Influence of the Fluorophore Mobility on Distance Measurements by Gas-Phase FRET.

Gas-phase Förster resonance energy transfer (FRET) combines mass spectrometry and fluorescence spectroscopy for the conformational analysis of mass-selected biomolecular ions. In FRET, fluorophore pairs are typically covalently attached to a biomolecule using short linkers, which affect the mobility of the dye and the relative orientation of the transition dipole moments of the donor and acceptor. Intramolecular interactions may further influence the range of motion. Yet, little is known about this factor, despite the importance of intramolecular interactions in the absence of a solvent. In this study, we applied transition metal ion FRET (tmFRET) to probe the mobility of a single chromophore pair (Rhodamine 110 and Cu2+) as a function of linker lengths to assess the relevance of intramolecular interactions. Increasing FRET efficiencies were observed with increasing linker length, ranging from 5% (2 atoms) to 28% (13 atoms). To rationalize this trend, we profiled the conformational landscape of each model system using molecular dynamics (MD) simulations. We captured intramolecular interactions that promote a population shift toward smaller donor-acceptor separation for longer linker lengths and induce a significant increase in the acceptor's transition dipole moment. The presented methodology is a first step toward the explicit consideration of a fluorophore's range of motion in the interpretation of gas-phase FRET experiments.

Determining the gas-phase structures of α-helical peptides from shape, microsolvation, and intramolecular distance data.

Mass spectrometry is a powerful technique for the structural and functional characterization of biomolecules. However, it remains challenging to accurately gauge the gas-phase structure of biomolecular ions and assess to what extent native-like structures are maintained. Here we propose a synergistic approach which utilizes Förster resonance energy transfer and two types of ion mobility spectrometry (i.e., traveling wave and differential) to provide multiple constraints (i.e., shape and intramolecular distance) for structure-refinement of gas-phase ions. We add microsolvation calculations to assess the interaction sites and energies between the biomolecular ions and gaseous additives. This combined strategy is employed to distinguish conformers and understand the gas-phase structures of two isomeric α-helical peptides that might differ in helicity. Our work allows more stringent structural characterization of biologically relevant molecules (e.g., peptide drugs) and large biomolecular ions than using only a single structural methodology in the gas phase.

Dilated cardiomyopathy mutation E525K in human beta-cardiac myosin stabilizes the interacting-heads motif and super-relaxed state of myosin.

The auto-inhibited, super-relaxed (SRX) state of cardiac myosin is thought to be crucial for regulating contraction, relaxation, and energy conservation in the heart. We used single ATP turnover experiments to demonstrate that a dilated cardiomyopathy (DCM) mutation (E525K) in human beta-cardiac myosin increases the fraction of myosin heads in the SRX state (with slow ATP turnover), especially in physiological ionic strength conditions. We also utilized FRET between a C-terminal GFP tag on the myosin tail and Cy3ATP bound to the active site of the motor domain to estimate the fraction of heads in the closed, interacting-heads motif (IHM); we found a strong correlation between the IHM and SRX state. Negative stain electron microscopy and 2D class averaging of the construct demonstrated that the E525K mutation increased the fraction of molecules adopting the IHM. Overall, our results demonstrate that the E525K DCM mutation may reduce muscle force and power by stabilizing the auto-inhibited SRX state. Our studies also provide direct evidence for a correlation between the SRX biochemical state and the IHM structural state in cardiac muscle myosin. Furthermore, the E525 residue may be implicated in crucial electrostatic interactions that modulate this conserved, auto-inhibited conformation of myosin.

Structural Studies of a Stapled Peptide with Native Ion Mobility-Mass Spectrometry and Transition Metal Ion Förster Resonance Energy Transfer in the Gas Phase.

