Following a Folding Transition with Capillary Electrophoresis and Ion Mobility Spectrometry.

Ion mobility spectrometry (IMS) is increasingly used to describe solution-phase phenomena and has recently been used to establish the presence of multiple intermediates during the folding of a model polypeptide, polyproline. These observations, however, are made on gas-phase structures. Capillary electrophoresis (CE) is a complementary solution-phase technique, also based on the separation of charged species as a function of size and charge. Here, both ion mobility and capillary electrophoresis are used to follow the folding transition of a 13-mer polyproline peptide from the all-cis polyproline I (PPI) conformation to the all-trans polyproline II (PPII) conformation upon immersion in aqueous solvent. Synchronous folding processes are observed using both techniques. Eight conformers are observed using ion mobility. Although only five peaks are observed using capillary electrophoresis, these peaks can be modeled as sums of the observed IMS conformers; this is strong evidence that ion mobility is sampling solution-phase structures. CE measurements provide the first direct evidence that multiple folding intermediates are present in solution.

"Wet" Versus "Dry" Folding of Polyproline.

When the all-cis polyproline-I helix (PPI, favored in 1-propanol) of polyproline-13 is introduced into water, it folds into the all-trans polyproline-II (PPII) helix through at least six intermediates [Shi, L., Holliday, A.E., Shi, H., Zhu, F., Ewing, M.A., Russell, D.H., Clemmer, D.E.: Characterizing intermediates along the transition from PPI to PPII using ion mobility-mass spectrometry. J. Am. Chem. Soc. 136, 12702-12711 (2014)]. Here, we show that the solvent-free intermediates refold into the all-cis PPI helix with high (>90%) efficiency. Moreover, in the absence of solvent, each intermediate appears to utilize the same small set of pathways observed for the solution-phase PPII → PPI transition upon immersion of PPIIaq in 1-propanol. That folding in solution (under conditions where water is displaced by propanol) and folding in vacuo (where energy required for folding is provided by collisional activation) occur along the same pathway is remarkable. Implicit in this statement is that 1-propanol mimics a "dry" environment, similar to the gas phase. We note that intermediates with structures that are similar to PPIIaq can form PPII under the most gentle activation conditions-indicating that some transitions observed in water (i.e., "wet" folding, are accessible (albeit inefficient) in vacuo. Lastly, these "dry" folding experiments show that PPI (all cis) is favored under "dry" conditions, which underscores the role of water as the major factor promoting preference for trans proline. Graphical Abstract ᅟ.

Ion Mobility-Mass Spectrometry Reveals the Energetics of Intermediates that Guide Polyproline Folding.

Proline favors trans-configured peptide bonds in native proteins. Although cis/trans configurations vary for non-native and unstructured states, solvent also influences these preferences. Water induces the all-cis right-handed polyproline-I (PPI) helix of polyproline to fold into the all-trans left-handed polyproline-II (PPII) helix. Our recent work has shown that this occurs via a sequential mechanism involving six resolved intermediates [Shi, L., Holliday, A.E., Shi, H., Zhu, F., Ewing, M.A., Russell, D.H., Clemmer, D.E.: Characterizing intermediates along the transition from PPI to PPII using ion mobility-mass spectrometry. J. Am. Chem. Soc. 136, 12702-12711 (2014)]. Here, we use ion mobility-mass spectrometry to make the first detailed thermodynamic measurements of the folding intermediates, which inform us about how and why this transition occurs. It appears that early intermediates are energetically favorable because of the hydration of the peptide backbone, whereas late intermediates are enthalpically unfavorable. However, folding continues, as the entropy of the system increases upon successive formation of each new structure. When PPII is immersed in 1-propanol, the PPII→PPI transition occurs, but this reaction occurs through a very different mechanism. Early on, the PPII population splits onto multiple pathways that eventually converge through a late intermediate that continues on to the folded PPI helix. Nearly every step is endothermic. Folding results from a stepwise increase in the disorder of the system, allowing a wide-scale search for a critical late intermediate. Overall, the data presented here allow us to establish the first experimentally determined energy surface for biopolymer folding as a function of solution environment.

Configurationally-Coupled Protonation of Polyproline-7.

