H backbone 3 Free

N-H backbone 2 N-Hπ w indole

N-H backbone 1 Free

O-H of Ser Doubly H-bonded

NH2 antisym -

O-H of Tyr H-bonded

Ablated WYSp Mass Spec

WYSp

WYSp ndash 30 amu

Oil in chamber

Smaller fragments

IonizationFluorescence Lab

Chambers Jet and MS

Dye Laser YAG Laser

PMT camera amp Monochrometer

Excimer LaserDye Laser

Mini YAG

Electronics

Computers amp Oscilloscope

Ablation

UV laser Chambers and Detection

Timing

Display and Storage

UVUV Hole Burning

3rd harmonic pumps coumarin 540A

General Valve nozzle 300 μm

~10-6 mbar

3 ndash 6 bar Ar

C7 C7

Initial Assignment of WYp

C10

Conformer A

Conformer B

(2) C7 C10

N-Hπ (2) N-Hπ

Free N-H of indole

Free N-H of indole

Bound O-H Bound O-H

Conformer A

Conformer B

Experimental amp Calculated Spectra

Conformer A

Conformers A amp B

Calculated 2Double C7M05-2X6-31gScaled by 09373

Calculated 1Straight ChainM05-2X6-31g (dp)Scaled by 09373

Calculations in progress

Results WYSp

Acetyl-Tryptophan(W)-Tyrosine(Y)-Serine(S)-Amide Same two UV chromophores

WYSp UVIR

Experimental

Red shifting due to H-bonds

Calculated 1M05-2X6-31g

Scaled by 09373NH2 sym NH2 antisym

O-H of Tyr

O-H of Ser

N-H of indole

(3) Backbone N-H

Calculations in progress

Conclusions

Presence of serine changes backbone configuration

Larger molecules tend to form fewer conformers Tryptamine = 7 WYp = 2 WYSp = 1

Peptide acts as a folding nucleus due to H-bonds

Ongoing and Future Work

Adapt ablation for Pittsburgh labs vibrationally and rotationally resolved spectroscopy

WYSLp Identified parent molecule via mass spec Signal did not last long amp high ablation power needed Significant fragmentation

Related Molecules [[[Tyr-Ser]-Leu]-Ala] (narrower spectrum without Trp)

Acknowledgements

Sophie Herbeaux (undergraduate Universiteacute Paris-Sud) for assistance in the lab

CEA (France) University of Pittsburgh and NSF for financial support of the project and my visit to the Laboratoire Francis Perrin

Tentative Assignment of WYSp

N-H of indole Bound

N-H backbone 3 Free

N-H backbone 2 N-Hπ w indole

N-H backbone 1 Free

O-H of Ser Doubly H-bonded

NH2 antisym -

O-H of Tyr H-bonded

Ablated WYSp Mass Spec

WYSp

WYSp ndash 30 amu

Oil in chamber

Smaller fragments

IonizationFluorescence Lab

Chambers Jet and MS

Dye Laser YAG Laser

PMT camera amp Monochrometer

Excimer LaserDye Laser

Mini YAG

Electronics

Computers amp Oscilloscope

Ablation

UV laser Chambers and Detection

Timing

Display and Storage

UVUV Hole Burning

3rd harmonic pumps coumarin 540A

General Valve nozzle 300 μm

~10-6 mbar

3 ndash 6 bar Ar

Experimental amp Calculated Spectra

Conformer A

Conformers A amp B

Calculated 2Double C7M05-2X6-31gScaled by 09373

Calculated 1Straight ChainM05-2X6-31g (dp)Scaled by 09373

Calculations in progress

Results WYSp

Acetyl-Tryptophan(W)-Tyrosine(Y)-Serine(S)-Amide Same two UV chromophores

WYSp UVIR

Experimental

Red shifting due to H-bonds

Calculated 1M05-2X6-31g

Scaled by 09373NH2 sym NH2 antisym

O-H of Tyr

O-H of Ser

N-H of indole

(3) Backbone N-H

Calculations in progress

Conclusions

Presence of serine changes backbone configuration

Larger molecules tend to form fewer conformers Tryptamine = 7 WYp = 2 WYSp = 1

Peptide acts as a folding nucleus due to H-bonds

Ongoing and Future Work

Adapt ablation for Pittsburgh labs vibrationally and rotationally resolved spectroscopy

