Biomolecule Dynamics and Interactions Probed by Fluorescence

The three-dimensional structures and dynamics of proteins and other biomolecules play a central role in determining their unique functions in living organisms and their specific interactions with other molecules. One major challenge of life science is to grasp how a given sequence of amino acid residues gives rise to the native structure and function. We have developed a probe of the conformational dynamics of unsolvated proteins and peptides that is based on modulation of the fluorescence of a covalently attached dye through intramolecular quenching by tryptophan (Trp) or other residues. These gas-phase measurements are used in combination with solution measurements and theoretical calculations to advance our understanding of how the solvent environment affects the behavior of biomolecules and non-covalent complexes.

For the gas-phase experiments, intact, unsolvated biomolecule ions are formed by electrospray ionization (ESI) and transferred into a radio-frequency Paul trap. A particular ion of interest is isolated by ejecting the unwanted ions which occur at lower or higher mass-to-charge ratios from the trap.

The ions under study are then thermalized by collisions with a helium buffer gas. Laser-induced fluorescence is excited by 532 nm light from a frequency-doubled Nd:YAG laser, isolated by bandpass filters, and detected by gallium arsenide photomultipliers.

The excitation and detection optics have been configured to reduce background noise from scattering to nearly zero. Fluorescence with a signal-to-noise ratio of ~ 400 is routinely measured from ~200 ions in a small volume of the trapped ion cloud (~2x10^-5 cm³).

This methodology has been applied to Trp-cage, a miniprotein which is fully structured in aqueous solution at low temperatures.¹ The fluorescence of Trp-cage 3+ ions is constant between 303 and 363 K and decreases by ~75% between 363 and 438 K.² The decrease in fluorescence with increasing temperature is consistent with a conformational change which results in more quenching interactions between the Trp side chain and the fluorescent dye.

The associated enthalpy change for this ion is 51% higher than that in solution,³ most likely a consequence of increased intramolecular hydrogen bonding in the absence of solvent. Hydrogen bond donors and acceptors which are solvated by water molecules in solution can form new intramolecular hydrogen bonds upon desolvation.

Molecular dynamics (MD) simulations indicate a decrease in the number of hydrogen bonds with increasing temperature which agrees well with the experimentally derived change in enthalpy.

A salt bridge between lysine, aspartic acid, and arginine (+ - +) is important for Trp-cage stability in solution, however, replacing negatively charged aspartic acid with uncharged asparagine (D9N mutation) does not significantly change the unfolding enthalpy in gas phase. This indicates that the salt bridge is not a major contributor to the conformational stability of these unsolvated ions.⁴

Measurements⁵ have also been made of small (≤12 residues) poly(Pro) and poly(Gly-Ser) peptides to isolate the effects of chain length, amino acid composition, and charge on the measured fluorescence. For the 2+ ions, the longer peptides, Pro₁₀ and (GlySer)₅, emit higher intensity fluorescence than the corresponding shorter peptides, Pro₄ and (GlySer)₂, at all temperatures studied. This suggests dynamics which are dominated by Coulomb repulsion between the two positive charges. For singly charged ions, in which Coulomb repulsion is absent, the dynamics appear to be characterized by the backbone flexibility, i.e., poly(Pro) is less flexible than poly(Gly-Ser). MD simulations in progress will provide more detailed information on the polypeptide dynamics which lead to the differences in measured fluorescence.

Ongoing projects aim to use these methods to investigate secondary structure formation and the dynamic interactions between the components of non-covalent complexes formed between peptides and other molecules of biological and/or therapeutic importance.

References cited

1. Neidigh, J. W.; Fesinmeyer, R. M.; Andersen, N. H. Nat. Struct. Biol. 2002, 9, 425.
2. Iavarone, A. T.; Parks, J. H. J. Am. Chem. Soc. 2005, 127, 8606.
3. Qiu, L.; Pabit, S. A.; Roitberg, A. E.; Hagen, S. J. J. Am. Chem. Soc. 2002, 124, 12952.
4. Iavarone, A. T.; Patriksson, A.; Van der Spoel, D.; Parks, J. H. J. Am. Chem. Soc., submitted.
5. Iavarone, A. T.; Meinen, J.; Schulze, S.; Parks, J. H. Int. J. Mass Spectrom., in press.