Intrinsic Probe Research Articles

Energy-efficient separation of a ternary azeotropic mixture via azeotropic reflux-induced sequences based on the intensified pressure-swing distillation

The recovery and separation of chloroform, acetonitrile and ethanol from waste water involving two azeotropes and a distillation boundary has emerged as a pivotal concern in the pursuit of environmental protection and resource conservation. Herein, the energy-efficient separation design of the ternary azeotropic mixture based on a triple-column pressure-swing distillation (TCPSD) process with five azeotropic reflux induced sequences is comprehensively investigated. The suitable operating pressure range of the distillation columns and the potential optimal distillation sequence is investigated based on the thermodynamic intrinsic probe. According to the optimal distillation sequence, further process intensification strategies are proposed to improve the thermodynamic efficiency encompassing the heat integration pressure-swing distillation (HI-PSD), heat pump assisted pressure-swing distillation (HP-PSD), and a combination of heat pump assisted and heat integration pressure-swing distillation (HPHI-PSD). It unveils the economic preeminence of the HI-PSD process, characterized by the lowest total annual cost (TAC) in a shorter payback period (3 to 7 years). Conversely, both the HP-PSD and HPHI-PSD processes exhibit superior environmental performance on CO2 emissions of 111.46 kg/h and 119.78 kg/h respectively, with the HP-PSD demonstrating prolonged economic viability and the HPHI-PSD showcasing heightened thermodynamic efficiency of a 65.12 % improvement. This study potentially provides a theoretical basis for devising industrial separation schemes for analogous pressure-sensitive ternary azeotropic systems.

Unveiling the Structural and Dynamic Characteristics of Concentrated LiNO3 Aqueous Solutions through Ultrafast Infrared Spectroscopy and Molecular Dynamics Simulations.

Highly concentrated aqueous electrolytes have attracted a significant amount of attention for their potential applications in lithium-ion batteries. Nevertheless, a comprehensive understanding of the Li+ solvation structure and its migration within electrolyte solutions remains elusive. This study employs linear vibrational spectroscopy, ultrafast infrared spectroscopy, and molecular dynamics (MD) simulations to elucidate the structural dynamics in LiNO3 solutions by using intrinsic and extrinsic vibrational probes. The N-O stretching vibrations of NO3- exhibit a distinct spectral splitting, attributed to its asymmetric interaction with the surrounding solvation structure. Analysis of the vibrational relaxation dynamics of intrinsic and extrinsic probes, in combination with MD simulations, reveals cage-like networks formed through electrostatic interactions between Li+ and NO3-. This microscopic heterogeneity is reflected in the intertwined arrangement of ions and water molecules. Furthermore, both vehicular transport and structural diffusion assisted by solvent rearrangement for Li+ were analyzed, which are closely linked with the bulk concentration.

Distinguishing the transitions of fluorescence spectra of tryptophan-134 and 213 in BSA induced by bindings of UV filters, oxybenzone-3, and avobenzone

Abstract Oxybenzone-3 (OBZ) and avobenzone (ABZ), commercially available ultraviolet-light filters for sunscreens, are known to induce photosensitizing allergy as an adverse effect, similar to an analgesic ketoprofen (KTP) due to their benzophenone moiety. The present study focused on OBZ and ABZ's protein binding compared to the related analgesics, KTP, diclofenac (DCF), and ibuprofen (IBP). The bovine serum albumin (BSA) was used as a protein model, measuring the fluorescent spectral peak shifts (i) and Stern–Volmer analysis (i) of its intrinsic tryptophans. Moreover, their adsorption types (iii) were verified using the singular value decomposition (SVD) computation of fluorescence spectra. For (i), (ii), and (iii), KTP and DCF caused a no-shift peak, an ordinary dynamic quenching, and a simple Langmuir adsorption. We found OBZ exhibiting (i) red-shift and (ii) including static quenching, ABZ suggesting (i) blue-shift and (iii) binding to multiple bind sites, and IBP indicating (i) blue-shift and (iii) multivalent bindings. Integrating the results, it can be understood that OBZ interacts with subdomain IA (around W134) in BSA, while ABZ interacts with subdomain IIA (around W213) in BSA. Moreover, IBP is bound to BSA with a cooperative effect, certified by Hill's plot. OBZ and ABZ had their individual binding sites on a protein, suggesting the exchange between OBZ and ABZ might reduce their own adverse effect. The present study verified the effectiveness of the SVD computation in distinguishing the details of the adsorption manner of ligands around the intrinsic fluorescent probes.

