Toward determining amyloid fibril structures using experimental constraints from Raman spectroscopy.

We present structural models for three different amyloid fibril polymorphs prepared from amylin20-29 (sequence SNNFGAILSS) and amyloid-ß25-35 (Aß25-35) (sequence GSNKGAIIGLM) peptides. These models are based on the amide C=O bond and Ramachandran ψ-dihedral angle data from Raman spectroscopy, which were used as structural constraints to guide molecular dynamics (MD) simulations. The resulting structural models indicate that the basic structural motif of amylin20-29 and Aß25-35 fibrils is extended ß-strands. Our data indicate that amylin20-29 forms both antiparallel and parallel ß-sheet fibril polymorphs, while Aß25-35 forms a parallel ß-sheet fibril structure. Overall, our work lays the foundation for using Raman spectroscopy in conjunction with MD simulations to determine detailed molecular-level structural models of amyloid fibrils in a manner that complements gold-standard techniques, such as solid-state nuclear magnetic resonance and cryogenic electron microscopy.

A facile approach to create sensitive and selective Cu(ii) sensors on carbon fiber microelectrodes.

A facile platform derived from deposition of ethynyl linkers on carbon fiber microelectrodes has been developed for sensitive and selective sensing of Cu(ii). This study is the first to demonstrate the successful anodic deposition of ethynyl linkers, specifically 1,4-diethynylbenzene, onto carbon fiber microelectrodes. Multi-scan deposition of DEB on these microelectrodes resulted in an increased sensitivity and selectivity towards Cu(ii) that persists amidst other divalent interferents and displays sustained performance over four days while stored at room temperature. This method can be extended to other ethynyl terminal moieties, thereby creating a versatile chemical platform that will enable improved sensitivity and selectivity for a new frontier of biomarker measurement.

Stimulated Raman versus Inverse Raman: Investigating Depletion Mechanisms for Super-Resolution Raman Microscopy.

Super-resolution fluorescence microscopy has been critical in elucidating the nanoscale structure of biological systems. However, fluorescent labels bring difficulties such as perturbative labeling steps and photobleaching. Thus, label-free super-resolution techniques are of great interest, like our group's 2016 stimulated Raman scattering (SRS) technique, stimulated Raman depletion microscopy (SRDM). Inspired by stimulated emission depletion microscopy, SRDM uses a toroidally shaped beam to deplete the signal formed on the edges of the focal spot, resulting in SRS signal being detected from only a subdiffraction limited region. In initial works, the cause of the depletion was not thoroughly characterized. Here, we conclusively demonstrate suppression mechanisms in SRDM, while also contrasting approaches to super-resolution Raman microscopy on the Stokes and anti-Stokes sides of the spectrum. By monitoring the depletion of both the SRS and inverse Raman scattering (IRS) signal at a range of depletion powers, we observed other four-wave coherent Raman pathways that correspond to the introduction of the femtosecond depletion beam. In addition, we showed the depletion of the IRS signal, paving the way for a super-resolution imaging technique based on IRS, inverse raman depletion microscopy (IRDM). Combined, SRDM and IRDM offer label-free super-resolution imaging over a large spectral range to accommodate a variety of different sample constraints.

Molecular Insights into the Binding of Linear Polyethylenimines and Single-Stranded DNA Using Raman Spectroscopy: A Quantitative Approach.

Establishing how polymeric vectors such as polyethylenimine (PEI) bind and package their nucleic acid cargo is vital toward developing more efficacious and cost-effective gene therapies. To develop a molecular-level picture of DNA binding, we examined how the Raman spectra of PEIs report on their local chemical environment. We find that the intense Raman bands located in the 1400-1500 cm-1 region derive from vibrations with significant CH2 scissoring and NH bending character. The Raman bands that derive from these vibrations show profound intensity changes that depend on both the local dielectric environment and hydrogen bonding interactions with the secondary amine groups on the polymer. We use these bands as spectroscopic markers to assess the binding between low molecular weight PEIs and single-stranded DNA (ssDNA). Analysis of the Raman spectra suggest that PEI primarily binds via electrostatic interactions to the phosphate backbone, which induces the condensation of the ssDNA. We additionally confirm this finding by conducting molecular dynamics simulations. We expect that the spectral correlations determined here will enable future studies to investigate important gene delivery activities, including how PEI interacts with cellular membranes to facilitate cargo internalization into cells.

Quinine copolymer reporters promote efficient intracellular DNA delivery and illuminate a protein-induced unpackaging mechanism.

