RESUMEN
Chymotrypsin inhibitor 2 (CI-2) is a well-studied, textbook example of a cooperative, two-state, native â denatured folding transition. A recent hybrid ion mobility spectrometry (IMS)/mass spectrometry (MS) thermal denaturation study of CI-2 (the well-studied truncated 64-residue model) in water reported evidence that this two-state transition involves numerous (â¼41) unique native and non-native (denatured) solution conformations. The characterization of so many, often low-abundance, states is possible because of the very high dynamic range of IMS-MS measurements of ionic species that are produced upon electrospraying CI-2 solutions from a variable temperature electrospray ionization source. A thermodynamic analysis of these states revealed large changes in enthalpy (ΔH) and entropy (ΔS) at different temperatures, and it was suggested that such variation might arise because of temperature-dependent conformational changes of the protein in response to changes in the conformational entropy and the dielectric permeability of water, which drops from a value of ε â¼ 79 at 24 °C to â¼ 60 at 82 °C. Herein, we examine how adding methanol to water influences the distributions of CI-2 conformers and their ensuing stabilities. The dielectric constant of a 60:40 water:methanol (MeOH) drops from ε â¼ 60 at 24 °C to â¼ 51 at 64 °C. Although the same set of conformers observed in water appears to be present in 60:40 water:MeOH, the abundance of each is substantially altered by the presence of methanol. Relative free energy values (ΔG) and thermodynamic values [ΔH and ΔS and heat capacities (ΔCp)] are derived from a Gibbs-Helmholtz analysis. A comparison of these data from water and water:MeOH systems allows rare insight into how variations in solvation and temperature affect many-state protein equilibria. While these studies confirm that variations in solvent dielectric constant with temperature affect the distributions of conformers that are observed, our findings suggest that other solvent differences may also affect abundances.
RESUMEN
Peptides with penultimate proline residues undergo trans â cis isomerization of the Phe1-Pro2 peptide bond followed by spontaneous bond cleavage at the Pro2-Xxx3 bond (where Xxx is another amino acid residue), leading to cleavage of the Pro2-Xxx3 bond and formation of a diketopiperazine (DKP). In this paper, ion mobility spectrometry and mass spectrometry techniques were used to study the dissociation kinetics of nine peptides [Phe1-Pro2-Glyn-Lysn+3 (n = 1-9)] in ethanol. Shorter (n = 1-3) peptides are found to be more stable than longer (n = 4-9) peptides. Alanine substitution studies indicate that, when experiments are initiated, the Phe1-Pro2 bond of the n = 9 peptide exists exclusively in the cis configuration, while the n = 1-8 peptides appear to exist initially with both cis- and trans-Phe1-Pro2 configured bonds. Molecular dynamics simulations indicate that intramolecular hydrogen bonding interactions stabilize conformations of shorter peptides, thus inhibiting DKP formation. Similar stabilizing interactions appear less frequently in longer peptides. In addition, in smaller peptides, the N-terminal amino group is more likely to be charged compared to the same group in longer peptides, which would inhibit the dissociation through the DKP formation mechanism. Analysis of temperature-dependent kinetics measurements provides insight about the mechanism of bond cleavage. The analysis gives the following transition state thermochemistry: ΔG⧧ values range from 94.6 ± 0.9 to 101.5 ± 1.9 kJ·mol-1, values of ΔH⧧ range from 89.1 ± 0.9 to 116.7 ± 1.5 kJ·mol-1, and ΔS⧧ values range from -25.4 ± 2.6 to 50.8 ± 4.2 J·mol-1·K-1. Proposed mechanisms and thermochemistry are discussed.
Asunto(s)
Dicetopiperazinas , Péptidos , Enlace de Hidrógeno , Cinética , ProlinaRESUMEN
The thermal stabilities of endogenous, intact proteins and protein assemblies in complex mixtures were characterized in parallel by means of variable-temperature electrospray ionization coupled to mass spectrometry (vT-ESI-MS). The method is demonstrated by directly measuring the melting transitions of seven proteins from a mixture of proteins derived from ribosomes. A proof-of-concept measurement of a fraction of an Escherichia coli lysate is provided to extend this approach to characterize the thermal stability of a proteome. As the solution temperature is increased, proteins and protein complexes undergo structural and organizational transitions; for each species, the folded â unfolded and assembled â disassembled populations are monitored based on changes in vT-ESI-MS charge state distributions and masses. The robustness of the approach illustrates a step toward the proteome-wide characterization of thermal stabilities and structural transitions-the stabilitome.