Structure and dynamics regulate protein function, but much less is known about how biomolecule-solvent interactions affect the structure-function relationship. Even less is known about the thermodynamics of biomolecule-solvent interactions and how such interactions influence conformational entropy. When transferred from propanol into 40:60 propanol:water under acidic conditions, a remarkably slow protonation reaction coupled with the conversion of the polyproline-I helix (PPI, having all cis-configured peptide bonds) into polyproline-II (PPII, all trans) helix is observed in this work. Kinetics and equilibrium measurements as a function of temperature allow determination of the thermochemistry and insight into how proton transfer is regulated in this system. For the proton-transfer process, PPI(+)(PrOH) + H3O(+) → PPII(2+)(PrOH/aq) + H2O, we determine ΔG = -20 ± 19 kJ·mol(-1), ΔH = -75 ± 14 kJ·mol(-1), and ΔS= -188 ± 48 J·mol(-1)·K(-1) for the overall reaction, and values of ΔG(⧧) = 91 ± 3 kJ·mol(-1), ΔH(⧧) = 84 ± 9 kJ·mol(-1), and ΔS(⧧) = -23 ± 31 J·mol(-1)·K(-1) for the transition state. For a minor process, PPI(+)(PrOH) → PPII(+)(PrOH/aq) without protonation, we determine ΔG = -9 ± 20 kJ·mol(-1), ΔH = 64 ± 14 kJ·mol(-1), and ΔS= 247 ± 50 J·mol(-1)·K(-1). This thermochemistry yields ΔG = -10 ± 29 kJ·mol(-1), ΔH = -139 ± 20 kJ·mol(-1), and ΔS= -435 ± 70 J·mol(-1)·K(-1) for PPII(+)(PrOH/aq) + H3O(+) → PPII(2+)(PrOH/aq) +H2O. The extraordinarily slow proton transfer appears to be an outcome of configurational coupling through a PPI-like transition state.

Characterizing intermediates along the transition from polyproline I to polyproline II using ion mobility spectrometry-mass spectrometry.

Polyproline exists predominately as the all-cis polyproline I (PPI) helix in aliphatic alcohols, whereas the all-trans polyproline II (PPII) helix is favored in aqueous solutions. Previous ion mobility spectrometry-mass spectrometry (IMS-MS) work demonstrates that the gas-phase conformations of polyproline ions can be related to the corresponding PPI and PPII helices in solution [J. Phys. Chem. B 2004, 108, 4885]. Here, we use IMS-MS to examine the detailed intermediate steps associated with the process of Polyproline-13 (Pro13) conversion from the PPI helix to the PPII helix upon solvent exchange. Collision cross section distributions of Pro13 [M + 2H](2+) ions obtained at different transition times indicate the presence of two major conformers, identified as the PPI and PPII helices, and six conformers that appear as subpopulations of polyproline. Further analysis shows a transition mechanism with sequential cis-trans isomerizations followed by a parallel process to establish PPII and two smaller subpopulations at equilibrium. Temperature-dependent studies are used to obtain Arrhenius activation parameters for each step of the mechanism, and molecular dynamics simulations provide insight about the structures of the intermediates. It appears that prolines sequentially flip from cis to trans starting from the N-terminus. However, after the first few transitions, possible steps take place at the center of the peptide chain; subsequently, several pathways appear to be accessible at the same time. Our results reflect the existence of stable subpopulations in polyprolines and provide new insight into the structural changes during the transition process of polyproline peptides converting from PPI to PPII in aqueous solution.

Oscillations of chiral preference in proline clusters.

Ion mobility/mass spectrometry techniques are used to study the chiral preferences of small proline clusters (containing 2 to 23 proline monomers) produced by electrospray ionization. By varying the composition of the electrospray solution from enantiomerically pure (100% L or 100% D) to racemic (50:50 L:D), it is possible to delineate which cluster sizes prefer homochiral (resolved) or heterochiral (antiresolved) compositions. The results show a remarkable oscillation in chiral preference. Singly protonated clusters, [xPro+H](+) (where x corresponds to the number of prolines), favor homochiral assemblies (for x = 4, 6, 11 and 12); heterochiral structures are preferred (although the preferences are not as strong) for x = 5 and 7. Larger, doubly protonated clusters [xPro+2H](2+) favor homochiral assemblies for x = 18, 19, and 23 and heterochiral structures for x = 14, 16, 17, 20, 21, and 22. Some of the variations that are observed can be rationalized through simple structures that would lead to especially stable geometries. It is suggested that some antiresolved clusters, such as [22Pro+2H](2+), may be comprised of resolved D- and L-proline domains.

Chirality and packing in small proline clusters.