Polymeric vehicles that efficiently package and controllably release nucleic acids enable the development of safer and more efficacious strategies in genetic and polynucleotide therapies. Developing delivery platforms that endogenously monitor the molecular interactions, which facilitate binding and release of nucleic acids in cells, would aid in the rational design of more effective vectors for clinical applications. Here, we report the facile synthesis of a copolymer containing quinine and 2-hydroxyethyl acrylate that effectively compacts plasmid DNA (pDNA) through electrostatic binding and intercalation. This polymer system poly(quinine-co-HEA) packages pDNA and shows exceptional cellular internalization, transgene expression, and low cytotoxicity compared to commercial controls for several human cell lines, including HeLa, HEK 293T, K562, and keratinocytes (N/TERTs). Using quinine as an endogenous reporter for pDNA intercalation, Raman imaging revealed that proteins inside cells facilitate the unpackaging of polymer-DNA complexes (polyplexes) and the release of their cargo. Our work showcases the ability of this quinine copolymer reporter to not only facilitate effective gene delivery but also enable diagnostic monitoring of polymer-pDNA binding interactions on the molecular scale via Raman imaging. The use of Raman chemical imaging in the field of gene delivery yields unprecedented insight into the unpackaging behavior of polyplexes in cells and provides a methodology to assess and design more efficient delivery vehicles for gene-based therapies.

Far-Field Super-Resolution Vibrational Spectroscopy.

Potential label-free alternatives to super-resolution fluorescence techniques have been the focus of considerable research due to the challenges intrinsic in the reliance on fluorescent tags. In this Feature, we discuss efforts to develop super-resolution techniques based on vibrational spectroscopies and address possible sample applications as well as future potential resolution enhancements.

New Insights into Quinine-DNA Binding Using Raman Spectroscopy and Molecular Dynamics Simulations.

Quinine's ability to bind DNA and potentially inhibit transcription and translation has been examined as a mode of action for its antimalarial activity. UV absorption and fluorescence-based studies have lacked the chemical specificity to develop an unambiguous molecular-level picture of the binding interaction. To address this, we use Raman spectroscopy and molecular dynamics (MD) to investigate quinine-DNA interactions. We demonstrate that quinine's strongest Raman band in the fingerprint region, which derives from a symmetric stretching mode of the quinoline ring, is highly sensitive to the local chemical environment and pH. The frequency shifts observed for this mode in solvents of varying polarity can be explained in terms of the Stark effect using a simple Onsager solvation model, indicating that the vibration reports on the local electrostatic environment. However, specific chemical interactions between the quinoline ring and its environment, such as hydrogen bonding and π-stacking, perturb the frequency of this mode in a more complicated but predictable manner. We use this vibration as a spectroscopic probe to investigate the binding interaction between quinine and DNA. We find that, when the quinoline ring is protonated, quinine weakly intercalates into DNA by forming π-stacking interactions with the base pairs. The Raman spectra indicate that quinine can intercalate into DNA with a ratio reaching up to roughly one molecule per 25 base pairs. Our results are confirmed by MD simulations, which also show that the quinoline ring adopts a t-shaped π-stacking geometry with the DNA base pairs, whereas the quinuclidine head group weakly interacts with the phosphate backbone in the minor groove. We expect that the spectral correlations determined here will enable future studies to probe quinine's antimalarial activities, such as disrupting hemozoin biocrystallization, which is hypothesized to be, among other things, one of its primary modes of action against Plasmodium parasites.

Interaction Enthalpy of Side Chain and Backbone Amides in Polyglutamine Solution Monomers and Fibrils.

We determined an empirical correlation that relates the amide I vibrational band frequencies of the glutamine (Q) side chain to the strength of hydrogen bonding, van der Waals, and Lewis acid-base interactions of its primary amide carbonyl. We used this correlation to determine the Q side chain carbonyl interaction enthalpy (Δ Hint) in monomeric and amyloid-like fibril conformations of D2Q10K2 (Q10). We independently verified these Δ Hint values through molecular dynamics simulations that showed excellent agreement with experiments. We found that side chain-side chain and side chain-peptide backbone interactions in fibrils and monomers are more enthalpically favorable than are Q side chain-water interactions. Q10 fibrils also showed a more favorable Δ Hint for side chain-side chain interactions compared to backbone-backbone interactions. This work experimentally demonstrates that interamide side chain interactions are important in the formation and stabilization of polyQ fibrils.

