Kinetic Unfolding Mechanism of the Inducible Nitric Oxide Synthase Oxygenase Domain Determined by Time-Resolved Electrospray Mass Spectrometry

Abstract

The inducible nitric oxide synthase core oxygen domain (iNOS(COD)) is a homodimeric protein complex of ca. 100 kDa. In this work, the subunit disassembly and unfolding of the protein following a pH jump from 7.5 to 2.8 were monitored by on-line rapid mixing in conjunction with electrospray (ESI) time-of-flight mass spectrometry. Various protein species become populated during the denaturation process. These can be distinguished by their ligand binding behavior, and by the different charge states that they produce during ESI. Detailed intensity-time profiles were obtained for all of these species, and the kinetics were subjected to a global analysis which allows a model of the denaturation process to be developed. The data are described well by three relaxation times (tau(1) = 0.36 s, tau(2) = 0.62 s, and tau(3) = 3.3 s), each of which has a characteristic amplitude spectrum. The initial step of the reaction is the disruption of the iNOS(COD) dimer, to generate heme-bound monomeric species in various degrees of unfolding. This first step is accompanied by the loss of two tetrahydrobiopterin cofactors. Subsequent heme loss generates monomeric apoproteins exhibiting various degrees of unfolding. In addition, the formation of proteins that are bound to two heme groups is observed. A subpopulation of holo monomers undergoes substantial unfolding while retaining contact with the heme cofactor. Together with previous studies, the results of this work suggest that the occurrence of complex reaction mechanisms involving several short-lived intermediates is a common feature for the denaturation of large multiprotein complexes.

A versatile microfluidic chip for millisecond time-scale kinetic studies by electrospray mass spectrometry

“…Fits are shown as solid lines in Figure 5. The data were analyzed using an implicit global analysis strategy based on that in reference [14]. The concentration versus time dependences for each cytochrome c charge state, C(a, m/z), were assumed to correspond to a sum of exponentials plus an offset.…”

“…While folding intermediates are quite common [13,37,38], unfolding intermediates are less commonly detected, particularly in a small proteins [14,35]. Cytochrome c may be an exception due to its covalently bound heme which could act as a structural nucleus during unfolding.…”

“…Cytochrome c may be an exception due to its covalently bound heme which could act as a structural nucleus during unfolding. Alternatively, the complex unfolding landscapes predicted by some computer models [39] and observed for large proteins [14] may apply equally to small proteins. The microfluidic reactor introduced here can help to shed light on this question by facilitating the detection of transient unfolding intermediates, especially those that cannot be observed by conventional optical methods.…”

“…P(a) was calculated as described in the theory section. In keeping with a global analysis strategy described previously [14], C(a) was assumed to have the form of a linear combination of exponentials plus an offset. Satisfactory fits to all of the kinetic data were acquired using two exponentials with rate constants k 1obs ϭ 32 s -1 Ϯ 5 and k 2obs ϭ 7.5 Ϯ 0.1 s Ϫ1 .…”

Microfluidic Devices for Studying Kinetics

“…Different reaction times are observed by moving a fused silica inner capillary within the outer capillary, which serves as the electrospray source. When the inner capillary is flushed with the end of the outer capillary, the observed reaction time corresponds to the dead time of the device, that is, the mixing time (typically around 7 ms) plus electrospray desolvation that requires typically less than 1 ms. 36 This approach successfully combines the analytical power of ESI-MS with continuous millisecond timescale analysis, making it a powerful tool, particularly for protein folding [37][38][39] and enzyme kinetic 40 studies; however, it shares the limitations of ESI-MS as a bioanalytical method (e.g., low salt tolerance 41 ).…”