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Title:
Forster Resonance Energy Transfer in Olfactory Peptides
Creator:
Martinez, Roman
Melendrez Zerwekh, Andrew
J. Haider, Austin
Place of Publication:
Denver, CO
Publisher:
Metropolitan State University of Denver
Publication Date:

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Conference Papers ( sobekcm )

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Collected for Auraria Institutional Repository by the Self-Submittal tool. Submitted by Matthew Mariner.
General Note:
Faculty mentor: Joshua P. Martin
General Note:
Major: Biochemistry

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Auraria Institutional Repository
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Auraria Library
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All applicable rights reserved by the source institution and holding location.

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F ORSTER R ESONANCE E NERGY T RANSFER IN O LFACTORY P EPTIDES 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 cyanophenylalanine tryptophan 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 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 Acknowledgements and Funding The addition of the nitrile group to Phe increase in the molar absorptivity ( ) and fluorescence quantum yield ( F ) compared with the native Phe 2 and results in a c omparable molar absorptivity and fluorescence quantum yield to tryptophan. Olfactory Peptides (OFP) 2 cyanophenylalanine and 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. Nonradiative pathways ( ) for the relaxation of the electron compete with the fluorescence mechanism. Fluorescence can be impacted by the local environment of the fluorophore ( i.e. Energy transfer pathways, level of solvation, pH, ionic composition, etc.) 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 E FRET will be performed to determine r 2 cyanophenylalanine (2 Phe CN ) and tryptophan ( Trp ) are useful spectroscopic probes in peptide/protein structural studies. 1,2 Förster Resonance Energy Transfer (FRET) OFP Short OFP Long 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. r r The Olfactory marker protein (OMP) is a highly expressed, 19 kDa protein that plays a role in signal transduction in mature olfactory neurons. The disordered region ( 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 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 dynamics. Obtained From Gitti et al . 5 1 J.P. Martin, N.R. Fetto , and M.J. Tucker, Phys. Chem. 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 , ChemPhysChem , 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. Spectral Overlap of 2 Phe CN and Tryptophan 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 270 290 310 330 350 370 390 Normalized Emission Intensity x 10 (M ) Wavelength (nm) Trp Abs. 2 Phe CN Emission 2 Phe CN Abs. Trp Emission Spectral Overlap OFP Emission 240nm Excitation Urea is commonly used to denature 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 PheCN is observed in OFP Long. Further experiments will probe the cause. Solvent = Water Solvent = 20% (v/v) TFE 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. acceptor For OFP Long, the emission from 2 Phe CN and tryptophan are near equal, indicating low FRET efficiency. 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. 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, E FRET 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 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. 2 Phe CN Trp