Bond-Specific Dissociation Following Excitation Energy Transfer for Distance Constraint Determination in the Gas Phase

Abstract

Herein, we report chemistry that enables excitation energy transfer (EET) to be accurately measured via action spectroscopy on gaseous ions in an ion trap. It is demonstrated that EET between tryptophan or tyrosine and a disulfide bond leads to excited state, homolytic fragmentation of the disulfide bond. This phenomenon exhibits a tight distance dependence, which is consistent with Dexter exchange transfer. The extent of fragmentation of the disulfide bond can be used to determine the distance between the chromophore and disulfide bond. The chemistry is well suited for the examination of protein structure in the gas phase because native amino acids can serve as the donor/acceptor moieties. Furthermore, both tyrosine and tryptophan exhibit unique action spectra, meaning that the identity of the donating chromophore can be easily determined in addition to the distance between donor/acceptor. Application of the method to the Trpcage miniprotein reveals distance constraints that are consistent with a native-like fold for the +2 charge state in the gas phase. This structure is stabilized by several salt bridges, which have also been observed to be important previously in proteins that retain native-like structures in the gas phase. The ability of this method to measure specific distance constraints, potentially at numerous positions if combined with site-directed mutagenesis, significantly enhances our ability to examine protein structure in the gas phase.

Structural Effects of Solvation by 18-Crown-6 on Gaseous Peptides and TrpCage after Electrospray Ionization

“…All peptides were modified with PM prior to use via a procedure described previously [20]. Modification was followed by lyophilization to remove all solvent before resuspension in 10:90, 50:50, or 80:20 MeOH/H 2 O solutions.…”

“…Various forms of spectroscopy can also be used to obtain information on biomolecules [18,19]. Peptides and proteins are also amenable to examination by excitation energy-transfer (EET) where distance constraint information is obtained and used to guide simulations toward relevant structures [20][21][22][23].…”

Near-UV Photodissociation of Tryptic Peptide Cation Radicals. Scope and Effects of Amino Acid Residues and Radical Sites

“…P hotodissociation is one of the tools for accomplishing dissociations of gas-phase ions [1][2][3][4] that more recently has been widely applied to biomolecular ions [5][6][7][8][9][10][11][12][13]. Photodissociation in the far UV (157 nm [14,15]) and middle UV (193 nm [16]) regions of the spectrum has been used to dissociate peptide ions.…”

Characterization of the Cysteine Content in Proteins Utilizing Cysteine Selenylation with 266 nm Ultraviolet Photodissociation (UVPD)

“…Based on absorbance profiles of amino acids in solution, it is anticipated that the photoabsorption cross-section for tryptophan in the gas phase is likely greater than that of tyrosine at 266 nm, and the photoabsorption cross-section for phenylalanine is expected to be rather low at 266 nm (these remarks are derived from solution profiles, not the gas phase [35]). Excitation energy transfer has been shown to occur between tryptophan or tyrosine and disulfide bonds, ultimately resulting in homolytic cleavage of the disulfide bond via an excited state [36]. We speculate that a similar phenomenon may occur for the NPSP-tagged proteins.…”

“…We speculate that a similar phenomenon may occur for the NPSP-tagged proteins. For example, lysozyme and α-lactalbumin contain multiple tryptophan residues, which may enhance S-Se bond cleavage from an excited state induced by absorption of 266 nm photons (similar to that shown for disulfide bonds [36]). The lack of tryptophan residues in ribonuclease A may explain the inhibition of tag loss for the protein.…”