Asunto(s)
Proteínas Ribosómicas , Espectrometría de Masa por Ionización de Electrospray , Escherichia coli , Proteoma , TemperaturaRESUMEN
We present simple considerations of how differences in time scales of motions of protons, the lightest and fastest chemical moiety, and the much longer time scales associated with the dynamics of proteins, among the heaviest and slowest analytes, may allow many protein conformations from solution to be kinetically trapped during the process of electrospraying protein solutions into the gas phase. In solution, the quantum nature of protons leads them to change locations by tunneling, an instantaneous process; moreover, the Grotthuss mechanism suggests that these small particles can respond nearly instantaneously to the dynamic motions of proteins that occur on much longer time scales. A conformational change is accompanied by favorable or unfavorable variations in the free energy of the system, providing the impetus for solvent â protein proton exchange. Thus, as thermal distributions of protein conformations interconvert, protonation states rapidly respond, as specific acidic and basic sites are exposed or protected. In the vacuum of the mass spectrometer, protons become immobilized in locations that are specific to the protein conformations from which they were incorporated. In this way, conformational states from solution are preserved upon electrospraying them into the gas phase. These ideas are consistent with the exquisite sensitivity of electrospray mass spectra to small changes of the local environment that alter protein structure in solution. We might remember this approximation for the protonation of proteins in solution with the colloquial expression-protons are fast and smart; proteins are slow and dumb.
Asunto(s)
Proteínas/análisis , Proteínas/química , Protones , Espectrometría de Masa por Ionización de Electrospray/métodos , Cinética , Conformación ProteicaRESUMEN
The understanding and prediction of the solubility of biomolecules, even of the simplest ones, reflect an open question and unmet need. Short aromatic tripeptides are among the most highly aggregative biomolecules. However, in marked contrast, Ala-Phe-Ala (AFA) was surprisingly found to be non-aggregative and could be solubilized at millimolar concentrations. Here, aiming to uncover the underlying molecular basis of its high solubility, we explore in detail the solubility, aggregation propensity, and atomic-level structure of the tripeptide. We demonstrate an unexpectedly high water solubility of AFA reaching 672 mM, two orders of magnitude higher than reported previously. The single crystal structure reveals an anti-parallel ß sheet conformation devoid of any aromatic interactions. This study provides clear mechanistic insight into the structural basis of solubility and suggests a simple and feasible tool for its estimation, bearing implications for design of peptide drugs, peptides materials, and advancement of peptide nanotechnology.
RESUMEN
Ion mobility spectrometry (IMS) and mass spectrometry (MS) techniques were used to monitor diketopiperazine (DKP) formation from the peptide FPG8K at multiple defined temperatures in methanol, ethanol, propanol, and water, with the motivation to study the effect of solvent polarity on spontaneous solution dissociation. The reaction rate increases with decreasing solvent polarity. The observed rates of trans â cis isomerization of Phe1-Pro2 and the cis-Pro2 isomer dissociation result in the cis isomer growing in abundance relative to the trans isomer throughout the reaction in all solvents. Analysis of rate constants derived from the data using a sequential unimolecular kinetics model that includes hidden intermediate states yields transition state thermodynamic values for both trans â cis isomerization of Phe1-Pro2 and dissociation. The measured thermochemistry appears to be closely correlated with these solvents' dielectric constants: a lower solvent dielectric constant accelerates the reaction by reducing the enthalpic barrier, albeit with slight entropic restriction.
Asunto(s)
Dicetopiperazinas , Isomerismo , Cinética , Solventes , TermodinámicaRESUMEN
The heterogeneity associated with glycosylation of the 66 N-glycan sites on the protein trimer making up the spike (S) region of the SARS-CoV-2 virus has been assessed by charge detection mass spectrometry (CDMS). CDMS allows simultaneous measurement of the mass-to-charge ratio and charge of individual ions, so that mass distributions can be determined for highly heterogeneous proteins such as the heavily glycosylated S protein trimer. The CDMS results are compared to recent glycoproteomics studies of the structure and abundance of glycans at specific sites. Interestingly, average glycan masses determined by "top-down" CDMS measurements are 35-47% larger than those obtained from the "bottom-up" glycoproteomics studies, suggesting that the glycoproteomic measurements underestimated the abundances of larger, more-complex glycans. Moreover, the distribution of glycan masses determined by CDMS is much broader than the distribution expected from the glycoproteomics studies, assuming that glycan processing on each trimer is not correlated. The breadth of the glycan mass distribution therefore indicates heterogeneity in the extent of glycan processing of the S protein trimers, with some trimers being much more heavily processed than others. This heterogeneity may have evolved as a way of further confounding the host's immune system.