The chiral composition of amino acid clusters may be related to the origin of chirality in biological systems. Here, we use ion mobility/mass spectrometry techniques to investigate the gas phase structures of singly charged proline clusters containing two to six monomers. Using deuterated L-proline (L(D7)) and different electrospray solution compositions varying from enantiopure (50:50 L:L(D7)) to racemic (50:50 L(D7):D), it is possible to study collision cross sections of L-, D-, and mixed xL:xD-proline clusters (where x refers to the number of monomers). These results show that [2Pro+H](+) and [3Pro+H](+) clusters, previously shown (Holliday et al. J. Phys. Chem. A 2012, DOI: 10.1021/jp302677n) to have a very small heterochiral preference, have similar collision cross sections for homochiral and heterochiral proline assemblies. The [4Pro+H](+) and [6Pro+H](+) clusters that exhibit homochiral preference have smaller collision cross sections for homochiral clusters and larger collision cross sections for heterochiral clusters. The [5Pro+H](+) cluster with heterochiral preference has a smaller collision cross section for its heterochiral compositions than for its homochiral compositions. These results suggest that the packing efficiency of subunits within each cluster influences the stability and prevalence of proline multimers as either homochiral or mixed L- and D- clusters.

Collisional activation of [14Pro+2H]2+ clusters: chiral dependence of evaporation and fission processes.

Ion mobility/mass spectrometry techniques are used to investigate the dissociation of the small proline cluster [14Pro+2H](2+) produced by electrospray ionization. While this cluster is known to prefer heterochiral compositions (i.e., mixed L- and D-compositions, J. Phys. Chem. A, submitted for publication), it is possible to produce homochiral forms by electrospraying solutions containing only L- or D-proline. Differences in the measured cross sections for [14Pro+2H](2+) produced from enantiomerically pure (100% l or 100% d) or racemic (50:50 l/d) solutions indicate that homochiral and heterochiral clusters have different structures. Upon low-energy collisional activation, both the heterochiral and homochiral doubly charged structures evaporate neutral proline monomers, resulting in the formation of [xPro+2H](2+) ions (where x = 9-13). At higher activation energies, there is evidence that these smaller clusters (primarily [10Pro+2H](2+)) fission to produce [xPro+H](+) (where x = 1-6). Analysis of product ion intensities reveals a strong chiral preference associated with fissioning. Products of evaporation also show a chiral dependence but to a lesser extent.

Differences in defensive volatiles of the forked fungus beetle, Bolitotherus cornutus, living on two species of fungus.

Forked fungus beetles, Bolitotherus cornutus, feed, mate, and live on the brackets of several species of shelf fungus that grow on decaying logs. In response to the specific threat stimulus of mammalian breath, B. cornutus beetles produce a volatile defensive secretion. We tested beetles collected from different host fungi to determine whether defensive secretion blends varied with host type. Using solid phase microextraction and gas chromatography-mass spectrometry, we detected large amounts of the alkylated benzoquinones, methyl-p-benzoquinone (toluquinone) and ethyl-p-benzoquinone, and smaller quantities of p-benzoquinone, 3-methylphenol (m-cresol), 3-ethylphenol, 2-methylhydroquinone, and 2-ethylhydroquinone in secretions. Volatile composition did not differ between male and female beetles. Secretions did differ between beetles collected from two species of fungus, Ganoderma applanatum and Fomes fomentarius, with the relative amount of p-benzoquinone secreted being the most important factor. Other relationships among the volatile components are discussed.

Field distribution in an electrospray ionization source determined by finite element method.

Three-dimensional computer models of electrospray ionization sources were constructed in COMSOL Multiphysics to solve the static electric fields using finite element methods. The magnitude of the electric field strength for onset of electrospray and optimum signal was calculated under various conditions. The modification of the electric field distribution in the ion source by an atmospheric pressure ion lens was also investigated by plotting the equipotential surfaces, electric field lines and trajectories of charged droplets. Both the calculated and the experimental results demonstrate that the changes in the ion signal detected by the mass spectrometer are attributable to the focusing effect of the ion lens when appropriate voltages are applied on the sprayer and ion lens. The optimum signal was found by setting the sprayer voltage from 3000 to 5000 V while scanning the ion lens voltage. The calculated strengths of the electric field at the sprayer tip for optimum signals are similar although the applied voltages at the sprayer and ion lens are significantly different.

Functionalized mesoporous silicates for the removal of ruthenium from reaction mixtures.

[reaction: see text] The removal of residual ruthenium after olefin metathesis reactions and asymmetric hydrogenations has been described using amine-functionalized mesoporous silicates. Aminopropyltriethoxy silane-derivatized silicates were found to be the most effective. More than 99.99% of RuCl(3) could be removed in a single treatment. Scavenging of Ru after metathesis reactions using a Grubbs generation 1 catalyst gave a product with concentrations of less than 10 microg/5 mg after two treatments with amine-modified silica. Most importantly, no chromatography was required.