Native mass spectrometry has emerged as an important tool for gas-phase structural biology. However, the conformations that a biomolecular ion adopts in the gas phase can differ from those found in solution. Herein, we report a synergistic, native ion mobility-mass spectrometry (IM-MS) and transition metal ion Förster resonance energy transfer (tmFRET)-based approach to probe the gas-phase ion structures of a nonstapled peptide (nsp; Ac-CAARAAHAAAHARARA-NH2) and a stapled peptide (sp; Ac-CXARAXHAAAHARARA-NH2). The stapled peptide contains a single hydrocarbon chain connecting the peptide backbone in the i and i + 4 positions via a Grubbs ring-closure metathesis. Fluorescence lifetime measurements indicated that the Cu-bound complexes of carboxyrhodamine 6g (crh6g)-labeled stapled peptide (sp-crh6g) had a shorter donor-acceptor distance (rDA) than the labeled nonstapled peptide (nsp-crh6g). Experimental collision cross-section (CCS) values were then determined by native IM-MS, which could separate the conformations of Cu-bound complexes of nsp-crh6g and sp-crh6g. Finally, the experimental CCS (i.e., shape) and rDA (i.e., distance) values were used as constraints for computational studies, which unambiguously revealed how a staple reduces the elongation of the peptide ions in the gas phase. This study demonstrates the superiority of combining native IM-MS, tmFRET, and computational studies to investigate the structure of biomolecular ions.

Adapting a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer for Gas-Phase Fluorescence Spectroscopy Measurement of Trapped Biomolecular Ions.

Gas-phase fluorescence spectroscopy is still in its infancy, which demands further instrumental developments. In this study, a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS), equipped with a lab-developed data acquisition system, was coupled to a tunable femtosecond laser and a state-of-the-art optical system for fluorescence studies of mass-selected ions. For excitation, a laser beam was focused (beam size < 1.0 mm) into the cylindrical ICR cell. A wire mesh replaced the back trapping plate, allowing ∼10% of the fluorescence emitted from trapped ions to be collected by a lens installed beside the wire mesh. The collected fluorescence light was then transmitted outside of the mass spectrometer via fiber optics. A novel accumulation during detection (ADD) scheme was developed to increase the duty cycle of gas-phase fluorescence spectroscopy experiments. With ADD, >90% duty cycle for mass spectrometry and fluorescence experiments could be achieved. This instrument was able to perform fluorescence experiments on various ions, from simple rhodamine dyes to large biomolecules (i.e., peptides and proteins) labeled with dyes of various optical properties. A fluorescence lifetime measurement of trapped rhodamine 6G cations was also performed, yielding a value of 5.97 ± 0.23 ns. This setup has a broad mass range and decent fluorescence spectroscopy performance (i.e., the emission spectrum of rhodamine 6G can be acquired with good S/N in a minute). Finally, this setup also allows more challenging gas-phase fluorescence spectroscopy experiments, for example, of low quantum yield fluorophores and large biomolecules in their native state that appear at high m/z, which may not be doable with quadrupole ion traps (QIT).

Transition Metal Ion FRET in the Gas Phase: A 10-40 Å Range Molecular Ruler for Mass-Selected Biomolecular Ions.

Structural studies of mass-selected biomolecules in the gas phase can reveal their intrinsic properties and provide useful benchmarks for biomolecular modeling. Here, we report the first evidence of transition metal ion FRET (tmFRET) in the gas phase and its application to measure short (10-40 Å) biomolecular backbone distances. The measured FRET efficiencies in rhodamine dye (donor) labeled helical peptides complexed with Cu2+ ions (acceptor) decreased with increasing donor - acceptor distances, confirming the occurrence of tmFRET. The distances estimated for similar peptide sequences from the FRET efficiencies were consistently longer in the gas phase compared to those reported in solution, indicating an expanded structure and a possible loss of helicity.

Fluorescence-based Techniques to Study the Structure and Dynamics of Mass-selected Biomolecular Ions.

Laser-induced fluorescence studies on mass-selected biomolecules are a promising route to understand their properties in the gas phase and probe their intrinsic properties in a solvent-free environment. Fluorescence has been used to investigate the conformation and dynamics of gaseous biomolecular ions. With Förster Resonance Energy Transfer (FRET), it is now possible to obtain sensitive intramolecular distance information from large biomolecules, like proteins, with high chemical specificity. With growing interest and applications, gas-phase fluorescence measurements can shed greater light on the characteristics of proteins in the gas phase. Compared to the solution phase measurements, gas-phase fluorescence can also help understand the influence of solvent interactions on the protein structure and function.

Fluorescence-Based Detection of the Desolvation Process of Protein Ions Generated in an Aqueous Electrospray Plume.