Protein vicinal thiols as intrinsic probes of brain redox states in health, aging, and ischemia

The nature of brain redox metabolism in health, aging, and disease remains to be fully established. Reversible oxidations, to disulfide bonds, of closely spaced (vicinal) protein thiols underlie the catalytic maintenance of redox homeostasis by redoxin enzymes, including thioredoxin peroxidases (peroxiredoxins), and have been implicated in redox buffering and regulation. We propose that non-peroxidase proteins containing vicinal thiols that are responsive to physiological redox perturbations may serve as intrinsic probes of brain redox metabolism. Using redox phenylarsine oxide (PAO)-affinity chromatography, we report that PAO-binding vicinal thiols on creatine kinase B and alpha-enolase from healthy rat brains were preferentially oxidized compared to other selected proteins, including neuron-specific (gamma) enolase, under conditions designed to trap in vivo protein thiol redox states. Moreover, measures of the extents of oxidations of vicinal thiols on total protein, and on creatine kinase B and alpha-enolase, showed that vicinal thiol-linked redox states were stable over the lifespan of rats and revealed a transient reductive shift in these redox couples following decapitation-induced global ischemia. Finally, formation of disulfide-linked complexes between peroxiredoxin-2 and brain proteins was demonstrated on redox blots, supporting a link between protein vicinal thiol redox states and the peroxidase activities of peroxiredoxins. The implications of these findings with respect to underappreciated aspects of brain redox metabolism in health, aging, and ischemia are discussed.

Studies on α-amylase inhibition by acarbose and quercetin using fluorescence, circular dichroism, docking, and dynamics simulations

This study looked at the effects of acarbose (ACA) and quercetin (QUE) on α-amylase activity, employing QUE and ACA to measure enzyme activity. The study observed that both drugs suppressed α-amylase activity, with greater inhibition reported at higher concentrations. The use of tryptophan residues as an intrinsic fluorescence probe permitted the observation of conformational changes in α-amylase, with CD measurements utilized to explore the secondary structure in the presence of QUE and ACA. Docking studies revealed an effective interaction between α-amylase, quercetin and acarbose, with a higher binding energy. Finally, a trajectory analysis was done to establish the stability and volatility of these complexes. These findings have potential significance for the development of new α-amylase-related therapeutics.

Dynamics of Formamide-Water Mixtures Investigated by Linear and Nonlinear Infrared Spectroscopy.

Formamide (FA) exhibits complete miscibility with water, offering a simplified model for exploring the solvation dynamics of peptide linkages in biophysical processes. Its liquid state demonstrates a three-dimensional hydrogen bonding network akin to water, reflecting solvent-like behavior. Analyzing the microscopic structure and dynamics of FA-water mixtures is expected to provide crucial insights into hydrogen bonding dynamics─a key aspect of various biophysical phenomena. This study is focused on the dynamics of FA-water mixtures using linear and femtosecond infrared spectroscopies. By using the intrinsic OD stretch and extrinsic probe SCN-, the local vibrational behaviors across various FA-water compositions were systematically investigated. The vibrational relaxation of OD stretch revealed a negligible impact of FA addition on the vibrational lifetime of water molecules, underscoring the mixture's water-like behavior. However, the reorientational dynamics of OD stretch slowed with increasing FA mole fraction (XFA), plateauing beyond XFA > 0.5. This suggests a correlation between OD's reorientational time and the strength of the hydrogen bond network, likely tied to the solution's changing dielectric constant. Conversely, the vibrational relaxation dynamics of SCN- was strongly correlated with XFA, highlighting a competition between water and FA molecules in solvating SCN-. Moreover, a linear relationship between rising viscosity and the prolonged correlation time of SCN-'s slow dynamics indicates that the solution's macroscopic viscosity is dictated by the extended structures formed between FA and water molecules. The relation between the reorientation dynamics of the SCN- and the macroscopic viscosity in aqueous FA-water mixture solutions was analyzed by using the Stokes-Einstein-Debye equations. The direct viscosity-diffusion coupling is observed, which can be attributed to the homogeneous dynamics feature in FA-water mixture solutions. The inclusion of these intrinsic and extrinsic probes not only enhances the comprehensiveness of our analysis but also provides valuable insights into various aspects of the dynamics within the FA-water system. This investigation sheds light on the fundamental dynamics of FA-water mixtures, emphasizing their molecular-level homogeneity in this binary mixture solution.

