Uncoupled analysis of secondary and tertiary protein structure by circular dichroism and electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) applied to protein conformational studies is a powerful new method that seems to provide specific information about protein tertiary structure. In this study, we analyzed the effect of trifluoroethanol (TFE) on a myoglobin peptide and cytochrome c (cyt c) at low pH by circular dichroism (CD) and ESI-MS. These experiments show that coil-to-helix transition per se does not affect ESI mass spectra, confirming that this technique is insensitive to the local conformation of the polypeptidic chain and, rather, reports on the tertiary contacts characterizing different protein conformations. This property makes ESI-MS an excellent method, complementary to CD, for the characterization of protein conformational changes. Fluorinated alcohols have been suggested to induce molten globule formation in acid-unfolded cyt c. The experiments described here show that TFE does not induce major changes in the ESI mass spectrum of cyt c at pH 2.2, indicating that no stabilization of compact, globular structures is detectable under the conditions employed. On the other hand, even low concentrations of TFE (2-5%) are shown to destabilize the folded state of the protein around the mid-point of its acid-induced unfolding transition.     

Thermal unfolding of proteins probed by laser spray mass spectrometry

The stability and conformational changes of cytochrome c (cyt c) at different temperatures and pH have been well examined so far by using various analytical methods. We have found that laser spray mass spectrometry enables much faster and more convenient monitoring of those changes of cyt c compared with other methods. The results correlated well with circular dichroism (CD) experiments under relatively acidic conditions, which destabilize the protein. Laser spray mass spectra of cyt c at various pH were obtained at different levels of laser power. Bimodal charge-state distributions of the protein were observed in laser spray mass spectra, indicating the two-state model of structural change; the lower charges correspond to the folded state, the higher charges to the unfolded state. Based on this result, the presumed denaturation curve of the protein was plotted as a function of laser power, and laser power by which 50% of the protein was assumed to be denatured, E50%, as obtained at each pH. We also examined the melting temperatures, Tm, of cyt c at various values of pH by using CD spectroscopy. The correlation coefficient between E50% and Tm for cyt c was 0.999, demonstrating an excellent correlation. Furthermore, laser spray analysis of ubiquitin, which is found to be more thermally stable than cyt c, gave a higher E50% than cyt c. These results indicate that laser spray mass spectrometry can be an extremely convenient method for probing thermal stabilities and dynamic conformational changes of proteins with subtle structural differences caused by slight changes in pH.     

Detection of the equilibrium folding intermediate of beta-lactoglobulin in the presence of trifluoroethanol by mass spectrometry

Nano-electrospray ionization mass spectrometry (nano-ESI-MS) was used to monitor the effect of trifluoroethanol (TFE) on the conformational properties of beta-lactoglobulin (BLG). TFE stabilizes protein secondary structure, particularly alpha-helices. However, it also acts as a denaturant above critical concentrations. In the case of BLG, TFE at low concentrations is known to induce formation of an equilibrium intermediate that contains non-native helical structure. Such an intermediate is thought to form also under physiological conditions, playing a role in BLG folding in vivo by preventing aggregation. This well-characterized system was chosen in order to test species distributions obtained by nano-ESI-MS. BLG spectra at increasing concentrations of TFE at pH 2 indicate transient accumulation of a conformer whose charge-state distribution (CSD) falls between that of the native and that of the denatured protein, indicating that the TFE-induced, partially folded form can be selectively monitored by this technique. The condition of its maximum accumulation corresponds to 16% TFE, in excellent agreement with results from solution experiments. In contrast, titrations with methanol or acetonitrile (ACN) reveal apparent two-state transitions from native to fully unfolded BLG. At 10% TFE, the protein appears to be still fully folded at room temperature but, if unfolding is elicited by the combination with other denaturing agents, e.g. heat or low concentrations of ACN, it proceeds via formation of the intermediate. Thus, TFE can also induce formation of the BLG intermediate in synergism with generic denaturing agents. This study indicates good agreement between ESI-MS and other biophysical methods monitoring protein conformational transitions in the presence of TFE.     

