Globular proteins, such as cytochrome c (cyt c), display an organized native conformation, maintained by a hydrogen bond interaction network. In the present work, the structural interrogation of kinetically trapped intermediates of cyt c was performed by correlating the ion-neutral collision cross section (CCS) and charge state with the starting solution conditions and time after desolvation using collision induced activation (CIA), time resolved hydrogen/deuterium back exchange (HDX) and trapped ion mobility spectrometry - mass spectrometry (TIMS-MS). The high ion mobility resolving power of the TIMS analyzer allowed the identification of new ion mobility bands, yielding a total of 63 mobility bands over the +6 to +21 charge states and 20 mobility bands over the −5 to −10 charge states. Mobility selected HDX rates showed that for the same charge state, conformers with larger CCS present faster HDX rates in both positive and negative ion mode, suggesting that the charge sites and neighboring exchange sites on the accessible surface area define the exchange rate regardless of the charge state. Complementary molecular dynamic simulations permitted the generation of candidate structures and a mechanistic model of the folding transitions from native (N) to molten globule (MG) to kinetic intermediates (U) pathways. Our results suggest that cyt c major structural unfolding is associated with the distancing of the N- and C- terminal helices and subsequent solvent exposure of the hydrophobic, heme-containing cavity.

In the present work, we demonstrate the potential and versatility of TIMS for the analysis of proteins, DNA-protein complexes and protein-protein complexes in their native and denatured states. In addition, we show that accurate CCS measurement are possible and in good agreement with previously reported CCS values using other IMS analyzers (<5% difference). The main challenges for the analysis of high mass proteins and protein complexes in the mobility and m/z domain are described. That is, the analysis of high molecular weight systems in their native state may require the use of higher electric fields or a compromise in the TIMS mobility resolution by reducing the bath gas velocity in order to effectively trap at lower electric fields. This is the first report of CCS measurements of high molecular weight biomolecules and biomolecular complexes (~ 150 kDa) using TIMS-MS.

Flavin adenine dinucleotide (FAD) is involved in important metabolic reactions where the biological function is intrinsically related to changes in conformation. In the present work, FAD conformational changes were studied in solution and in gas phase by measuring the fluorescence decay time and ion-neutral collision cross sections (CCS, in a trapped ion mobility spectrometer, TIMS) as a function of the solvent conditions (i.e., organic content) and gas-phase collisional partner (i.e., N2 doped with organic molecules). Changes in the fluorescence decay suggest that FAD can exist in four conformations in solution, where the abundance of the extended conformations increases with the organic content. TIMS-MS experiments showed that FAD can exist in the gas phase as deprotonated (M = C27H31N9O15P2) and protonated forms (M = C27H33N9O15P2) and that multiple conformations (up to 12) can be observed as a function of the starting solution for the [M + H]+ and [M + Na]+molecular ions. In addition, changes in the relative abundances of the gas-phase structures were observed from a “stack” to a “close” conformation when organic molecules were introduced in the TIMS cell as collision partners. Candidate structures optimized at the DFT/B3LYP/6-31G(d,p) were proposed for each IMS band, and results showed that the most abundant IMS band corresponds to the most stable candidate structure. Solution and gas-phase experiments suggest that the driving force that stabilizes the different conformations is based on the interaction of the adenine and isoalloxazine rings that can be tailored by the “solvation” effect created with the organic molecules.

Nicotinamide adenine dinucleotide (NAD) is found in all living cells where the oxidized (NAD) and reduced (NADH) forms play important roles in many enzymatic reactions. However, little is known about NAD and NADH conformational changes and kinetics as a function of the cell environment. In the present work, an analytical workflow is utilized to study NAD and NADH dynamics as a function of the organic content in solution using fluorescence lifetime spectroscopy and in the gas-phase using trapped ion mobility spectrometry coupled to mass spectrometry (TIMS-MS) and infrared multiple photon dissociation (IRMPD) spectroscopy. NAD solution time decay studies showed a two-component distribution, assigned to changes from a "close" to "open" conformation with the increase of the organic content. NAD gas-phase studies using nESI-TIMS-MS displayed two ion mobility bands for NAD protonated and sodiated species, while four and two ion mobility bands were observed for NADH protonated and sodiated species, respectively. Changes in the mobility profiles were observed for NADH as a function of the starting solution conditions and the time after desolvation, while NAD profiles showed no dependence. IRMPD spectroscopy of NAD and NADH protonated species in the 800-1800 and 3200-3700 cm spectral regions showed common and signature bands between the NAD forms. Candidate structures were proposed for NAD and NADH kinetically trapped intermediates of the protonated and sodiated species, based on their collision cross sections and IR profiles. Results showed that NAD and NADH species exist in open, stack, and closed conformations and that the driving force for conformational dynamics is hydrogen bonding of the N-H-O and O-H-O forms with ribose rings.

DREAM (also known as K+ channel interacting protein 3 and calsenilin) is a calcium binding protein and an active modulator of KV4 channels in neuronal cells as well as a novel Ca2+-regulated transcriptional modulator. DREAM has also been associated with the regulation of Alzheimer’s disease through the prevention of presenilin-2 fragmentation. Many interactions of DREAM with its binding partners (Kv4, calmodulin, DNA, and drugs) have been shown to be dependent on calcium. Therefore, understanding the structural changes induced by binding of metals to DREAM is essential for elucidating the mechanism of signal transduction and biological activity of this protein. Here, we show that the fluorescence emission and excitation spectra of the calcium luminescent analogue, Tb3+, are enhanced upon binding to the EF-hands of DREAM due to a mechanism of energy transfer between Trp and Tb3+. We also observe that unlike Tb3+-bound calmodulin, the luminescence lifetime of terbium bound to DREAM decays as a complex multiexponential (τaverage ~ 1.8 ms) that is sensitive to perturbation of the protein structure and drug (NS5806) binding. Using isothermal calorimetry, we have determined that Tb3+ binds to at least three sites with high affinity (Kd = 1.8 μM in the presence of Ca2+) and displaces bound Ca2+ through an entropically driven mechanism (ΔH ~ 12 kcal mol−1, and TΔS ~ 22 kcal mol−1). Furthermore, the hydrophobic probe 1,8-ANS shows that Tb3+, like Ca2+, triggers the exposure of a hydrophobic surface on DREAM, which modulates ligand binding. Analogous to Ca2+ binding, Tb3+ binding also induces the dimerization of DREAM. Secondary structural analyses using far-UV circular dichroism and trapped ion mobility spectrometry–mass spectrometry reveal that replacement of Ca2+ with Tb3+ preserves the folding state with minimal changes to the overall structure of DREAM. These findings pave the way for further investigation of the metal binding properties of DREAM using lanthanides as well as the study of DREAM–protein complexes by lanthanide resonance energy transfer or nuclear magnetic resonance.