More Like this

1. Salt bridges govern the structural heterogeneity of heme protein interactions and porphyrin networks: microperoxidase-11

   https://doi.org/10.1039/d0ra04956e  

   Porter, J. ; Dit Fouque, K. Jeanne ; Miksovska, J. ; Fernandez-Lima, F. ( September 2020 , RSC Advances)

   null (Ed.)

   In this work, a proteolytic digest of cytochrome c (microperoxidase 11, MP-11) was used as a model to study the structural aspects of heme protein interactions and porphyrin networks. The MP-11 structural heterogeneity was studied as a function of the starting pH ( e.g. , pH 3.1–6.1) and concentration ( e.g. , 1–50 μM) conditions and adduct coordination. Trapped ion mobility spectrometry coupled to mass spectrometry (TIMS-MS) showed the MP-11 structural dependence of the charge state distribution and molecular ion forms with the starting pH conditions. The singly charged ( e.g. , [M] + , [M − 2H + NH 4 ] + , [M − H + Na] + and [M − H + K] + ) and doubly charged ( e.g. , [M + H] 2+ , [M − H + NH 4 ] 2+ , [M + Na] 2+ and [M + K] 2+ ) molecular ion forms were observed for all solvent conditions, although the structural heterogeneity ( e.g. , number of mobility bands) significantly varied with the pH value and ion form. The MP-11 dimer formation as a model for heme-protein protein interactions showed that dimer formation is favored toward more neutral pH and favored when assisted by salt bridges ( e.g. , NH 4 + , Na + and K + vs. H + ). Inspection of the dimer mobility profiles (2+ and 3+ charge states) showed a high degree of structural heterogeneity as a function of the solution pH and ion form; the observation of common mobility bands suggest that the different salt bridges can stabilize similar structural motifs. In addition, the salt bridge influence on the MP-11 dimer formations was measured using collision induced dissociation and showed a strong dependence with the type of salt bridge ( i.e. , a CE 50 of 10.0, 11.5, 11.8 and 13.0 eV was observed for [2M + H] 3+ , [2M − H + NH 4 ] 3+ , [2M + Na] 3+ and [2M + K] 3+ , respectively). Measurements of the dimer equilibrium constant showed that the salt bridge interactions increase the binding strength of the dimeric species. 

   more » « less
2. The effects of solution additives and gas‐phase modifiers on the molecular environment and conformational space of common heme proteins

   https://doi.org/10.1002/rcm.8347  

   Butcher, David ; Miksovska, Jaroslava ; Ridgeway, Mark E. ; Park, Melvin A. ; Fernandez‐Lima, Francisco ( February 2019 , Rapid Communications in Mass Spectrometry)

   The molecular environment is known to impact the secondary and tertiary structures of biomolecules both in solution and in the gas phase, shifting the equilibrium between different conformational and oligomerization states. However, there is a lack of studies monitoring the impacts of solution additives and gas‐phase modifiers on biomolecules characterized using ion mobility techniques.

   The effect of solution additives and gas‐phase modifiers on the molecular environment of two common heme proteins, bovine cytochrome c and equine myoglobin, is investigated as a function of the time after desolvation (e.g., 100–500 ms) using nanoelectrospray ionization coupled to trapped ion mobility spectrometry with detection by time‐of‐flight mass spectrometry. Organic compounds used as additives/modifiers (methanol, acetonitrile, acetone) were either added to the aqueous protein solution before ionization or added to the ion mobility bath gas by nebulization.

   Changes in the mobility profiles are observed depending on the starting solution composition (i.e., in aqueous solution at neutral pH or in the presence of organic content: methanol, acetone, or acetonitrile) and the protein. In the presence of gas‐phase modifiers (i.e., N₂doped with methanol, acetone, or acetonitrile), a shift in the mobility profiles driven by the gas‐modifier mass and size and changes in the relative abundances and number of IMS bands are observed.

   We attribute the observed changes in the mobility profiles in the presence of gas‐phase modifiers to a clustering/declustering mechanism by which organic molecules adsorb to the protein ion surface and lower energetic barriers for interconversion between conformational states, thus redefining the free energy landscape and equilibria between conformers. These structural biology experiments open new avenues for manipulation and interrogation of biomolecules in the gas phase with the potential to emulate a large suite of solution conditions, ultimately including conditions that more accurately reflect a variety of intracellular environments.