Characterization of β2‐microglobulin conformational intermediates associated to different fibrillation conditions

β2‐Microglobulin (β2m) is the light chain of the class‐I major histocompatibility complex, being also the causing agent of dialysis‐related amyloidosis, which results from its accumulation as amyloid material in the skeletal joints. This study describes conformational properties of β2m under two distinct, in vitro amyloidogenic conditions: neutral pH in the presence of 20% 2,2,2‐trifluoroethanol (TFE) and acidic pH in the absence of TFE. Species distribution analysis by electrospray ionization–mass spectrometry (ESI‐MS) is combined with information obtained by ion mobility–mass spectrometry (IM‐MS), fluorescence and circular dichroism (CD) spectroscopy. It is shown that β2m populates quite different conformational ensembles under the two conditions, but both ensembles display a minor fraction of the population in a partially folded state. In spite of similar compactness, these two partially folded forms display different conformations: helical secondary structure is predominant in the species at pH 7.4, 20% TFE, while the low‐pH form is mainly random coil. As temperature is increased, the TFE intermediate looses helical structure becoming more similar to the low‐pH intermediate. The existence of different conformational ensembles may rationalize the different aggregation propensity displayed by β2m under the two fibrillation conditions analyzed here. Copyright © 2011 John Wiley & Sons, Ltd.     

Electrospray ionization mass spectra of acyl carrier protein are insensitive to its solution phase conformation

Electrospray ionization mass spectrometry (ESI-MS) can be used to monitor conformational changes of proteins in solution based on the charge state distribution (CSD) of the corresponding gas-phase ions, although relatively few studies of acidic proteins have been reported. Here, we have compared the CSD and solution structure of recombinant Vibrio harveyi acyl carrier protein (rACP), a small acidic protein whose secondary and tertiary structure can be manipulated by pH, fatty acylation, and site-directed mutagenesis. Circular dichroism and intrinsic fluorescence demonstrated that apo-rACP adopts a folded helical conformation in aqueous solution below pH 6 or in 50% acetonitrile/0.1% formic acid, but is unfolded at neutral and basic pH values. A rACP mutant, in which seven conserved acidic residues were replaced with their corresponding neutral amides, was folded over the entire pH range of 5 to 9. However, under the same solvent conditions, both wild type and mutant ACPs exhibited similar CSDs (6(+)-9(+) species) at all pH values. Covalent attachment of myristic acid to the phosphopantetheine prosthetic group of rACP, which is known to stabilize a folded conformation in solution, also had little influence on its CSD in either positive or negative ion modes. Overall, our results are consistent with ACP as a "natively unfolded" protein in a dynamic conformational equilibrium, which allows access to (de)protonation events during the electrospray process.     

Electrochemistry of unfolded cytochrome c in neutral and acidic urea solutions

The present investigation reports the first experimental measurements of the reorganization energy of unfolded metalloprotein in urea solution. Horse heart cytochrome c (cyt c) has been found to undergo reversible one-electron transfer reactions at pH 2 in the presence of 9 M urea. In contrast, the protein is electrochemically inactive at pH 2 under low-ionic strength conditions in the absence of urea. Urea is shown to induce ligation changes at the heme iron and lead to practically complete loss of the alpha-helical content of the protein. Despite being unfolded, the electron-transfer (ET) kinetics of cyt c on a 2-mercaptoethanol-modified Ag(111) electrode remain unusually fast and diffusion controlled. Acid titration of ferric cyt c in 9 M urea down to pH 2 is accompanied by protonation of one of the axial ligands, water binding to the heme iron (pK(a) = 5.2), and a sudden protein collapse (pH < 4). The formal redox potential of the urea-unfolded six-coordinate His18-Fe(III)-H(2)O/five-coordinate His18-Fe(II) couple at pH 2 is estimated to be -0.083 V vs NHE, about 130 mV more positive than seen for bis-His-ligated urea-denatured cyt c at pH 7. The unusually fast ET kinetics are assigned to low reorganization energy of acid/urea-unfolded cyt c at pH 2 (0.41 +/- 0.01 eV), which is actually lower than that of the native cyt c at pH 7 (0.6 +/- 0.02 eV), but closer to that of native bis-His-ligated cyt b(5) (0.44 +/- 0.02 eV). The roles of electronic coupling and heme-flattening on the rate of heterogeneous ET reactions are discussed.     

