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FORSTER RESONANCE ENERGY TRANSFER IN OLFACTORY PEPTIDES Roman J. Martinez, Austin J. Haider, Andrew Melendrez Zerwekh, and Joshua P. Martin Department of Chemistry, Metropolitan State University of Denver Introduction We report Forster Resonance Energy Transfer studies of a 2 -cyanophenylalaninetryptophan donor-acceptor pair within peptide chains. Two olfactory peptides (OFP) are synthesized as sequence homologues of a 12 residue, disordered region in an olfactory marker protein. In these peptides, tryptophan occupies the N-terminus and 2 cyanophenylalanine is substituted in place of phenylalanine at two different distances from tryptophan; in OFP Long the 2 -cyanophenylalanine is at the C-terminus, whereas in OFP Short the nitrile derivatized amino acid is only two residues away from the tryptophan. While phenylalanine is native to the peptide, 2 -cyanophenylalanine exhibits a larger fluorescence quantum yield providing better spectroscopic selectivity. Further, addition of the nitrile group to phenylalanine has been reported to change the peptide structure only minimally, thus preserving the native protein structure. As such, OFP Long and Short allow for comparison of energy transfer efficiency between the donor and acceptor fluorophores at two distances. Additionally, examining OFP Long and Short in solvents that either promote or inhibit secondary structures provide model systems in which spectroscopic techniques are used to determine structural perturbations induced by environmental changes. Our results further demonstrate the potential of 2 -cyanophenylalanine as a site-specific probe of protein structure and dynamics. Fluorescence Theory 2 -cyanophenylalanine and Tryptophan 2 -cyanophenylalanine (2 -Phe. CN) and tryptophan (Trp) are useful spectroscopic probes in peptide/protein structural studies. 1, 2 → The small size of the nitrile group results in only slight perturbation 3, 4 of peptide/protein geometries allowing for studies of native structures and Förster Resonance Energy Transfer (FRET) studies 1, 2 at short distances. • Fluorescence can be impacted by the local environment of the fluorophore (i. e. Energy transfer pathways, level of solvation, p. H, ionic composition, etc. ) Solvent = 20% (v/v) TFE Spectral Overlap of 2 -Phe. CN and Tryptophan 1. 6 Trp Abs. 1. 4 2 -Phe. CN Emission 0. 8 1. 2 ε x 10− 3 (M− 1∙cm− 1) 1. 0 Trp Emission 2 -Phe. CN Abs. 0. 8 0. 6 0. 4 Spectral Overlap 0. 6 0. 4 0. 2 0. 0 270 320 Wavelength (nm) 370 Normalized Emission Intensity • Nonradiative pathways (�) for the relaxation of the electron compete with the fluorescence mechanism. Trp • Significant spectral overlap of the tryptophan absorption spectrum and the 2 -Phe. CN emission spectrum. No overlap vice versa. • FRET pair: donor = 2 -Phe. CN and acceptor = Trp • Selective excitation of 2 -Phe. CN at 240 nm results in emission from tryptophan at 365 nm. • Preliminary calculations indicate the R 0 of 2 -Phe. CN and Tryptophan is 15. 2 ± 0. 2 Å. Olfactory Peptides (OFP) • The Olfactory marker protein (OMP) is a highly expressed, 19 k. Da protein that plays a role in signal transduction in mature olfactory neurons. The disordered region (Ω-loop 3) has been associated with a high level of conformational flexibility. 5 • The olfactory peptides (OFP Short and OFP Long) utilized in this work are sequence homologues of the Ω-loop 3 with a synthetic 2 -Phe. CN residue in place of a phenylalanine residue. • An optical probe in the form of the 2 -Phe. CN − Trp FRET pair in the Ω-loop 3 would allow for intricate study of OMP’s dynamics. Förster Resonance Energy Transfer (FRET) • Excitation of a donor chromophore results in distance-dependent energy transfer to an acceptor fluorophore • Variance in the distance between the acceptor and donor, r, changes the efficiency of the energy transfer, EFRET • The distance at 50% efficiency is called the Forster Distance, R 0, and can be calculated from overlap of donor absorbance spectra and acceptor emission spectra Solvent = Water → The addition of the nitrile group to Phe results in a “significant” increase in the molar absorptivity (ε) and fluorescence quantum yield (ΦF) compared with the native Phe 2 and results in a comparable molar absorptivity and fluorescence quantum yield to tryptophan. • Electrons in the fluorophore are photoexcited to the S 1 or S 2 electronic states. • Radiative relaxation of the excited electron from the S 1 state results in the emission of a photon, i. e. fluorescence. 2 -Phe. CN OFP Emission – 240 nm Excitation Obtained From Gitti et al. 5 OFP Long OFP Short r r J. P. Martin, N. R. Fetto, and M. J. Tucker, Phys. Chem. Phys. , 2016, 18, 20750. 2 M. J. Tucker, R. Oyola, and F. Gai, J. Phys. Chem. B, 2005, 109, 4788. 3 Z. Getahun, C. -y. Huang, T. Wang, B. De Leon, W. F. Degrado, and F. Gai, J. Am. Chem. Soc. , 2003, 125, 405. 4 R. Adhikary, J. Zimmermann, P. E. Dawson, and F. E. Romesberg, Chem. Phys. Chem, 2014, 15, 849. 5 R. K. Gitti, N. T. Wright, J. W. Margolis, K. M. Varney, D. J. Weber and F. L. Margolis; Biochemistry, 2005, 44, 9673. 6 M. Buck, Q. Rev. Biophysics, 1998, 31(3), 297. 1 Solvent = 7 M Urea • Using water as the solvent mimics a more native environment for OFP. • The largest emission contribution in OFP Short is from tryptophan indicating high efficiency of FRET. → due to close proximity of donor and acceptor • For OFP Long, the emission from 2 -Phe. CN and tryptophan are near equal, indicating low FRET efficiency. → due to larger distance between donor and acceptor • Using 2, 2, 2 -trifluroethanol (TFE) as a solvent that promotes secondary structure in proteins. 6 • Compared to water, the secondary structure in TFE improved FRET efficiency in both OFP Short and OFP Long as the contribution from 2 -Phe. CN has decreased. • Thus, secondary structures of both OFPs in TFE must force 2 -Phe. CN and tryptophan within closer proximity than in H 2 O. • Urea is commonly used to denature proteins forming a “straight chain. ” Therefore, FRET efficiency should decrease in Urea as donor and acceptor are further apart. • This trend is observed in OFP Short, where the 2 -Phe. CN contribution is the lowest of all solvents. • However, no significant contribution from 2 -Phe. CN is observed in OFP Long. Further experiments will probe the cause. Conclusions and Future Directions • Further experimental validation of the R 0 values for OFP Short and OFP Long will be performed in the same solvent environments. Additionally, Molecular Dynamics simulations will be performed to provide a theoretical value of R 0. • A series of peptides, moving the 2 -Phe. CN away from the tryptophan by one residue in each variation, will be used to investigate potential loops present in the structure. These peptides will be examined in the same solvent environments. • Equally, temperature dependence studies of the EFRET will be performed to determine r experimentally, providing an “intrinsic-ruler” in a disordered region of OMP. Acknowledgements and Funding • We would like to gratefully acknowledge Natalie R. Fetto and Dr. Matthew J. Tucker (University of Nevada, Reno) for the synthesis of the olfactory peptides and their continued collaboration. • Funding: CLAS Mini-Grant