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Investigating protein folding and unfolding in electrospray nanodrops upon rapid mixing using theta-glass emitters.

ABSTRACT: Theta-glass emitters are used to rapidly mix two solutions to induce either protein folding or unfolding during nanoelectrospray (nanoESI). Mixing acid-denatured myoglobin with an aqueous ammonium acetate solution to increase solution pH results in protein folding during nanoESI. A reaction time and upper limit to the droplet lifetime of 9 ± 2 μs is obtained from the relative abundance of the folded conformer in these rapid mixing experiments compared to that obtained from solutions at equilibrium and a folding time constant of 7 μs. Heme reincorporation does not occur, consistent with the short droplet lifetime and the much longer time constant for this process. Similar mixing experiments with acid-denatured cytochrome c and the resulting folding during nanoESI indicate a reaction time of between 7 and 25 μs depending on the solution composition. The extent of unfolding of holo-myoglobin upon rapid mixing with theta-glass emitters is less than that reported previously ( Fisher et al. Anal. Chem. 2014 , 86 , 4581 - 4588 ), a result that is attributed to the much smaller, ∼1.5 μm, average o.d. tips used here. These results indicate that the time frame during which protein folding or unfolding can occur during nanoESI depends both on the initial droplet size, which can be varied by changing the emitter tip diameter, and on the solution composition. This study demonstrates that protein folding or unfolding processes that occur on the ∼10 μs time scale can be readily investigated using rapid mixing with theta-glass emitters combined with mass spectrometry.

SUBMITTER: Mortensen DN

PROVIDER: S-EPMC4303338 | biostudies-other | 2015 Jan

REPOSITORIES: biostudies-other

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Project description:To monitor the development of tertiary structural contacts during folding, a unique tryptophan residue was introduced at seven partially buried locations (residues 15, 27, 61, 76, 91, 102 and 121) of a tryptophan-free variant of staphylococcal nuclease (P47G/P117G/H124L/W140H). Thermal unfolding measurements by circular dichroism indicate that the variants are destabilized, but maintain the ability to fold into a native-like structure. For the variants with Trp at positions 15, 27 and 61, the intrinsic fluorescence is significantly quenched in the native state due to close contact with polar side-chains that act as intramolecular quenchers. All other variants exhibit enhanced fluorescence under native conditions consistent with burial of the tryptophan residues in an apolar environment. The kinetics of folding was observed by continuous and stopped-flow fluorescence measurements over refolding times ranging from 100 micros to 10 s. The folding kinetics of all variants is quantitatively described by a mechanism involving a major pathway with a series of intermediate states and a minor parallel channel. The engineered tryptophan residues in the beta-barrel and the N-terminal part of the alpha-helical domain become partially shielded from the solvent at an early stage (<1 ms), indicating that this region undergoes a rapid collapse. For some variants, a major increase in fluorescence coincides with the rate-limiting step of folding on the 100 ms time scale, indicating that these tryptophan residues are buried only during the late stages of folding. Other variants exhibit a transient increase in fluorescence during the 10 ms phase followed by a decrease during the rate-limiting phase. These observations are consistent with burial of these probes in a collapsed, but loosely packed intermediate, followed by the rate-limiting formation of the densely packed native core, which brings the tryptophan residues into close contact with intramolecular quenchers.