WYSLp Identified parent molecule via mass spec Signal did not last long amp high ablation power needed Significant fragmentation

Related Molecules [[[Tyr-Ser]-Leu]-Ala] (narrower spectrum without Trp)

Acknowledgements

Sophie Herbeaux (undergraduate Universiteacute Paris-Sud) for assistance in the lab

CEA (France) University of Pittsburgh and NSF for financial support of the project and my visit to the Laboratoire Francis Perrin

Tentative Assignment of WYSp

N-H of indole Bound

N-H backbone 3 Free

N-H backbone 2 N-Hπ w indole

N-H backbone 1 Free

O-H of Ser Doubly H-bonded

NH2 antisym -

O-H of Tyr H-bonded

Ablated WYSp Mass Spec

WYSp

WYSp ndash 30 amu

Oil in chamber

Smaller fragments

IonizationFluorescence Lab

Chambers Jet and MS

Dye Laser YAG Laser

PMT camera amp Monochrometer

Excimer LaserDye Laser

Mini YAG

Electronics

Computers amp Oscilloscope

Ablation

UV laser Chambers and Detection

Timing

Display and Storage

UVUV Hole Burning

3rd harmonic pumps coumarin 540A

General Valve nozzle 300 μm

~10-6 mbar

3 ndash 6 bar Ar

Results WYSp

Acetyl-Tryptophan(W)-Tyrosine(Y)-Serine(S)-Amide Same two UV chromophores

WYSp UVIR

Experimental

Red shifting due to H-bonds

Calculated 1M05-2X6-31g

Scaled by 09373NH2 sym NH2 antisym

O-H of Tyr

O-H of Ser

N-H of indole

(3) Backbone N-H

Calculations in progress

Conclusions

Presence of serine changes backbone configuration

Larger molecules tend to form fewer conformers Tryptamine = 7 WYp = 2 WYSp = 1

Peptide acts as a folding nucleus due to H-bonds

Ongoing and Future Work

Adapt ablation for Pittsburgh labs vibrationally and rotationally resolved spectroscopy

WYSLp Identified parent molecule via mass spec Signal did not last long amp high ablation power needed Significant fragmentation

Related Molecules [[[Tyr-Ser]-Leu]-Ala] (narrower spectrum without Trp)

Acknowledgements

Sophie Herbeaux (undergraduate Universiteacute Paris-Sud) for assistance in the lab

CEA (France) University of Pittsburgh and NSF for financial support of the project and my visit to the Laboratoire Francis Perrin

Tentative Assignment of WYSp

N-H of indole Bound

N-H backbone 3 Free

N-H backbone 2 N-Hπ w indole

N-H backbone 1 Free

O-H of Ser Doubly H-bonded

NH2 antisym -

O-H of Tyr H-bonded

Ablated WYSp Mass Spec

WYSp

WYSp ndash 30 amu

Oil in chamber

Smaller fragments

IonizationFluorescence Lab

Chambers Jet and MS

Dye Laser YAG Laser

PMT camera amp Monochrometer

Excimer LaserDye Laser

Mini YAG

Electronics

Computers amp Oscilloscope

Ablation

UV laser Chambers and Detection

Timing

Display and Storage

UVUV Hole Burning

3rd harmonic pumps coumarin 540A

General Valve nozzle 300 μm

~10-6 mbar

3 ndash 6 bar Ar

WYSp UVIR

Experimental

Red shifting due to H-bonds

Calculated 1M05-2X6-31g

Scaled by 09373NH2 sym NH2 antisym

O-H of Tyr

O-H of Ser

N-H of indole

(3) Backbone N-H

Calculations in progress

Conclusions

Presence of serine changes backbone configuration

Larger molecules tend to form fewer conformers Tryptamine = 7 WYp = 2 WYSp = 1

Peptide acts as a folding nucleus due to H-bonds

Ongoing and Future Work

Adapt ablation for Pittsburgh labs vibrationally and rotationally resolved spectroscopy