Asunto(s)
Espectrometría de Masas , Polisacáridos/metabolismo , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células HEK293 , Humanos , Dominios ProteicosRESUMEN
Thermal denaturation of holomyoglobin (hMb) in solution (10 mM ammonium acetate at pH = 4.5, 6.8, and 9.0) was monitored by ion mobility spectrometry (IMS) and mass spectrometry (MS) techniques to characterize the stability and investigate structural changes involved in unfolding. We utilize two experimental approaches to induce thermal denaturation: a variable-temperature electrospray ionization (vT-ESI) source that heats the bulk solution in the ESI emitter, and a variable-power 10.6 µm CO2 laser that rapidly heats nanodroplets produced by ESI. These two approaches sample different time scales of the denaturation process; long time scales (seconds to minutes) where the system is at equilibrium using the vT-ESI approach and shorter time scales (µs) by rapid droplet heating in which the system is in a pre-equilibrium state. Increasing the solution temperature (from 28 to 95 °C in the vT-ESI experiments) shifts the charge state distribution from low charge states ([M + 7H]7+ to [M + 9H]9+) to more highly charged species. This is accompanied by loss of the heme group to yield the apomyoglobin (aMb) species, indicating that the protein has unfolded. Monitoring the formation of aMb and the shift in average charge states of aMb and hMb with solution temperature allows for relative quantitation of their individual stabilities, highlighting the stabilizing effects of heme binding. We compare the degree of unfolding induced by heating the bulk solution (using vT-ESI) to the laser droplet heating approach and find that the rapid nature of the laser heating approach allows for transient pre-equilibrium states to be sampled.
RESUMEN
Chymotrypsin inhibitor 2 (CI-2) is a classic model for two-state cooperative protein folding and is one of the most extensively studied systems. Alan Fersht, a pioneer in the field of structural biology, has studied the wild-type (wt) and over 100 mutant forms of CI-2 with traditional analytical and biochemical techniques. Here, we examine wt CI-2 and three mutant forms (A16G, K11A, L32A) to demonstrate the utility of variable-temperature (vT) electrospray ionization (ESI) paired with ion mobility spectrometry (IMS) and mass spectrometry (MS) to map the free energy folding landscape. As the solution temperature is increased, the abundance of each of the six ESI charge states for wt CI-2 and each mutant is found to vary independently. These results require that at least six unique types of CI-2 solution conformers are present. Ion mobility analysis reveals that within each charge state there are additional conformers having distinct solution temperature profiles. A model of the data at â¼30 different temperatures for all four systems suggests the presence of 41 unique CI-2 solution conformations. A thermodynamic analysis of this system yields values of ΔCp as well as ΔG, ΔH, and ΔS for each state at every temperature studied. Detailed energy landscapes derived from these data provide a rare glimpse into Anfinsen's thermodynamic hypothesis and the process of thermal denaturation, normally thought of as a cooperative two-state transition involving the native state and unstructured denatured species. Specifically, as the temperature is varied, the entropies and enthalpies of different conformers undergo dramatic changes in magnitude and relative order to maintain the delicate balance associated with equilibrium.
Asunto(s)
Proteínas Mutantes/química , Péptidos/química , Proteínas de Plantas/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Secuencia de Aminoácidos , Modelos Químicos , Transición de Fase , Conformación Proteica , Pliegue de Proteína , Temperatura , TermodinámicaRESUMEN
Thermally induced structural transitions of the quaternary structure of the hemoglobin tetramer (human) in aqueous solution (150 mM ammonium acetate) were investigated using a variable temperature electrospray ionization (vt-ESI) technique in combination with ion mobility spectrometry (IMS) and mass spectrometry (MS) measurements. At low solution temperatures (28 to â¼40 °C), a heterotetrameric (α2ß2) complex is the most abundant species that is observed. When the solution temperature is increased, this assembly dissociates into heterodimers (holo αß forms) before ultimately forming insoluble aggregates at higher temperatures (>60 °C). In addition to the holo αß forms, a small population of αß dimers containing only a single heme ligand and having a dioxidation modification mapping to the ß subunit are observed. The oxidized heterodimers are less stable than the unmodified holo-heterodimer. The Cys93 residue of the ß subunit is the primary site of dioxidation. The close proximity of this post translational modification to both the αß subunit interface and the heme binding site suggests that this modification is coupled to the loss of the heme and decreased protein stability. Changes in the charge state and collision cross sections of these species indicate that the tetramers and dimers favor less compact structures at elevated temperatures (prior to temperatures where dissociation dominates).
