Fluorescence lifetime probe of biomolecular conformations

Abstract

Methods have been developed to measure the fluorescence lifetime versus temperature of trapped biomolecular ions derivatized with a fluorescent dye. Previous measurements for different sequences of polyproline peptides demonstrated that quenching rates are related to conformations and their spatial fluctuations. This paper presents the results of extending these methods to study the conformational dynamics of larger biomolecules. Vancomycin-peptide noncovalent complexes in the 1ϩ charge state were studied as a function of temperature for different W-KAA peptide chiralities (L-LDD, D-LDD, L-DLL). Fluorescence-quenching rates, k q , were found to be stereoselective for these different chiralities with relative magnitudesThe variation in fluorescent quenching resulting from switching the chirality of the single Trp residue was readily detectable. Molecular dynamics analysis of complexes formed by W-KAA (L-LDD) and W-KAA(L-DLL) indicates that increased flexibility in the (L-DLL) complex is correlated with reduced quenching rates. Fluorescence measurements were also performed for the Trp-cage protein comparing quenching rates in the 1ϩ, 2ϩ, and 3ϩ charge states for which k q. Measurements of a sequence including a single-point mutation infer the presence of a salt-bridge structure in the 1ϩ charge state and its absence in both the 2ϩ and 3ϩ states. Molecular dynamics structures of Trp-cage indicate that a salt bridge in the 1ϩ charge state produces more compact conformations leading to larger quenching rates based on the quenching mechanism. In both these experimental studies the fluorescence-quenching rates were consistent with changes in structure induced by either intermolecular or intramolecular interactions. (J Am Soc Mass Spectrom 2010, 21, 707-718) © 2010 American Society for Mass Spectrometry P revious measurements [1] of the lifetime of a fluorescent dye covalently bound to polypeptide ions exhibited a quenching rate that depends both on the separation of the dye to a Trp side chain and on the strength of electrostatic fields in the vicinity of the dye. Quenching rates were found to depend on specific peptide sequences giving rise to different conformations and spatial fluctuations characterized by each conformation. Consequently, lifetime measurements provide an opportunity to extract information sensitive to local molecular arrangements and, perhaps more importantly, changes in that arrangement determined by intramolecular and intermolecular interactions. This paper applies lifetime measurements to study two larger biomolecules: the vancomycin-peptide noncovalent complex and the Trp-cage protein. The structures of these biomolecules have been identified in NMR solution measurements; and each has been studied previously in gas-phase measurements. Results of gas-phase studies of the vancomycin-peptide complex infer that specific changes in the peptide ligand chirality lead to different hydrogen binding networks and thus to different conformations. Gas-phase measurements and calculations of Trp-cage protein i...

Fluorescence lifetimes of rhodamine dyes in vacuo

“…Isolation of a particular m/z inside the ion trap ensures that both the charge state of the sample is known and, importantly, that contaminants are removed from the ion population under investigation. Several laboratories have implemented fluorescence excitation and detection capabilities into trapping mass spectrometry set-ups in order to individually probe mass-selected populations of large ions formed by ESI or MALDI using laser-induced fluorescence emission [28][29][30][31][32][33][34][35] as well as fluorescence lifetime measurements [36][37][38][39][40][41][42].…”

“…A lifetime of 6.0 ± 1.5 ns was reported, with the relatively large uncertainty primarily due to the use of a slow photomultiplier tube as detector. Subsequent work in the Parks group reported fluorescence lifetimes of dye-labeled biomolecules, formed by electrospray ionization, stored and mass selected in a 3-D quadrupole ion trap (also known as a Paul trap) [38][39][40]. The Parks group has used TCSPC to monitor the gas-phase dynamics of several peptides, peptide-vancomycin complexes and a small protein by monitoring dynamic quenching between BoDIPY dye labels and tryptophan residues in the gaseous biomolecules.…”

Time-Resolved Photodetachment Anisotropy: Gas-Phase Rotational and Vibrational Dynamics of the Fluorescein Anion

“…For example, time-resolved fluorescence anisotropy (TR-FA) and imaging have enabled the direct probing of biophysical processes, [1][2][3] while alignment experiments in the gas-phase have opened up countless avenues for probing molecular-frame processes. [4][5][6] Some of the experimental tools are transferable between solution and gas phases and fluorescence measurements have elegantly demonstrated their potential power in large isolated molecular systems, [7][8][9][10][11][12][13][14] including distance measurements using Förster resonance energy transfer. [14][15][16][17][18][19][20] TR-FA in particular has been very informative in biophysics and consequently, it also has great potential to become an important analytical tool in structure and function determination in the gas-phase, however, this premise has yet to be fully realized.…”

Relationship between Conformational Dynamics and Electron Transfer in a Desolvated Peptide. Part II. Temperature Dependence

“…7,8 An early review of this research is given in ref 9. Measurements have included quenching in Trp−cage protein, 4,6,8 polyproline peptide sequences, 5,7 and noncovalent vancomycin−peptide complexes. 8 Fluorescence lifetime data were found to be specifically sensitive to peptide sequence 7,9 and intramolecular interactions.…”

“…Measurements have included quenching in Trp−cage protein, 4,6,8 polyproline peptide sequences, 5,7 and noncovalent vancomycin−peptide complexes. 8 Fluorescence lifetime data were found to be specifically sensitive to peptide sequence 7,9 and intramolecular interactions. 8 These experiments verified that interactions between the dye and a Trp residue were required to observe quenching of the dye fluorescence.…”