WYSLp Identified parent molecule via mass spec Signal did not last long amp high ablation power needed Significant fragmentation

Related Molecules [[[Tyr-Ser]-Leu]-Ala] (narrower spectrum without Trp)

Acknowledgements

Sophie Herbeaux (undergraduate Universiteacute Paris-Sud) for assistance in the lab

CEA (France) University of Pittsburgh and NSF for financial support of the project and my visit to the Laboratoire Francis Perrin

Tentative Assignment of WYSp

N-H of indole Bound

N-H backbone 3 Free

N-H backbone 2 N-Hπ w indole

N-H backbone 1 Free

O-H of Ser Doubly H-bonded

NH2 antisym -

O-H of Tyr H-bonded

Ablated WYSp Mass Spec

WYSp

WYSp ndash 30 amu

Oil in chamber

Smaller fragments

IonizationFluorescence Lab

Chambers Jet and MS

Dye Laser YAG Laser

PMT camera amp Monochrometer

Excimer LaserDye Laser

Mini YAG

Electronics

Computers amp Oscilloscope

Ablation

UV laser Chambers and Detection

Timing

Display and Storage

UVUV Hole Burning

3rd harmonic pumps coumarin 540A

General Valve nozzle 300 μm

~10-6 mbar

3 ndash 6 bar Ar

Conclusions

Presence of serine changes backbone configuration

Larger molecules tend to form fewer conformers Tryptamine = 7 WYp = 2 WYSp = 1

Peptide acts as a folding nucleus due to H-bonds

Ongoing and Future Work

Adapt ablation for Pittsburgh labs vibrationally and rotationally resolved spectroscopy

WYSLp Identified parent molecule via mass spec Signal did not last long amp high ablation power needed Significant fragmentation

Related Molecules [[[Tyr-Ser]-Leu]-Ala] (narrower spectrum without Trp)

Acknowledgements

Sophie Herbeaux (undergraduate Universiteacute Paris-Sud) for assistance in the lab

CEA (France) University of Pittsburgh and NSF for financial support of the project and my visit to the Laboratoire Francis Perrin

Tentative Assignment of WYSp

N-H of indole Bound

N-H backbone 3 Free

N-H backbone 2 N-Hπ w indole

N-H backbone 1 Free

O-H of Ser Doubly H-bonded

NH2 antisym -

O-H of Tyr H-bonded

Ablated WYSp Mass Spec

WYSp

WYSp ndash 30 amu

Oil in chamber

Smaller fragments

IonizationFluorescence Lab

Chambers Jet and MS

Dye Laser YAG Laser

PMT camera amp Monochrometer

Excimer LaserDye Laser

Mini YAG

Electronics

Computers amp Oscilloscope

Ablation

UV laser Chambers and Detection

Timing

Display and Storage

UVUV Hole Burning

3rd harmonic pumps coumarin 540A

General Valve nozzle 300 μm

~10-6 mbar

3 ndash 6 bar Ar

Ongoing and Future Work

Adapt ablation for Pittsburgh labs vibrationally and rotationally resolved spectroscopy

WYSLp Identified parent molecule via mass spec Signal did not last long amp high ablation power needed Significant fragmentation

Related Molecules [[[Tyr-Ser]-Leu]-Ala] (narrower spectrum without Trp)

Acknowledgements

Sophie Herbeaux (undergraduate Universiteacute Paris-Sud) for assistance in the lab