Comparison of the self-assembly and cytocompatibility of conjugates of Fmoc (9-fluorenylmethoxycarbonyl) with hydrophobic, aromatic, or charged amino acids.

The self-assembly in aqueous solution of three Fmoc-amino acids with hydrophobic (aliphatic or aromatic, alanine or phenylalanine) or hydrophilic cationic residues (arginine) is compared. The critical aggregation concentrations were obtained using intrinsic fluorescence or fluorescence probe measurements, and conformation was probed using circular dichroism spectroscopy. Self-assembled nanostructures were imaged using cryo-transmission electron microscopy and small-angle X-ray scattering (SAXS). Fmoc-Ala is found to form remarkable structures comprising extended fibril-like objects nucleating from spherical cores. In contrast, Fmoc-Arg self-assembles into plate-like crystals. Fmoc-Phe forms extended structures, in a mixture of straight and twisted fibrils coexisting with nanotapes. Spontaneous flow alignment of solutions of Fmoc-Phe assemblies is observed by SAXS. The cytocompatibility of the three Fmoc-amino acids was also compared via MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] mitochondrial activity assays. All three Fmoc-amino acids are cytocompatible with L929 fibroblasts at low concentration, and Fmoc-Arg shows cell viability up to comparatively high concentration (0.63 mM).

Early events during the aggregation of Aβ16-22-derived switch-peptides tracked using Protein Charge Transfer Spectra

BackgroundUnderstanding Aβ aggregation and inhibiting it at early stages is of utmost importance in treating Alzheimer's and other related amyloidogenic diseases. However, majority of the techniques to study Aβ aggregation mainly target the late stages; while those used to monitor early stages are either expensive, use extrinsic dyes, or do not provide information on molecular level interactions. Here, we investigate the early events of Aβ16-22(KLVFFAE) aggregation using Aβ16-22 derived switch-peptides (SwPs) through a novel label-free approach employing Protein Charge Transfer Spectra (ProCharTS). ResultsWhen pH is increased from 2 to 7.2, the Aβ-derived switch peptides undergo controlled self-assembly, where the initial random coil peptides convert into β-sheet. We leveraged the intrinsic absorbance/luminescence arising from ProCharTS among growing peptide oligomers to observe the aggregation kinetics in real-time. In comparison to monomer, the lysine and glutamate headgroups in the peptide oligomer are expected to come in proximity enhancing ProCharTS intensity due to photoinduced electron transfer. With a combination of Aβ-derived switch-peptides and ProCharTS, we obtained structural insights on the early stages of Aβ-derived SwP aggregation in four unique peptides. Increase in scatter corrected ProCharTS absorbance (250–500 nm) and luminescence (320–720 nm) along with decreased mean luminescence lifetime (2.3–0.8 ns) characterize the initial stages of aggregation monitored for 1–96 h depending on the peptide. We correlated the results with Circular Dichroism (CD), 8-anilino-1-naphthalenesulfonic acid (ANS) and Thioflavin T (ThT) measurements. SignificanceWe demonstrate ProCharTS as an intrinsic analytical probe with following advantages over other conventional methods to track aggregation: it is a label-free probe; it's intensity can be measured using a UV–Vis spectrophotometer; it is more sensitive in detecting the early molecular events in aggregation compared to ANS and ThT; and it can provide information on specific contacts made between charged headgroups of Lysine/Glutamate in the oligomer.

Raman spectroscopic reporting of oxidative stress via intrinsic probes

Raman spectroscopic reporting of oxidative stress via intrinsic probes

Tyrosine fluorescence as an intrinsic probe for protein aggregation

Tyrosine fluorescence as an intrinsic probe for protein aggregation

Fluorescent Probes and Quenchers in Studies of Protein Folding and Protein-Lipid Interactions.