A new experimental setup to study laser-induced fluorescence from analytes at different locations in an electrospray plume has been developed. The high fluorescence collection efficiency (∼2%) of the setup, along with a sensitive charge coupled device (CCD) detector, enabled the study of low ion concentrations (down to ∼fM) in the plume. The use of small electrospray tip inner diameters (<1 µm) facilitated the fast desolvation of gaseous protein ions in an aqueous electrospray plume. Fluorescence spectra were acquired from specific locations along the plume axis in different aqueous electrospray plumes with three different analytes: a rhodamine dye and two proteins (ubiquitin and apomyoglobin) labeled with rhodamine dyes. To confirm the presence of gaseous ions, pure gas-phase fluorescence spectra were acquired in the vacuum of a modified ion trap mass spectrometer. These spectra were used to fit to confirm the presence of gaseous species in the corresponding spectra obtained from the electrospray plume. This study shows that with small inner diameter spray capillaries, gaseous protein ions generated at atmospheric pressure in an electrospray plume can be detected with fluorescence-based techniques. Fluorescence measurements can be used to study their structure in the electrospray plume, and the dynamics as they transition from solution to the gas phase and in the early stages after desolvation from charged droplets. Other techniques can also be applied to further study gaseous biomolecular structures under ambient conditions immediately after desolvation.

Breaking the Brightness Barrier: Design and Characterization of a Selected-Ion Fluorescence Measurement Setup with High Optical Detection Efficiency.

A quadrupole ion trap (QIT) mass spectrometer has been modified and coupled with tunable laser excitation and highly sensitive fluorescence detection systems to perform fluorescence studies on mass-selected ions. Gaseous ions, generated using nanoelectrospray ionization (nano-ESI), are trapped in the QIT that allows optical access for laser irradiation. The emitted fluorescence is collected from a 5.0 mm diameter hole drilled into the ring electrode of the QIT and is directed toward the detection setup. Due to the small inner diameter (7.07 mm) of the ring electrode and a relatively large opening for fluorescence collection, a fluorescence collection efficiency of 2.3% is achieved. After some losses in transmission, around 1.8% of the emitted fluorescence reaches the detectors, more than any other similar instrument reported in the literature. This improved fluorescence collection translates to a much shorter measurement time for a fluorescence signal. Another key feature of this setup is the ability to perform a variety of fluorescence experiments on trapped ions including excitation and emission spectroscopy, lifetime measurement, and ion imaging. The capabilities of the instrument are demonstrated by measuring fluorescence spectra of dyes and biomolecules labeled with dyes in a range of different excitation and emission wavelengths, quantum yields, m/z, and different polarities. A fluorescence lifetime measurement and ion image of trapped rhodamine 6G cations are also shown. With a wide array of functionality and high fluorescence detection performance, this setup provides an opportunity to study biomolecular structures and photophysics of fluorophores in well-controlled environments.

Cryo-EM structure of the inhibited (10S) form of myosin II.

Myosin II is the motor protein that enables muscle cells to contract and nonmuscle cells to move and change shape1. The molecule has two identical heads attached to an elongated tail, and can exist in two conformations: 10S and 6S, named for their sedimentation coefficients2,3. The 6S conformation has an extended tail and assembles into polymeric filaments, which pull on actin filaments to generate force and motion. In 10S myosin, the tail is folded into three segments and the heads bend back and interact with each other and the tail3-7, creating a compact conformation in which ATPase activity, actin activation and filament assembly are all highly inhibited7,8. This switched-off structure appears to function as a key energy-conserving storage molecule in muscle and nonmuscle cells9-12, which can be activated to form functional filaments as needed13-but the mechanism of its inhibition is not understood. Here we have solved the structure of smooth muscle 10S myosin by cryo-electron microscopy with sufficient resolution to enable improved understanding of the function of the head and tail regions of the molecule and of the key intramolecular contacts that cause inhibition. Our results suggest an atomic model for the off state of myosin II, for its activation and unfolding by phosphorylation, and for understanding the clustering of disease-causing mutations near sites of intramolecular interaction.

N-terminal domain replacement changes an archaeal monoacylglycerol lipase into a triacylglycerol lipase.