Asunto(s)
Hemoglobina A/análisis , Temperatura , Humanos , Espectrometría de Movilidad Iónica , Espectrometría de Masas , Estructura Secundaria de Proteína , SolucionesRESUMEN
The strong synergy arising from coupling two orthogonal analytical techniques such as ion mobility and mass spectrometry can be used to separate complex mixtures and determine structural information of analytes in the gas phase. A tandem study is performed using two systems with different gases and pressures to ascertain gas-phase conformations of homopolymer ions. Aside from spherical and stretched configurations, intermediate configurations formed by a multiply charged globule and a "bead-on-a-string" appendix are confirmed for polyethylene-glycol (PEG), polycaprolactone (PCL), and polydimethylsiloxane (PDMS). These intermediate configurations are shown to be ubiquitous for all charge states and masses present. For each charge state, configurations evolve in two distinctive patterns: an inverse evolution which occurs as an elementary charge attached to the polymer leaves the larger globule and incorporates itself into the appendage, and a forward evolution which reduces the globule without relinquishing a charge while leaving the appendix relatively constant. Forward evolutions are confirmed to form self-similar family shapes that transcend charge states for all polymers. Identical structural changes occur at the same mass over charge regardless of the system, gas or pressure strongly suggesting that conformations are only contingent on number of charges and chain length, and start arranging once the ion is at least partially ejected from the droplet, supporting a charge extrusion mechanism. Configurational changes are smoother for PDMS which is attributed to the larger steric hindrance caused by protruding pendant groups. This study has implications in the study of the configurational space of more complex homopolymers and heteropolymers. Graphical Abstract.
RESUMEN
Ubiquitin confined within nanodroplets was irradiated with a variable-power CO2 laser. Mass spectrometry analysis shows evidence for a protein "melting"-like transition within droplets prior to solvent evaporation and ion formation. Ion mobility spectrometry reveals that structures associated with early steps of denaturation are trapped because of short droplet lifetimes.
RESUMEN
Ion mobility and mass spectrometry techniques are coupled with a temperature-controlled electrospray ionization source to follow the structural transitions of ubiquitin in aqueous solution (pH = 3) at elevated solution temperatures (T = 26-96 °C). Changes in the charge state distribution are consistent with a two-state, cooperative unfolding transition having a melting temperature of Tm = 71 ± 2 °C, in agreement with prior measurements [ Wintrode , P. L. ; Makhatadze , G. I. ; Privalov , P. L. Proteins , 1994 , 18 , 246 - 253 ]. However, analysis of ion mobility distributions reveals the two-state transition is a composite of transitions involving at least nine unique species: three native or native-like structures; two that appear to be equilibrium intermediates (i.e., populations of new conformers that form at elevated temperatures but subsequently disappear at higher temperatures); and four products observed at high temperatures, including the well-characterized ubiquitin A state, and two solution species that are differentiated based on a cis- or trans-configured Glu18-Pro19 peptide bond. These nine states vary in abundances by factors as large as â¼103 over the range of solution temperatures. Although experimental melting transitions are conceived as a loss of well-defined structure leading to a random distribution of unstructured, denatured forms, the results provide evidence for new conformers having at least some well-defined structural elements are stabilized as temperature is increased.
Asunto(s)
Temperatura , Ubiquitina/química , Conformación Proteica , Desnaturalización Proteica , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Chiral analysis of constituents in solution-phase reaction mixtures can be performed by tandem mass spectrometry using the kinetic method to determine the enantiomeric excess (ee). Simply diluting an aliquot of a reaction mixture, adjusting the pH, and adding reagents necessary to form a chiral cluster ion allows chiral analysis. The product of a stereospecific N-selective alkylation reaction, 2-(3-(2-methoxyethoxy)-5-oxo-1,6-naphthyridin-6(5H)-yl)propanoic acid, was monitored for ee during the course of reaction, and it showed the expected inversion without ee erosion. Base-catalyzed racemization of the reaction product showed the expected decrease in ee as the reaction proceeded. The base-catalyzed racemization of ibuprofen was monitored on-line, providing near real-time data on ee.