Testing the role of solvent surface tension in protein ionization by electrospray

This work was aimed at probing the influence of solvent surface tension on protein ionization by electrospray. In particular, we were interested in testing the previously suggested hypothesis that the charge-state distributions (CSDs) of proteins in electrospray ionization mass spectrometry (ESI-MS) are controlled by the surface tension of the least volatile solvent component. In the attempt to minimize uncontrolled conformational effects, we used acid-sensitive proteins (cytochrome c and myoglobin) at low pH or highly stable proteins (ubiquitin and lysozyme) in the presence of low concentrations of organic solvents. A first set of experiments compared the effect of 1- and 2-propanol. These two alcohols have similar chemico-physical properties but values of vapor pressure below and above that of water, respectively. Both compounds have much lower surface tension than water. The solvents employed allowed testing of the influence of surface tension on protein spectra obtained from similarly denaturing solutions. The compared solvent conditions gave rise to very similar spectra for each tested protein. We then investigated the effect of the addition of dimethyl sulfoxide to acid-unfolded proteins. We observed enhanced ionization in the presence of acetic or formic acid, consistent with the previously described supercharging effect, but almost no shift of the CSD in the presence of HCl. Finally, we analyzed thermally denatured cytochrome c, to obtain reference spectra of the unfolded protein in high-surface-tension solutions. Also in this case, the CSD of the unfolded protein was shifted towards lower m/z values relative to low-surface-tension systems. In contrast to the other results reported here, this effect is consistent with an influence of solvent surface tension on CSD. The magnitude of the effect, however, is much smaller than predicted by the Rayleigh equation. The results presented here are not easy to reconcile with the hypothesis that the maximum charge state exhibited by proteins in ESI-MS reflects the Rayleigh-limit charge of the precursor droplet. The data are discussed with reference to models for the mechanism of electrospray ionization.     

Conformational analysis of dynorphin A (1-13) using hydrogen-deuterium exchange and tandem mass spectrometry

Trifluoroethanol (TFE)-induced conformational changes in dynorphin A (1-13) were investigated using charge-state distribution (CSD) and hydrogen-deuterium exchange (HDX), combined with electrospray ionization (ESI) mass spectrometry (MS). Individual amino acids involved in secondary structural elements were identified by collision-induced dissociation-tandem mass spectrometry (MS/MS). It was observed that dynorphin A (1-13) largely exists in an unfolded conformation and a folded structure in increasing concentrations of TFE. In 50% TFE, it forms an alpha-helix that encompasses residues 1-9 and remains flexible from residues 10 to 13.     

Folding of the 25 residue Abeta(12-36) peptide in TFE/water: temperature-dependent transition from a funneled free-energy landscape to a rugged one

The free-energy landscape of the Alzheimer beta-amyloid peptide Abeta(12-36) in a 40% (v/v) 2,2,2-trifluoroethanol (TFE)/water solution was determined by using multicanonical molecular dynamics simulations. Simulations using this enhanced conformational sampling technique were initiated from a random unfolded polypeptide conformation. Our simulations reliably folded the peptide to the experimental NMR structure, which consists of two linked helices. The shape of the free energy landscape for folding was found to be strongly dependent on temperature: Above 325 K, the overall shape was funnel-like, with the bottom of the funnel coinciding exactly with the NMR structure. Below 325 K, on the other hand, the landscape became increasingly rugged, with the emergence of new conformational clusters connected by low free-energy pathways. Finally, our simulations reveal that water and TFE solvate the polypeptide in different ways: The hydrogen bond formation between TFE and Abeta was enhanced with decreasing temperature, while that between water and Abeta was depressed.     

