Synthesis of 5-Cyano-Tryptophan as a Two-Dimensional Spectroscopic Reporter of Structure (2018)
Farzaneh Chalyavi, Philip H. Gilmartin, Andrew J. Schmitz, Michael W. Fennie, and Matthew J. Tucker

A concise synthesis of protected 5-cyanotryptophan (Trp5CN) has been developed for 2D IR spectroscopic investigations within either peptides or proteins. To assess the potential of differently substituted cyanotryptophans, severalmodel cyano-indole systems were characterized using IR spectroscopy. Upon assessment of their spectroscopic properties, Trp5CN was integrated into a model peptide sequence, Trp5CN-Gly-Phe4CN, to elucidate its structure. This peptide demonstrates the capability of this probe to capture structural information by 2D IR spectroscopy. The 2D IR spectrum of the peptide in water was simulated to reveal a unique spectral signature resulting from the presence of dipolar coupling. The coupling strength between cyano labels was determined to be 1.4 cm-1 by matching the slopes along the max contour for the simulated and experimental spectrum. Using transition dipole coupling, a distance between the two probes of 13 angstroms was calculated.

 
 

Selective Excitation of Cyanophenylalanine Fluorophores for MultiSite Binding Studies (2017)
Natalie R. Fetto, Wenqiang Cao, Ian S. Wallace, and Matthew J. Tucker

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.

 
 

Interspecies Bombolitins Exhibit Structural Diversity upon Membrane Binding, Leading to Cell Specificity (2019)
Matthew G. Roberson, Devin K. Smith, Simon M. White, Ian S. Wallace, Matthew J. Tucker

Bombolitins, a class of peptides produced by bees of the genus Bombus, target and disrupt cellular membranes, leading to lysis. Antimicrobial peptides exhibit various mechanisms of action resulting from the interplay between peptide structure, lipid composition, and cellular target membrane selectivity. Herein, two bombolitins displaying significant amino-acid-sequence similarity, BII and BL6, were assessed for antimicrobial activity as well as correlated dodecylphosphocholine (DPC) micelle binding and membrane-induced peptide conformational changes. Infrared and circular dichroism spectroscopies were used to assess the structure-function relationship of each bombolitin, and the results indicate that BII forms a rigid and helically ordered secondary structure upon binding to DPC micelles, whereas BL6 largely lacks secondary structural order. Moreover, the binding affinity of each peptide to DPC micelles was determined, revealing that BL6 displayed a difference in binding affinity by over two orders of magnitude. Further investigations into the growth-inhibitory activity of the two bombolitins were performed against Escherichia coli and Saccharomyces cerevisiae. Interestingly, BII specifically targeted S. cerevisiae, whereas BL6 more effectively inhibited E. coli growth. Overall, the antimicrobial selectivity and specificity of BII and BL6 are largely dependent on the primary as well as secondary structural content of the peptides and the membrane composition.