Aloke Das

Indian Institute of Science Education and Research, Pune

Mimicking trimeric interactions in the aromatic side chains of the proteins: A gas phase study of indole...(pyrrole)2 heterotrimer

Introduction

Polypeptide (Building block of proteins)

Backbone

Side chain

Phenylalanine Tryptophan Tyrosine Histidine

Four aromatic amino acid residues

Introduction S. K. Burley and G. A. Petsko; Science 229, 23 (1985):

“About 60 percent of the aromatic side chains in proteins are involved in aromatic pairs, 80 percent of which form networks of three or more interacting aromatic side chains”

Higher order aromatic clusters (trimers, tetramers etc.) are present in about 50% of the proteins, which have been crystallized to date.

Analysis of high resolution crystal structures of 34 proteins

Detailed survey of the crystal structures in the Protein Data Bank (PDB). Total number of the protein structures studied were 18547

Lanzarotti et al.: J. Chem. Inf. Model. 51, 1623 (2011):

Example:

Protein: L-ribulose-5-phosphate 4-epimerase (PDB ID: 1JDI) Trp…Phe…Phe

Motivation

Aromatic trimeric interactions are very important in

(i)Stabilization of protein structures(ii)Protein-protein recognition(iii)Protein-ligand binding

Literature study of the aromatic-aromatic interactions at the molecular level beyond the dimer is limited to mostly aromatic hydrocarbons.

Most extensive aromatic trimeric study:

Benzene trimer(Cyclic symmetric structure)

Model for trimeric

interactions amongphenylalanine residues in proteins

Phenylalanine

JCP 85, 3739 (1981)JCP 110, 5758 (1999)JCP 98, 8361 (1993)JPCA 105, 1904 (2001)JPCA 109, 10475 (2005)

There are no study on the molecular level interactions in the aromatic trimers containing indole and imidazole, which are present in the side chains of tryptophan and histidine residues, respectively!!!

Motivation

Indole…(pyrrole)2 trimer

Tryptophan is the most effective π-hydrogen bond acceptors among all the amino acid residues in the proteins. [Review of π-hydrogen bonding in proteins based on 593 crystal structures, Steiner and Koellner, J. Mol. Biol. 305, 535 (2001)]

Study of this heterotrimer is quite interesting

Effect of asymmetry in the cyclic trimeric structure

π-hydrogen bonding is generally the backbone of trimericaromatic interactions in the proteins

Trp Phe Tyr His

With any N-H, O-H, S-H 17.6 5.8 8.8 0.7

With peptide N-H 3.2 0.9 1.6 0.3

With side chain N-H 4.5 1.0 1.6 0.4

With side chain O-H 0.6 0.2 0.4 -

With Cα-H 14.2 7.5 8.3 4.8

Efficiencies of π-hydrogen bond acceptors in the proteins (JMB 305, 535, 2001)

Motivation

Indole…(pyrrole)2 trimer

Gas phase studies for the determination of π-hydrogen bond accepting strength of indole is scarce in the literature.

Indole complexes studied as a π-hydrogen bond donor:

Indole...benzene(JPCA 115, 9485, 2011)

Indole...furan(JPCA 116, 1368, 2012)

Indole...pyrrole(IJQM 92, 516, 2003)

Pyrrole...benzene(PCCP 13, 14110, 2011)

Present study

Indole as both π-hydrogen bond acceptor and donor

Experimental setup and theoretical methods

Home-built Jet-cooled Laser Desorption REMPI (Resonantly Enhanced Multiphoton Ionization)Time OF Flight Mass Spectrometer

UV/VIS Laser(Nd:YAG pumped dye laser)

IR Laser(Nd:YAG pumped IR OPO)

Theory: Dispersion corrected DFT calculations using M05-2X, M06-2X, DFT-D functionals (Gaussian09 software).