Fluorescence spectroscopy provides numerous methodological tools for structural and functional studies of biological macromolecules and their complexes. All fluorescence-based approaches require either existence of an intrinsic probe or an introduction of an extrinsic one. Moreover, studies of complex systems often require an additional introduction of a specific quencher molecule acting in combination with a fluorophore to provide structural or thermodynamic information. Here, we review the fundamentals and summarize the latest progress in applications of different classes of fluorescent probes and their specific quenchers, aimed at studies of protein folding and protein-membrane interactions. Specifically, we discuss various environment-sensitive dyes, FRET probes, probes for short-distance measurements, and several probe-quencher pairs for studies of membrane penetration of proteins and peptides. The goals of this review are: (a) to familiarize the readership with the general concept that complex biological systems often require both a probe and a quencher to decipher mechanistic details of functioning and (b) to provide example of the immediate applications of the described methods.

Fluorous Tagged Peptides for Intracellular Delivery and Biomedical Imaging.

Fluorous tagged peptides have shown promising features for biomedical applications such as drug delivery and multimodal imaging. The bioconjugation of fluoroalkyl ligands onto cargo peptides greatly enhances their proteolytic stability and membrane penetration via a proposed "fluorine effect". The tagged peptides also efficiently deliver other biomolecules such as DNA and siRNA into cells via a co-assembly strategy. The fluoroalkyl chains on peptides with antifouling properties enable efficient gene delivery in the presence of serum proteins. Besides intracellular biomolecule delivery, the amphiphilic peptides can be used to stabilized perfluorocarbon-filled microbubbles for ultrasound imaging. The fluorine nucleus on fluoroalkyls provides intrinsic probes for background-free magnetic resonance imaging. Labeling of fluorous tags with radionuclide 18 F also allows tracing the biodistribution of peptides via positron emission tomography imaging. This mini-review will discuss properties and mechanism of the fluorous tagged peptides in these applications.

Allosteric and substrate site effects of relevant amino acids on structural stability and flexibility of glutamate dehydrogenase-1 (GDH-1).

Mammalian GDH-1, which converts glutamate to alpha-ketoglutarate and ammonia while reducing NAD(P), is constitutively expressed in virtually all cells of the body. The extensive literature on GDH-1 suggests that functionally relevant GDH-1 structures may be distributed over a conformational flexibility landscape: polyhexamer, hexamer, trimer, monomer, monomer (unfolded), monomer (misfolded), polymer (disassembled). We have used both intrinsic (tryptophan) and extrinsic (ANS) fluorescent probes to study the dependence of the reversible hexamer to trimer dissociation on the concentrations of the denaturing agents urea and guanidine hydrochloride (GdnHCl). Dissociation at each denaturant concentration increases with increase in concentration and/or temperature, and these dependencies are shown to be affected by amino acids which bind to allosteric and/or substrate binding sites. Dissociation of the hexamer is accompanied by a large increase in the fluorescence of ANS due to binding to increase in exposed hydrophobic microdomains on the protein. In contrast, tryptophan fluorescence decreases due to increased exposure of tryptopan in the protein to the aqueous environment. Measurement were made of the influence of several known substrates and allosteric modifiers (i.e. a selection physiologically relevant monocarboxylic amino acids) on the GDH-1 hexamer to trimer transition. The observed effect of adding each amino acid is consistent with independent measurements of its binding affinity. From the temperature dependence of the fluorescence intensity changes for ANS we obtained activation parameters for the hexamer-trimer transitions. Our data are consistent with the hexamer/trimer transition in GDH-1 having significant regulatory function in vivo, perhaps contribution to/complementing allosteric regulation of the enzyme activity.

Focused Engineering of Pyrrolysyl-tRNA Synthetase-Based Orthogonal Translation Systems for the Incorporation of Various Noncanonical Amino Acids.

The expansion of the genetic code has become a valuable tool for molecular biology, biochemistry, and biotechnology. The pyrrolysyl-tRNA synthetase (PylRS) variants with their cognate tRNAPyl derived from methanogenic archaea of the genus Methanosarcina are the most popular tools for ribosomally mediated site-specific and proteome-wide statistical incorporation of noncanonical amino acids (ncAAs) into proteins. The incorporation of ncAAs can be used for numerous biotechnological and even therapeutically relevant applications. Here we present a protocol of engineering PylRS for novel substrates with unique chemical functionalities. These functional groups can act as intrinsic probes, especially in complex biological environments such as mammalian cells, tissues, and even whole animals.