Interaction Between Cytochrome c and the Hapten 2,4-Dinitro-fluorobenzene by Electrospray Ionization Mass Spectrometry

Allergic contact dermatitis is a delayed hypersensitivity reaction, which results from skin exposure to low molecular weight chemicals such as haptens. To clarify the pathogenic mechanism, electrospray ionization mass spectrometry (ESI-MS) and hydrogen/deuterium (H/D) exchange, as well as UV spectroscopy, were applied to determine the interaction between the model protein cytochrome c (cyt c) and the hapten 2,4-dinitro-fluorobenzene (DNFB). The ESI-MS results demonstrate that the conformation of cyt c can change from native folded state into partially unfolded state with the increase of DNFB. The equilibrium state H/D exchange followed by ESI-MS further confirms the above results. UV spectroscopy indicates that the strongfield coordination between iron of heme (prosthetic group) and His18 or Met80 of cyt c is not obviously affected by the hapten.     

Ultrafast Heme Dynamics of Ferric Cytochrome c in Different Environments: Electronic,Vibrational, and Conformational Relaxation

The excited‐state dynamics of ferric cytochrome c (Cyt c), an important electron‐transfer heme protein, in acidic to alkaline medium and in its unfolded form are investigated by using femtosecond pump–probe spectroscopy, exciting the heme and Tryptophan (Trp) to understand the electronic, vibrational, and conformational relaxation of the heme. At 390 nm excitation, the electronic relaxation of heme is found to be ≈150 fs at different pH values, increasing to 480 fs in the unfolded form. Multistep vibrational relaxation dynamics of the heme, including fast and slow processes, are observed at pH 7. However, in the unfolded form and at pH 2 and 11, fast phases of vibrational relaxation dominate, revealing the energy dissipation occurring through the covalent bond interaction between the heme and the nearest amino acids. A significant shortening of the excited‐state lifetime of Trp is observed at various pH values at 280 nm excitation due to resonance energy transfer to the heme. The longer time constant (25 ps) observed in the unfolded form is attributed to a complete global conformational relaxation of Cyt c.     

pH-Induced changes in adsorbed cytochrome c. voltammetric and surface-enhanced resonance Raman characterization performed simultaneously at chemically modified silver electrodes

The influence of pH on the redox properties of cytochrome c (cyt c) adsorbed on roughened silver electrodes chemically modified with a self-assembled monolayer (SAM) of 11-mercapto-1-undecanoic acid (MUA) was studied with voltammetric techniques in combination with surface-enhanced resonance Raman scattering (SERRS). The experiments were performed simultaneously on the same electrode sample in a homemade spectroelectrochemical cell suitable for such applications. At pH 7.0 cyt c was found in its native state; at higher pH values (ranging from 8.0 to 9.0) the redox properties of the adsorbed protein varied considerably, featuring a redox behavior which does not resemble the one reported for the alkaline transition. Our results instead indicate the presence of an electrochemically inactive 6cLS species immobilized on MUA at pH 9.0. The pH-induced conformational changes observed for cyt c immobilized on the SAM of MUA were found to be repeatable and chemically reversible, meaning that the recovery of the electrochemical signal due to the native protein occurred instantaneously (on the second time scale) when the electrode was switched back to pH 7.0. The pH-induced changes observed were attributed to a conformational change involving a heme reorientation with respect to the electrode surface.     

