1. 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

2. Introduction Polypeptide (Building block of proteins) Backbone Side chain PhenylalanineTryptophanTyrosineHistidine Four aromatic amino acid residues

3. 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

4. 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 among phenylalanine 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!!!

5. 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 trimeric aromatic interactions in the proteins Efficiencies of π-hydrogen bond acceptors in the proteins (JMB 305, 535, 2001)

6. 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

7. 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).

8. Electronic spectra of Indole…(pyrrole) 2 trimer S0S0 S1S1 35240 cm -1 Indole S0S0 S1S1 35104 cm -1 Indole…pyrrole trimer D0D0 D0D0 h R2PI (Resonant two photon ionization) technique

9. UV-UV hole-burning spectrum of Indole…(pyrrole) 2 trimer UV-UV hole-burning spectroscopy  Only one conformer of the trimer is present in the experiment

10. IR spectra of indole…(pyrrole) 2 trimer RIDIR (Resonant Ion Dip Infrared) spectroscopy Sumit Kumar and Aloke Das, J. Chem. Phys. 136, 174302 (2012) Direct experimental evidence of cyclic asymmetric trimeric structure!!

11. Binding energies of various structures of indole…(pyrrole) 2 trimer IP2-1 IP2-2 Binding energies are in kcal/mol.

12. Symmetric or asymmetric structures of the trimers from geometrical parameters Geometrical parameters (indole) 2 …pyrrole trimer (pyrrole) 3 IP2-1  r N5-H10 (Å) 0.00940.0090  r N15-H20 (Å) 0.00760.0092  r N25-H30 (Å) 0.00930.0091  r C11-H16 (Å) 0.0009 b  py(A)-py(B) 50.761.0 b  py(B)-py(C) 52.261.0 b  py(C)-py(A) 69.461.0 d C11-H16…π (Å)2.96 d N25-H30…π (Å)2.302.28 d N5-H10…π (Å)2.28 d N15-H20…π (Å)2.412.28  (Debye)0.880.00 Cyclic symmetric structure Cyclic asymmetric structure (pyrrole) 3 Indole...(pyrrole) 2 trimer Calculation @ M05-2X/cc-pVTZ

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

14. Comparison of IR frequencies of indole…(pyrrole) 2 and (pyrrole) 3 N-H (cm -1 ) Theory (Expt) IR intensity (km/mol) Raman Intensity (km/mol) Assignment IP2-1 3412(3408)483105Asymmetric stretching 3398(3389)63892Asymmetric Stretching 3385(3376)81265Symmetric Stretching IP2-2 344433087Asymmetric Stretching 340160289Asymmetric Stretching 3389183232Symmetric Stretching (pyrrole) 3 3405(3393) b 63190Asymmetric Stretching 3405(3393) b 62890Asymmetric Stretching 3388(3376) b 2280Symmetric Stretching b Dauster et. al. Phys. Chem. Chem. Phys. 10, 2827 (2008) Indole…(pyrrole) 2 trimer: Cyclic asymmetric structure (Pyrrole) 3 : Cyclic symmetric structure (Pyrrole) 3 3376 3393 NH NH (Sym) NH (Asym) Zeroth order Coupled Davydov splitting 17 cm -1 NH 3376 3389 3408 NH (Sym) NH (Asym) 17 cm -1 32 cm -1 Zeroth orderCoupled Indole…(pyrrole) 2 Other examples: J. Chem. Phys. 105, 8965 (1996) J. Phys. Chem. 99, 5761 (1995)

15. Natural bond orbital analysis Natural bond orbitals of the IP2-1 structure of indole…(pyrrole) 2 trimer E i  j* is in kcal/mol. (2)

16. 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.

17. Acknowledgement Funding: Indian Institute of Science Education & Research (IISER) Pune Department of Science & Technology, India Sumit Kumar (Ph. D. Student)

18. Time of Flight mass spectrum of complexes of indole and pyrrole