Protein charge transfer spectra in a monomeric protein with no lysine.

UV-Visible absorption and luminescence originating from non-aromatic groups in proteins is being intensely investigated today. Earlier work has shown that non-aromatic charge clusters in a folded monomeric protein can collectively act like a chromophore. Incident light in the near UV-Visible wavelength causes photoinduced electron transfer from the Highest Occupied Molecular Orbital (HOMO) of the electron-rich donor (like a carboxylate anion) to the Lowest Unoccupied Molecular Orbital (LUMO) of the electron-deficient acceptor (like a protonated amine or the polypeptide backbone) in the protein giving rise to absorption spectra in the 250-800 nm range referred to as Protein Charge Transfer Spectra (ProCharTS). The transferred electron can relax back from the LUMO to fill up the hole in the HOMO by a charge recombination process, emitting weak ProCharTS luminescence. Previous studies in monomeric proteins displaying ProCharTS absorption/luminescence always involved lysine-containing proteins. The lysine (Lys) sidechain plays a dominant role in ProCharTS; however, experimental evidence for ProCharTS among proteins/peptides devoid of Lys is lacking. Recently, the absorption features of charged amino acids have been examined using time-dependent density functional theory calculations. In this study, we show that amino acids: arginine (Arg), histidine (His) and aspartate (Asp); homo-polypeptides: poly-arginine and poly-aspartate; and a protein: Symfoil PV2 that is rich in Asp, His and Arg, but lacks Lys, profusely display ProCharTS. The folded Symfoil PV2 protein displayed maximum ProCharTS absorptivity in the near UV-Vis region, in comparison to the homo-polypeptides and amino acids. Furthermore, features like overlapping ProCharTS absorption spectra, decreasing ProCharTS luminescence intensity with longer excitation wavelength, large Stokes shift, multiple excitation bands and multiple luminescence lifetime components appeared to be conserved across peptides, proteins and amino acids studied. Our results underscore the utility of ProCharTS as an intrinsic spectral probe to monitor the structure of any protein that is rich in charged amino acids.

Fluorescence microscopy imaging of a neurotransmitter receptor and its cell membrane lipid milieu.

Hampered by the diffraction phenomenon, as expressed in 1873 by Abbe, applications of optical microscopy to image biological structures were for a long time limited to resolutions above the ∼200nm barrier and restricted to the observation of stained specimens. The introduction of fluorescence was a game changer, and since its inception it became the gold standard technique in biological microscopy. The plasma membrane is a tenuous envelope of 4nm-10nm in thickness surrounding the cell. Because of its highly versatile spectroscopic properties and availability of suitable instrumentation, fluorescence techniques epitomize the current approach to study this delicate structure and its molecular constituents. The wide spectral range covered by fluorescence, intimately linked to the availability of appropriate intrinsic and extrinsic probes, provides the ability to dissect membrane constituents at the molecular scale in the spatial domain. In addition, the time resolution capabilities of fluorescence methods provide complementary high precision for studying the behavior of membrane molecules in the time domain. This review illustrates the value of various fluorescence techniques to extract information on the topography and motion of plasma membrane receptors. To this end I resort to a paradigmatic membrane-bound neurotransmitter receptor, the nicotinic acetylcholine receptor (nAChR). The structural and dynamic picture emerging from studies of this prototypic pentameric ligand-gated ion channel can be extrapolated not only to other members of this superfamily of ion channels but to other membrane-bound proteins. I also briefly discuss the various emerging techniques in the field of biomembrane labeling with new organic chemistry strategies oriented to applications in fluorescence nanoscopy, the form of fluorescence microscopy that is expanding the depth and scope of interrogation of membrane-associated phenomena.