BACKGROUND: Lipolytic enzymes of hyperthermophilic archaea generally prefer small carbon chain fatty acid esters (C2-C12) and are categorized as esterases. However, a few have shown activity with long-chain fatty acid esters, but none of them have been classified as a true lipase except a lipolytic enzyme AFL from Archaeglobus fulgidus. Thus, our main objective is to engineer an archaeal esterase into a true thermostable lipase for industrial applications. Lipases which hydrolyze long-chain fatty acid esters display an interfacial activation mediated by the lid domain which lies over active site and switches to open conformation at the oil-water interface. Lid domains modulate enzyme activities, substrate specificities, and stabilities which have been shown by protein engineering and mutational analyses. Here, we report engineering of an uncharacterized monoacylglycerol lipase (TON-LPL) from an archaeon Thermococcus onnurineus (strain NA1) into a triacylglycerol lipase (rc-TGL) by replacing its 61 N-terminus amino acid residues with 118 residues carrying lid domain of a thermophilic fungal lipase-Thermomyces lanuginosus (TLIP). RESULTS: TON-LPL and rc-TGL were cloned and overexpressed in E. coli, and the proteins were purified by Ni-NTA affinity chromatography for biochemical studies. Both enzymes were capable of hydrolyzing various monoglycerides and shared the same optimum pH of 7.0. However, rc-TGL showed a significant decrease of 10 °C in its optimum temperature (Topt). The far UV-CD spectrums were consistent with a well-folded α/ß-hydrolase fold for both proteins, but gel filtration chromatography revealed a change in quaternary structure from trimer (TON-LPL) to monomer (rc-TGL). Seemingly, the difference in the oligomeric state of rc-TGL may be linked to a decrease in temperature optimum. Nonetheless, rc-TGL hydrolyzed triglycerides and castor oil, while TON-LPL was not active with these substrates. CONCLUSIONS: Here, we have confirmed the predicted esterase activity of TON-LPL and also performed the lid engineering on TON-LPL which effectively expanded its substrate specificity from monoglycerides to triglycerides. This approach provides a way to engineer other hyperthermophilic esterases into industrially suitable lipases by employing N-terminal domain replacement. The immobilized preparation of rc-TGL has shown significant activity with castor oil and has a potential application in castor oil biorefinery to obtain value-added chemicals.

Understanding anomalous mobility of proteins on SDS-PAGE with special reference to the highly acidic extracellular domains of human E- and N-cadherins.

During SDS-PAGE experiments, proteins generally display electrophoretic mobility in keeping with their molecular weights; however, some proteins display anomalies in mobility. Here, we focus attention on the anomalies displayed by the highly acidic ∼110 residues-long, sequence-homologous, structurally-analogous, extracellular domains of human E- and N-cadherin. We report that there is a strong correlation between the acidity of each domain and the degree of the anomaly that it displays. The anomaly is only seen if the ratio of the numbers of negatively-charged and positively-charged residues is equal to or greater than the value of 1.50. The degree of the anomaly rises in proportion with this NC:PC ratio. Greater-than-expected anomalies are observed for domains containing dense clusters of negatively charged residues. A simple explanation for these observations is that highly acidic domains electrostatically repel SDS. This results in insufficient SDS binding, insufficient electromotive incentive and (consequently) lowered electrophoretic mobility. This explanation is in consonance with the current view that initial stages of SDS-protein engagement tend to be dominated by electrostatics. We discuss the current anomalies within the broader context of all conceivable explanations for such anomalies.

Multiple thermostable enzyme hydrolases on magnetic nanoparticles: An immobilized enzyme-mediated approach to saccharification through simultaneous xylanase, cellulase and amylolytic glucanotransferase action.