Cytochrome c–dimyristoylphosphatidylglycerol interactions studied by asymmetrical flow field-flow fractionation

Lipid membranes are well recognized ligands that bind peripheral and integral proteins in a specific manner and regulate their function. Cytochrome c (cyt c) is one of the partner peripheral protein that binds to the lipid membranes via electrostatic and hydrophobic interactions. In this study, asymmetrical flow field-flow fractionation (AsFlFFF) was used to compare the interactions of cyt c with the acidic phospholipid 1,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol (DMPG), oleic acid (OA), and sodium dodecyl sulfate (SDS). The influence of pH and the cyt c–lipid molar mass ratios were evaluated by monitoring the diffusion coefficients and particle diameter distributions obtained for the free and lipid-bound protein. The hydrodynamic particle diameter of cyt c (pI 10) was 4.1 nm at pH 11.4 and around 4.2 nm at pH 7.0 and 8.0. Standard molar mass marker proteins were used for calibration to obtain the molar masses of free cyt c and its complexes with lipids. AsFlFFF revealed the binding of cyt c to DMPG and to OA to be mainly electrostatic. In the absence of electrostatic interactions, minor complex formation occurred, possibly due to the extended lipid anchorage involving the hydrophobic cavity of cyt c and the hydrocarbon chains of DMPG or SDS. The possibility of the formation of the molten globule state of cyt c, induced by the interaction between cyt c and lipids, is discussed.Electronic Supplementary Material Supplementary material is available for this article at     

A tandem time-of-flight mass spectrometer: combination of a multi-turn time-of-flight and a quadratic-field mirror

A tandem time-of-flight (ToF) mass spectrometer consisting of a multi-turn time-of-flight (ToF) and a quadratic-field ion mirror has been designed and constructed. The instrument combines the unique capabilities of both ToF instruments, namely high-resolution and monoisotopic precursor ion selection from the multi-turn ToF and temporal focus for fragment ions with different kinetic energies from the quadratic-field mirror. The first tandem mass spectra for this unique combination of ToF systems are presented.     

Interactions of apo cytochrome C with alternating copolymers of maleic acid and alkene

Apo cytochrome c (apo cyt c) tends to aggregate at alkali pH. Poly(isobutylene-alt-maleic acid) (PIMA) is soluble molecularly, whereas poly(1-tetradecene-alt-maleic acid) (PTMA) forms particles that tend to dissociate by increasing pH and decreasing concentration. Dynamic light scattering and surface plasmon resonance are used to investigate the interactions of PIMA and PTMA with apo cyt c at different pH values to understand the mechanism of the interactions. When the positive or negative charges are in excess, the copolymer-protein complex particles can be stabilized by the charges on the surface. When the ratio of the positive to negative charges is close to the stoichiometric value, precipitation occurs. At pH 11.8, both PTMA and apo cyt c carry negative charges, but the hydrophobic interaction makes them form complexes. A competition exists between the interaction of the copolymer with apo cyt c and the self-aggregation of PTMA or apo cyt c alone. The interaction of PIMA or PTMA with apo cyt c at neutral and alkali pH destroys the aggregation of PTMA or apo cyt c and forms new complex particles.     

Dynamic spectroelectrochemical measurement for the conformational transition of cytochrome c induced by bromopyrogal red

The conformational transition of horse heart cytochrome c induced by bromopyrogal red (BPR) in very low concentration has been firstly investigated by dynamic spectroelectrochemical technique, both at the BPR adsorbed platinum gauze electrode and at a bare platinum gauze electrode in a solution containing BPR. The effect of BPR on the structure of cytochrome c was studied by UV-visible and Fourier transform IR spectroscopy. The unfolded cytochrome c behaves simply as an electron transfer protein with a formal potential of −142 mV vs. a normal hydrogen electrode. The difference between the formal potentials of the native and unfolded cytochrome c is coupled to a difference in conformational energy of the two states of about 40 kJ mol⁻¹, which agrees well with the result reported. The stability and slow refolding of the unfolded cytochrome c are discussed.     