High-Field (3.4 T) Electron Paramagnetic Resonance, 1H Electron-Nuclear Double Resonance, ESEEM, HYSCORE, and Relaxation Studies of Asphaltene Solubility Fractions of Bitumen for Structural Characterization of Intrinsic Carbon-Centered Radicals

Petroleum asphaltenes are considered the most irritating components of various oil systems, complicating the extraction, transportation, and processing of hydrocarbons. Despite the fact that the paramagnetic properties of asphaltenes and their aggregates have been studied since the 1950s, there is still no clear understanding of the structure of stable paramagnetic centers in petroleum systems. The paper considers the possibilities of various electron paramagnetic resonance (EPR) techniques to study petroleum asphaltenes and their solubility fractions using a carbon-centered stable free radical (FR) as an intrinsic probe. The dilution of asphaltenes with deuterated toluene made it possible to refine the change in the structure at the initial stage of asphaltene disaggregation. From the measurements of samples of bitumen, a planar circumcoronene-like model of FR structure and FR-centered asphaltenes is proposed. The results show that EPR-based approaches can serve as sensitive numerical tools to follow asphaltenes' structure and their disaggregation.

Adjacent and Nonadjacent Dipyrimidine Photoproducts as Intrinsic Probes of DNA Secondary and Tertiary Structure†.

While the photochemistry of duplex DNA has been extensively studied, the photochemistry of nonduplex DNA structures is largely unexplored. Because the structure and stereochemistry of DNA photoproducts depend on the secondary structure and conformation of the DNA precursor, they can serve as intrinsic probes of DNA structure. This review focuses on the structures and stereoisomers of pyrimidine dimer photoproducts arising from adjacent and nonadjacent pyrimidines in A, B and denatured DNA, bulge loops, G-quadruplexes and reverse Hoogsteen hairpins and methods for their detection.

Development of a Redox-Label-Doped Molecularly Imprinted Polymer on β-Cyclodextrin/Reduced Graphene Oxide for Electrochemical Detection of a Stress Biomarker.

Cortisol is a major stress biomarker involved in the regulation of metabolic and immune responses. Readily accessible assays with sufficient quantitative and temporal resolution can assist in prevention, early diagnosis, and management of chronic diseases. Whereas conventional assays are costly in terms of time, labor, and capital, an electrochemical approach offers the possibility of miniaturization and detection at the point-of-care. Here, we investigate the biosensor application of molecularly imprinted polypyrrole (PPy) doped with hexacyanoferrate (HCF) and coupled to reduced graphene oxide functionalized with β-cyclodextrin (β-CD). β-CD provides an inclusion site for lipophilic cortisol and was electrochemically grafted simultaneous with reduction of GO. Next, PPy was electrochemically deposited in presence of cortisol template with HCF dopant ions serving as intrinsic redox probe. Thus, the sensor response was evaluated via changes of redox peak current in cyclic voltammetry and demonstrated a broad logarithmic detection range (5 pg/mL to 5000 ng/mL, R2 = 0.995), with a sensitivity of 8.809 μA log–1 (ng/mL) cm–2 and LOD of 19.3 pM. The sensor was shown to be specific toward cortisol in reference to salivary cortisol concentration in saliva over structural analogues. The sensor was exhibited to determine cortisol in artificial saliva at normal and elevated levels. The good performance and facile electrochemical fabrication of this antibody- and external label-free interface are promising for the development of affordable point-of-care biosensors.

Predicting Membrane-Active Peptide Dynamics in Fluidic Lipid Membranes.

Understanding the interactions between peptides and lipid membranes could not only accelerate the development of antimicrobial peptides as treatments for infections but also be applied to finding targeted therapies for cancer and other diseases. However, designing biophysical experiments to study molecular interactions between flexible peptides and fluidic lipid membranes has been an ongoing challenge. Recently, with hardware advances, algorithm improvements, and more accurate parameterizations (i.e., force fields), all-atom molecular dynamics (MD) simulations have been used as a "computational microscope" to investigate the molecular interactions and mechanisms of membrane-active peptides in cell membranes (Chen et al., Curr Opin Struct Biol 61:160-166, 2020; Ulmschneider and Ulmschneider, Acc Chem Res 51(5):1106-1116, 2018; Dror et al., Annu Rev Biophys 41:429-452, 2012). In this chapter, we describe how to utilize MD simulations to predict and study peptide dynamics and how to validate the simulations by circular dichroism, intrinsic fluorescent probe, membrane leakage assay, electrical impedance, and isothermal titration calorimetry. Experimentally validated MD simulations open a new route towards peptide design starting from sequence and structure and leading to desirable functions.