Microbe-derived enzymes such as xylanases, cellulases and amylases, are efficient at hydrolyzing plant biomass. Efforts to harness the functionalities of these enzymes towards applications in energy and fuel biosciences, and food and nutrition, continue apace in many laboratories. Given that enzymes derived from mesophile proteomes undergo facile denaturation and/or degradation at ambient temperatures, and require frequent replenishment during bioprocessing, it is desirable that they be replaced by structurally-stable enzymes capable of functioning efficiently and resisting denaturation and degradation, immobilized on solid media to further add to stability and facilitate recovery and reuse. Towards these objectives, we used synthetic magnetic nanoparticles (MNPs) and immobilized upon their surfaces three different structurally-stable hydrolases: a thermostable xylanase (BSX) derived from Bacillus sp. NG-27, a cellulase (RMCel12A) derived from Rhodothermus marinus, and an amylase-cum-glucanotransferase (PfuAmyGT) derived from Pyrococcus furiosus. The MNPs were activated with glutaraldehyde and BSX, RMCel12A, and PfuAmyGT, respectively, were covalently immobilized with efficiencies of ~92%, 45% and 93%. The enzymes and the MNPs were fully characterized before and after immobilization, and the immobilized enzymes were found to be active at 50 °C against synthetic substrates as well as pre-treated biomass derived from corn cob and rice husk. The enzyme-coupled MNPs displayed high stability upon storage properties, high operational stability as well as high reusability (retaining 69, 48, and 50% residual activity after 13 uses for BSX, RMCel12A and PfuAmyGT, respectively). Experiments were also conducted with MNPs loaded simultaneously with all three enzymes. Such immobilized enzyme combinations on MNPs can be used in the saccharification of plant biomass.

Structural-Mechanical and Biochemical Functions of Classical Cadherins at Cellular Junctions: A Review and Some Hypotheses.

This article begins with a general review of cell adhesion molecules (CAMs) and narrows the focus down progressively to the cadherins (calcium binding-dependent CAMs), classifications of subfamilies of the cadherins, type I (E- and N-) cadherins, evolutionary relationships amongst cadherins, structural-mechanical and functional consequences of calcium binding to the cadherins, differential molecular interactions involving the extracellular (ecto) and intracellular (cytoplasmic) domains of the cadherins, multiple adherence-related homophilic and heterophilic interactions and associated functions of E- and N-cadherin in organismal development and disease and cadherin trafficking and membrane rafts. It ends by summarizing multiple perspectives and hypotheses concerning different aspects of cadherin structure, stability and function.

Spectroscopic Evidences for Strong Hydrogen Bonds with Selenomethionine in Proteins.

Careful protein structure analysis unravels many unknown and unappreciated noncovalent interactions that control protein structure; one such unrecognized interaction in protein is selenium centered hydrogen bonds (SeCHBs). We report, for the first time, SeCHBs involving the amide proton and selenium of selenomethionine (Mse), i.e., amide-N-H···Se H-bonds discerned in proteins. Using mass selective and conformer specific high resolution vibrational spectroscopy, gold standard quantum chemical calculations at CCSD(T), and in-depth protein structure analysis, we establish that amide-N-H···Se and amide-N-H···Te H-bonds are as strong as conventional amide-NH···O and amide-NH···O═C H-bonds despite smaller electronegativity of selenium and tellurium than oxygen. It is in fact, electronegativity, atomic charge, and polarizability of the H-bond acceptor atoms are at play in deciding the strength of H-bonds. The amide-N-H···Se and amide-N-H···Te H-bonds presented here are not only new additions to the ever expanding world of noncovalent interactions, but also are of central importance to design new force-fields for better biomolecular structure simulations.

Critical Assessment of the Strength of Hydrogen Bonds between the Sulfur Atom of Methionine/Cysteine and Backbone Amides in Proteins.

Gas-phase vibrational spectroscopy, coupled cluster (CCSD(T)), and dispersion corrected density functional (B97-D3) methods are employed to characterize surprisingly strong sulfur center H-bonded (SCHB) complexes between cis and trans amide NH and S atom of methionine and cysteine side chain. The amide N-H···S H-bonds are compared with the representative classical σ- and π-type H-bonded complexes such as N-H···O, N-H···O═C and N-H···π H-bonds. With the spectroscopic, theoretical, and structural evidence, amide N-H···S H-bonds are found to be as strong as the classical σ-type H-bonds, despite the smaller electronegativity of sulfur in comparison to oxygen. The strength of backbone-amide N-H···S H-bonds in cysteine and methionine containing peptides and proteins are also investigated and found to be of similar magnitudes as those observed in the intermolecular model complexes studied in this work. All such SCHBs also confirm that the electronegativities of the acceptors are not the sole criteria to predict the H-bond strength.