Electronic spectra of Indole…(pyrrole)2 trimer

35250352003515035100

(a)

(b)

000

(352

40 c

m-1

)

(351

04 c

m-1

)

000 23

85

28

40 51

36 63.5

Wavenumber (cm-1

)

(c)

000

S0

S1

35240 cm-1

Indole

S0

S135104 cm-1

Indole…pyrroletrimer

D0

D0

h

R2PI (Resonant twophoton ionization) technique

UV-UV hole-burning spectrum of Indole…(pyrrole)2 trimer

3525035200351503510035050

(a)

(b)

000

(351

04 c

m-1

)

Wavenumber (cm-1

)

S0 v = 0

S1

D0

h2 (UV)fixed

h2 (UV)

h1 (UV)tuned

100 ns

UV-UV hole-burningspectroscopy

Only one conformer of the trimer is present in the experiment

IR spectra of indole…(pyrrole)2 trimer

35503500345034003350

Wavenumber (cm-1

)

(a)

(b)

(c)3385

33983412

3389 34083376

3526

3389

3401

3444(d)

IP2-1

IP2-2

S0 v = 0

S1

D0

h2 (UV)fixed

h2 (UV)

h1 (IR)tuned

100 ns

RIDIR (Resonant Ion DipInfrared) spectroscopy

Sumit Kumar and Aloke Das, J. Chem. Phys. 136, 174302 (2012)

Direct experimental evidence of cyclicasymmetric trimeric structure!!

Binding energies of various structures of indole…(pyrrole)2 trimer

Theory

Indole…(pyrrole)2 trimer

IP2-1 IP2-2

ΔEe ΔEo Erel ΔEe ΔEo Erel

B97-D/cc-pVTZ -23.05 -20.51 0.000 -22.96 -20.45 0.049

B97-D/aVDZ -22.38 -20.02 0.000 -22.16 -19.84 0.140

B97X-D/cc-pVTZ -22.85 -20.54 0.000 -22.46 -20.47 0.079

B97X-D / aVDZ -22.84 -20.69 0.000 -22.39 -20.31 0.372

M05-2X /cc-pVTZ -20.24 -18.15 0.000 -19.83 -17.92 0.255

M05-2X/aVDZ -20.29 -18.25 0.000 -19.84 -17.99 0.230

M06-2X /cc-pVTZ -21.74 -19.46 0.000 -21.25 -19.16 0.304

M06-2X/aVDZ -22.35 -20.05 0.000 -21.84 -19.69 0.315

IP2-1 IP2-2

Binding energies arein kcal/mol.

Symmetric or asymmetric structures of the trimers from geometrical parameters

Geometrical parameters(indole)2…pyrrole trimer

(pyrrole)3 IP2-1

rN5-H10 (Å) 0.0094 0.0090

rN15-H20 (Å) 0.0076 0.0092

rN25-H30 (Å) 0.0093 0.0091

rC11-H16 (Å) 0.0009

bpy(A)-py(B) 50.7 61.0

bpy(B)-py(C) 52.2 61.0

bpy(C)-py(A) 69.4 61.0

dC11-H16…π (Å) 2.96

dN25-H30…π (Å) 2.30 2.28

dN5-H10…π (Å) 2.28 2.28

dN15-H20…π (Å) 2.41 2.28

(Debye) 0.88 0.00

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3231

3433

35

30 36A

B

CD

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30A

B

C

Cyclic symmetric structure

Cyclic asymmetric structure

(pyrrole)3

Indole...(pyrrole)2 trimer

Calculation @ M05-2X/cc-pVTZ

Assignment of the peaks in the IR spectrum of indole…(pyrrole)2 trimer

Asymmetric N-H stretching (3412 cm-1)

Asymmetric N-H stretching (3398 cm-1)

Symmetric N-H stretching (3385 cm-1)

Normal modes of two asymmetric and one symmetric N-H stretches in the IP2-1 structure of indole…(pyrrole)2 trimer at the M05-2X/cc-pVTZ level

Comparison of IR frequencies of indole…(pyrrole)2 and (pyrrole)3

N-H (cm-1)

Theory (Expt)

IR intensit

y(km/mol)

Raman Intensit

y(km/mol)

Assignment

IP2-1 3412 (3408) 483 105 Asymmetric stretching

3398 (3389) 638 92Asymmetric Stretching

3385 (3376) 81 265 Symmetric Stretching

IP2-2 3444 330 87Asymmetric Stretching

3401 602 89Asymmetric Stretching

3389 183 232 Symmetric Stretching

(pyrrole)3 3405 (3393)b 631 90Asymmetric Stretching

3405 (3393)b 628 90Asymmetric Stretching

3388 (3376)b 2 280 Symmetric Stretching

b Dauster et. al. Phys. Chem. Chem. Phys. 10, 2827 (2008)