Protein charge-state distributions in electrospray-ionization mass spectrometry do not appear to be limited by the surface tension of the solvent

According to a current model for protein electrospray, the charge-state distributions (CSDs) observed by electrospray-ionization mass spectrometry (ESI-MS) are controlled by the Rayleigh-limit charge of the droplets that generate the gas-phase protein ions. A testable prediction of this model is that the maximum charge state displayed by proteins in ESI-MS should respond to changes in the surface tension of the ESI droplets according to the Rayleigh equation. In this work, we subject this specific hypothesis to direct experimental testing. We show data obtained by time-of-flight (TOF) nano-ESI-MS with several different proteins in aqueous solutions containing 20-50% 1-propanol or 40% 1,2-propylene glycol. Both of these compounds have lower vapor pressure and lower surface tension than water. Propylene glycol also has a lower evaporation rate than water, providing an even more stringent test for surface tension effects in late ESI droplets. None of these cosolvents affects the CSDs of either folded or unfolded proteins as predicted by the Rayleigh-charge model. The only changes induced by 1-propanol can be ascribed to protein unfolding triggered above critical concentrations of the alcohol. Below such a threshold, no shift of the CSDs toward lower charge states is observed. The presence of 40% propylene glycol in the original protein solutions gives rise to CSDs that either are the same as those in the control samples or present much smaller changes than those calculated by the Rayleigh equation. Thus, the charge states of gas-phase protein ions produced by electrospray do not seem to be limited by the surface tension of the solvent. They rather appear to be quite protein-specific.     

Identification of non-covalent structure in apocytochrome c by hydrogen exchange and mass spectrometry

Apocytochrome c, the in vivo precursor to active cytochrome c, was analyzed by amide hydrogen exchange and mass spectrometry to search for fixed, non-covalent structure. The protein was incubated in H(2)O at pH 3.3 or 6.7 for various times, then exposed to D(2)O to initiate isotope labeling of unfolded regions. Following acid quenching of hydrogen exchange, the labeled apocytochrome c was digested with pepsin into fragments that were analyzed by directly coupled high-performance liquid chromatography/electrospray ionization mass spectrometry. The intermolecular distribution of deuterium and the deuterium levels in structurally distinctive populations were determined from the mass spectra of the peptic fragments. Spectra of peptic fragments derived from apocytochrome c incubated at pH 3.3 had single envelopes of isotope peaks with masses indicating that all of the amide hydrogens had been replaced with deuterium. These results showed that apocytochrome c at pH 3.3 offered little resistance to hydrogen exchange, indicating that it was unfolded with little fixed structure. However, mass spectra of peptic fragments including residues 81-94 of apocytochrome c incubated at pH 6.7 had two envelopes of isotope peaks, indicating that one population was unfolded and the other population was highly structured in this region. Mass spectra of peptic fragments including residues N-terminal to residue 81 indicated that this region of the protein remained unfolded with little fixed structure at pH 6.7.     

Mass spectrometry and hydrogen/deuterium exchange measurements of alcohol-induced structural changes in cellular retinol-binding protein type I

To bind and release its ligand, cellular retinol-binding protein type I (CRBP) needs to undergo conformational and dynamic changes to connect the inner, solvent-shielded cavity, where retinol is found to bind, and the outside medium. Retinol dissociation in vitro is favoured by water/alcohol mixtures whose moderately low dielectric constants mimic a property characteristic of the membrane microenvironment where this process occurs in vivo. Apo- and holo-CRBP, in either water/methanol or water/trifluoroethanol (TFE) mixtures, were analyzed at equilibrium by electrospray ionization with orthogonal quadrupole time-of-flight mass spectrometry (ESI-Q-TOFMS) to identify the alcohol-induced species. The questions were asked whether the presence of alcohols affects protein dynamics, as reflected by hydrogen/deuterium (H/D) exchange monitored by continuous-labelling experiments, and to which extent retinol dissociation influences the process. With increasing methanol, at pH near neutrality, apo-CRBP exhibits a progressively more compact conformation, resulting in reduced H/D exchange with respect to the native protein in water. Retinol dissociation from the holo-protein did not promote hydrogen replacement. Similarly, in the presence of the low TFE concentration sufficient to cause retinol dissociation, the hydrogen exchange of the resulting apo-protein was not exalted. However, in contrast with the alkanol, higher TFE concentrations induced a transition of apo-CRBP to a new alpha-helix conformation capable of exchanging all available hydrogen atoms.