    

   more » « less
3. Metal ions induced secondary structure rearrangements: mechanically interlocked lasso vs. unthreaded branched-cyclic topoisomers

   https://doi.org/10.1039/c8an00138c  

   Jeanne Dit Fouque, Kevin ; Moreno, Javier ; Hegemann, Julian D. ; Zirah, Séverine ; Rebuffat, Sylvie ; Fernandez-Lima, Francisco ( January 2018 , The Analyst)

   Metal ions can play a significant role in a variety of important functions in protein systems including cofactor for catalysis, protein folding, assembly, structural stability and conformational change. In the present work, we examined the influence of alkali (Na, K and Cs), alkaline earth (Mg and Ca) and transition (Co, Ni and Zn) metal ions on the conformational space and analytical separation of mechanically interlocked lasso peptides. Syanodin I, sphingonodin I, caulonodin III and microcin J25, selected as models of lasso peptides, and their respective branched-cyclic topoisomers were submitted to native nESI trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). The high mobility resolving power of TIMS permitted to group conformational families regardless of the metal ion. The lower diversity of conformational families for syanodin I as compared to the other lasso peptides supports that syanodin I probably forms tighter binding interactions with metal ions limiting their conformational space in the gas-phase. Conversely, the higher diversity of conformational families for the branched-cyclic topologies further supports that the metal ions probably interact with a higher number of electronegative groups arising from the fully unconstraint C-terminal part. A correlation between the lengths of the loop and the C-terminal tail with the conformational space of lasso peptides becomes apparent upon addition of metal ions. It was shown that the threaded C-terminal region in lasso peptides allows only for distinct interactions of the metal ion with either residues in the loop or tail region. This limits the size of the interacting region and apparently leads to a bias of metal ion binding in either the loop or tail region, depending whichever section is larger in the respective lasso peptide. For branched-cyclic peptides, the non-restricted C-terminal tail allows metal coordination by residues throughout this region, which can result in gas-phase structures that are sometimes even more compact than the lasso peptides. The high TIMS resolution also resulted in the separation of almost all lasso and branched-cyclic topoisomer metal ions ( r ∼ 2.1 on average). It is also shown that the metal incorporation ( e.g. , doubly cesiated species) can lead to the formation of a simplified IMS pattern (or preferential conformers), which results in baseline analytical separation and discrimination between lasso and branched-cyclic topologies using TIMS-MS. 

   more » « less
4. Tandem trapped ion mobility spectrometry

   https://doi.org/10.1039/c7an02054f  

   Liu, Fanny C. ; Ridgeway, Mark E. ; Park, Melvin A. ; Bleiholder, Christian ( January 2018 , The Analyst)

   There is currently a strong interest in the use of ion mobility spectrometry-mass spectrometry (IMS-MS) instrumentation for structural biology. In these applications, momentum transfer cross sections derived from IMS-MS measurements are used to reconstruct the three-dimensional analyte structure. Recent reports indicate that additional structural information can be extracted from measuring changes in cross sections in response to changes of the analyte structure. To further this approach, we constructed a tandem trapped IMS analyser (TIMS-TIMS) and incorporated it in a QqTOF mass spectrometer. TIMS-TIMS is constructed by coupling two TIMS analysers via an “interface region” composed of two apertures. We show that peptide oligomers (bradykinin) and native-like protein (ubiquitin) ions can be preserved through the course of an experiment in a TIMS-TIMS analyser. We demonstrate the ability to collisionally-activate as well as to trap mobility-selected ions, followed by subsequent mobility-analysis. In addition to inducing conformational changes, we show that we can fragment low charge states of ubiquitin at >1 mbar between the TIMS analysers with significant sequence coverage. Many fragment ions exhibit multiple features in their TIMS spectra, which means that they may not generally exist as the most stable isomer. The ability of TIMS-TIMS to dissociate mobility-selected protein ions and to measure the cross sections of their fragment ions opens new possibilities for IMS-based structure elucidation. 

   more » « less
5. Effective discrimination of gas-phase peptide conformers using TIMS-ECD-ToF MS/MS

   https://doi.org/10.1039/d1ay01461g  

   Jeanne Dit Fouque, K. ; Wellmann, M. ; Leyva Bombuse, D. ; Santos-Fernandez, M. ; Cintron-Diaz, Y. L. ; Gomez-Hernandez, M. E. ; Kaplan, D. ; Voinov, V. G. ; Fernandez-Lima, F. ( November 2021 , Analytical Methods)

   In the present work, four, well-studied, model peptides ( e.g. , substance P, bradykinin, angiotensin I and AT-Hook 3) were used to correlate structural information provided by ion mobility and ECD/CID fragmentation in a TIMS-q-EMS-ToF MS/MS platform, incorporporating an electromagnetostatic cell (EMS). The structural heterogeneity of the model peptides was observed by (i) multi-component ion mobility profiles (high ion mobility resolving power, R ∼115–145), and (ii) fast online characteristic ECD fragmentation patterns per ion mobility band (∼0.2 min). Particularly, it was demonstrated that all investigated species were probably conformers, involving cis / trans -isomerizations at X-Pro peptide bond, following the same protonation schemes, in good agreement with previous ion mobility and single point mutation experiments. The comparison between ion mobility selected ECD spectra and traditional FT-ICR ECD MS/MS spectra showed comparable ECD fragmentation efficiencies but differences in the ratio of radical (˙)/prime (′) fragment species (H˙ transfer), which were associated with the differences in detection time after the electron capture event. The analysis of model peptides using online TIMS-q-EMSToF MS/MS provided complementary structural information on the intramolecular interactions that stabilize the different gas-phase conformations to those obtained by ion mobility or ECD alone. 

   more » « less