IP2-1 IP2-2

indole…(pyrrole)2 trimer

(pyrrole)3

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D

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Indole…(pyrrole)2 trimer: Cyclic asymmetric structure(Pyrrole)3: Cyclic symmetric structure

(Pyrrole)3

3376

3393NH NH NH

NH (Sym)

NH (Asym)

Zeroth orderCoupled

Davydovsplitting17 cm-1

NH NH NH

3376

3389

3408

NH (Sym)

NH (Asym)

NH (Asym)

17 cm-1

32 cm-1

Zeroth order Coupled

Indole…(pyrrole)2

Other examples:

J. Chem. Phys. 105, 8965 (1996)

J. Phys. Chem. 99, 5761 (1995)

Natural bond orbital analysis

*C1-C2 N25-H30

E =i j 1.73 kcal/mol(2)

*C3-C4 N25-H30

E =i j 1.17 kcal/mol(2)

*C13-C14 N5-H10

E =i j 2.04 kcal/mol(2)

*C11-C12 N5-H10

E =i j 1.11 kcal/mol(2)

*C21-C22 N15-H20

E =i j 0.34 kcal/mol(2)

*C23-C24 N15-H20

E =i j 2.09 kcal/mol(2)* *

*

*

*

*

Natural bond orbitals of theIP2-1 structure of indole…(pyrrole)2 trimer

IP2-1 (pyrrole)3

Δq(H)pyrrole(A) 0.0412 0.0406

Δq(H)pyrrole(B) 0.0327 0.0406

Δq(H)pyrrole(C) 0.0390 0.0406

δ( πC1-C2 ) 1.8462 1.8470

δ( πC3-C4 ) 1.8477 1.8471

δ( πC11-C12) 1.8434 1.8470

δ( πC13-C14 ) 1.8517 1.8471

δ( πC21-C22) 1.6062 1.8470

δ( πC23-C24) 1.8611 1.8471

δ( σ* H10-N5 ) 0.0235 0.0234

δ( σ* H20-N15 ) 0.0227 0.0234

δ( σ* H30-N25 ) 0.0246 0.0234

E 2 *ji ( πpyrrole(A) σ*

H30-N25 ) 2.90 (1.73+1.17)

3.03 (1.49+1.54)

E 2 *ji ( πpyrrole(B) σ*

H10-N5 ) 3.15 (1.11+2.04)

3.04 (1.50+1.54)

E 2 *ji ( πpyrrole(C) σ*

H20-N15 ) 2.43 (2.09+0.34)

3.04 (1.50+1.54)

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Eij* is in kcal/mol.

(2)

Summary

For the first time, we have found a direct experimental evidence of a cyclic asymmetric structure of a heterocyclic aromatic heterotrimer bound by three N-H…π hydrogen bonding interactions.

Due to asymmetry in the cyclic ring structure, symmetric N-H stretching vibration is also weakly observed in the IR spectrum along with the two strong non-degenerate asymmetric N-H stretching vibrations.

Different strength of the three N-H…π hydrogen bonding interactions in the cyclic asymmetric structure of the trimer is revealed through the calculation of the relevant geometric parameters as well as the NBO analysis.

Excellent agreement between experimental and theoretical (Dispersion corrected DFT) IR frequencies as well as intensities of the N-H stretching vibrations in the trimer is noteworthy.

The current results have implication in quantitative understanding of the trimeric interactions present in the aromatic side chains of the proteins.

Acknowledgement

Funding: Indian Institute of Science Education & Research (IISER) Pune

Department of Science & Technology, India

Sumit Kumar (Ph. D. Student)

500400300200100Mass (a.m.u.)

[In

d]+

(In

d..

.H2O

)+

[In

d..

.(H

2O

) 2 ]+

(In

d..

.Py

)+

(In

d) 2

+

[(In

d) 2

...P

y]+

[In

d..

.(P

y) 2

]+

(In

d) 3

+

(In

d..

.Py

...H

2O

)+

[(In

d) 2

...P

y..

.H2O

]+

Time of Flight mass spectrum of complexes of indole and pyrrole