Article pubs.acs.org/JPCB
Selective Excitation of Cyanophenylalanine Fluorophores for MultiSite Binding Studies Natalie R. Fetto,† Wenqiang Cao,† Ian S. Wallace,†,‡ and Matthew J. Tucker*,† †
Department of Chemistry, University of Nevada, 1664 North Virginia Street, Reno, Nevada 89557, United States Department of Biochemistry and Molecular Biology, University of Nevada, 1664 North Virginia Street, Reno, Nevada 89557, United States
‡
ABSTRACT: Recently, it has been shown that nitrile-derivatized phenylalanines possess distinct fluorescent properties depending on the position of the cyano-group within the aromatic ring. These fluorophores have potential as probes for studying protein dynamics due to their sensitivity to local environment. Herein, we demonstrate that 2-cyanophenylalanine (Phe2CN) and Phe4CN can independently monitor multiple sites during the Ca2+ dependent binding of a skeletal muscle myosin light chain kinase (MLCK) peptide fragment to the protein calmodulin (CaM). These cyano-probes were incorporated at two different positions along the peptide chain and monitored simultaneously via selective excitation of the two chromophores. The peptide was labeled with Phe4CN at a residue known to bind to a hydrophobic binding pocket of CaM, while Phe2CN was designed to acquire dynamics external to the binding pocket. By selectively exciting each of the chromophores, it was determined that the fluorescence emission of Phe4CN located at position 581 of MLCK was quenched in the presence of CaM, while no significant change in Phe2CN emission was observed at exposed position 594. The CaM binding affinity (Kd) of the double labeled MLCK peptide was calculated to be approximately 64 nM, which is in agreement with previous measurements. These results indicate that multiple PheCN reporters within the same peptide can simultaneously detect variations in the local environment, and that these fluorophores could be utilized to investigate a wide variety of biological problems.
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INTRODUCTION Natural and non-natural amino acid chromophores, such as tryptophan (Trp) and 4-cyanophenylalanine (Phe4CN), have been widely utilized as fluorescent probes to investigate biological systems. For example, folding studies of ribosomal protein L9 showed quenching of Phe4CN fluorescence despite solvent exposure due to Förster resonance energy transfer (FRET).1 In calmodulin (CaM), selenomethionine (SeMet) quenching of Trp fluorescence was shown to be useful in finding distances in folding studies.2 Phe4CN and Trp have also been used as a donor−acceptor pair in one- and two-step FRET studies of villin headpiece subdomain (HP35).3 Both the infrared1,4−10 and fluorescence11−15 signals of Phe4CN are sensitive to changes in hydrogen bonding and electrostatics in the local environment,4 supporting applications for peptide/ protein folding16−19 and binding studies.4,20 Recently, other nitrile derivatives of phenylalanine, such as 2-cyanophenylalanine (Phe2CN) and 3-cyanophenylalanine (Phe3CN), were added to the library of fluorophores capable of detecting changes in hydration and pH.21 Fluorescence quantum yields of these molecules (Φ ∼ 0.14 and 0.13, respectively) are quite similar to both tryptophan22 and Phe4CN21 making them ideal for protein/peptide studies. Nitrile derivatives of phenylalanine are easily incorporated into peptides through solid phase peptide synthesis1 or site-specific mutagenesis,5,23,24 while © XXXX American Chemical Society
causing minimal perturbation to peptide structure and function.8,25 Incorporation of multiple natural amino acid probes into a single peptide is often rendered useless due to overlapping transitions in the absorption spectra.26 This spectral overlap does not allow excitation of a single chromophore, leading to complex emission profiles which limit the amount of information obtained for the system. However, Phe4CN and Phe2CN can be selectively excited to examine the fluorescence properties of each probe at two independent positions within a given system. Selective fluorophore excitation is possible due to differences in preferred electronic transitions of the two chromophores. Previous studies have suggested that Phe4CN favors the S2 ← S0 transition,27 which is centered around ∼230 nm in the absorption spectrum, while Phe2CN favors the S1 ← S0 transition, which is centered around ∼280 nm.21 These transitions are favored due to the relative orientation between the transition dipole moment and the permanent dipole moment, μp.21 As the position of the cyano-group on the ring changes, the μp is directed toward the nitrile group. In our molecular framework, the transition dipole moment for the S2 Received: August 23, 2017 Revised: September 25, 2017 Published: September 26, 2017 A
DOI: 10.1021/acs.jpcb.7b08442 J. Phys. Chem. B XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry B
Finally, the column was eluted in 10 × 1 mL fractions of elution buffer (50 mM Tris-HCl pH 7.5, 0.5 mM DTT, 1 mM egtazic acid (EGTA)). Sample purity was evaluated by SDS-PAGE. Fractions containing VU-1 calmodulin were dialyzed against 20 mM ammonium bicarbonate, and lyophilized. Absorption Measurements. All absorption spectra were measured with a commercial dual-beam UV−Vis spectrophotometer (PerkinElmer Lambda 25) using a 1 cm quartz cuvette. Fluorescence Measurements. The fluorescence spectra were obtained on a Floromax-3 (Jobin Yvon Horiba, Edison, NJ, USA) using a 1 cm quartz cuvette with an integration time of 1 s·nm−1, a resolution of 1 nm, and an excitation slit width of 2 nm. Optical densities of all samples were