CEA (France) University of Pittsburgh and NSF for financial support of the project and my visit to the Laboratoire Francis Perrin

Tentative Assignment of WYSp

N-H of indole Bound

N-H backbone 3 Free

N-H backbone 2 N-Hπ w indole

N-H backbone 1 Free

O-H of Ser Doubly H-bonded

NH2 antisym -

O-H of Tyr H-bonded

Ablated WYSp Mass Spec

WYSp

WYSp ndash 30 amu

Oil in chamber

Smaller fragments

IonizationFluorescence Lab

Chambers Jet and MS

Dye Laser YAG Laser

PMT camera amp Monochrometer

Excimer LaserDye Laser

Mini YAG

Electronics

Computers amp Oscilloscope

Ablation

UV laser Chambers and Detection

Timing

Display and Storage

UVUV Hole Burning

3rd harmonic pumps coumarin 540A

General Valve nozzle 300 μm

~10-6 mbar

3 ndash 6 bar Ar

Acknowledgements

Sophie Herbeaux (undergraduate Universiteacute Paris-Sud) for assistance in the lab

CEA (France) University of Pittsburgh and NSF for financial support of the project and my visit to the Laboratoire Francis Perrin

Tentative Assignment of WYSp

N-H of indole Bound

N-H backbone 3 Free

N-H backbone 2 N-Hπ w indole

N-H backbone 1 Free

O-H of Ser Doubly H-bonded

NH2 antisym -

O-H of Tyr H-bonded

Ablated WYSp Mass Spec

WYSp

WYSp ndash 30 amu

Oil in chamber

Smaller fragments

IonizationFluorescence Lab

Chambers Jet and MS

Dye Laser YAG Laser

PMT camera amp Monochrometer

Excimer LaserDye Laser

Mini YAG

Electronics

Computers amp Oscilloscope

Ablation

UV laser Chambers and Detection

Timing

Display and Storage

UVUV Hole Burning

3rd harmonic pumps coumarin 540A

General Valve nozzle 300 μm

~10-6 mbar

3 ndash 6 bar Ar

Tentative Assignment of WYSp

N-H of indole Bound

N-H backbone 3 Free

N-H backbone 2 N-Hπ w indole

N-H backbone 1 Free

O-H of Ser Doubly H-bonded

NH2 antisym -

O-H of Tyr H-bonded

Ablated WYSp Mass Spec

WYSp

WYSp ndash 30 amu

Oil in chamber

Smaller fragments

IonizationFluorescence Lab

Chambers Jet and MS

Dye Laser YAG Laser

PMT camera amp Monochrometer

Excimer LaserDye Laser

Mini YAG

Electronics

Computers amp Oscilloscope

Ablation

UV laser Chambers and Detection

Timing

Display and Storage

UVUV Hole Burning

3rd harmonic pumps coumarin 540A

General Valve nozzle 300 μm

~10-6 mbar

3 ndash 6 bar Ar

Ablated WYSp Mass Spec

WYSp

WYSp ndash 30 amu

Oil in chamber

Smaller fragments

IonizationFluorescence Lab

Chambers Jet and MS

Dye Laser YAG Laser

PMT camera amp Monochrometer

Excimer LaserDye Laser

Mini YAG

Electronics

Computers amp Oscilloscope

Ablation

UV laser Chambers and Detection

Timing

Display and Storage

UVUV Hole Burning

3rd harmonic pumps coumarin 540A

General Valve nozzle 300 μm

~10-6 mbar

3 ndash 6 bar Ar

IonizationFluorescence Lab

Chambers Jet and MS

Dye Laser YAG Laser

PMT camera amp Monochrometer

Excimer LaserDye Laser

Mini YAG

Electronics

Computers amp Oscilloscope

Ablation

UV laser Chambers and Detection

Timing

Display and Storage

UVUV Hole Burning

3rd harmonic pumps coumarin 540A

General Valve nozzle 300 μm

~10-6 mbar

3 ndash 6 bar Ar