Monomeric Polyglutamine Structures That Evolve into Fibrils.

We investigate the solution and fibril conformations and structural transitions of the polyglutamine (polyQ) peptide, D2Q10K2 (Q10), by synergistically using UV resonance Raman (UVRR) spectroscopy and molecular dynamics (MD) simulations. We show that Q10 adopts two distinct, monomeric solution conformational states: a collapsed ß-strand and a PPII-like structure that do not readily interconvert. This clearly indicates a high activation barrier in solution that prevents equilibration between these structures. Using metadynamics, we explore the conformational energy landscape of Q10 to investigate the physical origins of this high activation barrier. We develop new insights into the conformations and hydrogen bonding environments of the glutamine side chains in the PPII and ß-strand-like conformations in solution. We also use the secondary structure-inducing cosolvent, acetonitrile, to investigate the conformations present in low dielectric constant solutions with decreased solvent-peptide hydrogen bonding. As the mole fraction of acetonitrile increases, Q10 converts from PPII-like structures into α-helix-like structures and ß-sheet aggregates. Electron microscopy indicates that the aggregates prepared from these acetonitrile-rich solutions show morphologies similar to our previously observed polyQ fibrils. These aggregates redissolve upon the addition of water! These are the first examples of reversible fibril formation. Our monomeric Q10 peptides clearly sample broad regions of their available conformational energy landscape. The work here develops molecular-level insight into monomeric Q10 conformations and investigates the activation barriers between different monomer states and their evolution into fibrils.

Aluminum Film-Over-Nanosphere Substrates for Deep-UV Surface-Enhanced Resonance Raman Spectroscopy.

We report here the first fabrication of aluminum film-over nanosphere (AlFON) substrates for UV surface-enhanced resonance Raman scattering (UVSERRS) at the deepest UV wavelength used to date (λex = 229 nm). We characterize the AlFONs fabricated with two different support microsphere sizes using localized surface plasmon resonance spectroscopy, electron microscopy, SERRS of adenine, tris(bipyridine)ruthenium(II), and trans-1,2-bis(4-pyridyl)-ethylene, SERS of 6-mercapto-1-hexanol (as a nonresonant molecule), and dielectric function analysis. We find that AlFONs fabricated with the 210 nm microspheres generate an enhancement factor of approximately 104-5, which combined with resonance enhancement of the adsorbates provides enhancement factors greater than 106. These experimental results are supported by theoretical analysis of the dielectric function. Hence our results demonstrate the advantages of using AlFON substrates for deep UVSERRS enhancement and contribute to broadening the SERS application range with tunable and affordable substrates.

Polyglutamine Fibrils: New Insights into Antiparallel ß-Sheet Conformational Preference and Side Chain Structure.

Understanding the structure of polyglutamine (polyQ) amyloid-like fibril aggregates is crucial to gaining insights into the etiology of at least ten neurodegenerative disorders, including Huntington's disease. Here, we determine the structure of D2Q10K2 (Q10) fibrils using ultraviolet resonance Raman (UVRR) spectroscopy and molecular dynamics (MD). Using UVRR, we determine the fibril peptide backbone Ψ and glutamine (Gln) side chain χ3 dihedral angles. We find that most of the fibril peptide bonds adopt antiparallel ß-sheet conformations; however, a small population of peptide bonds exist in parallel ß-sheet structures. Using MD, we simulate three different potential fibril structural models that consist of either ß-strands or ß-hairpins. Comparing the experimentally measured Ψ and χ3 angle distributions to those obtained from the MD simulated models, we conclude that the basic structural motif of Q10 fibrils is an extended ß-strand structure. Importantly, we determine from our MD simulations that Q10 fibril antiparallel ß-sheets are thermodynamically more stable than parallel ß-sheets. This accounts for why polyQ fibrils preferentially adopt antiparallel ß-sheet conformations instead of in-register parallel ß-sheets like most amyloidogenic peptides. In addition, we directly determine, for the first time, the structures of Gln side chains. Our structural data give new insights into the role that the Gln side chains play in the stabilization of polyQ fibrils. Finally, our work demonstrates the synergistic power and utility of combining UVRR measurements and MD modeling to determine the structure of amyloid-like fibrils.

Photonic crystal protein hydrogel sensor materials enabled by conformationally induced volume phase transition.

Hydrogels that change volume in response to specific molecular stimuli can serve as platforms for sensors, actuators and drug delivery devices. There is great interest in designing intelligent hydrogels for tissue engineering, drug delivery, and microfluidics that utilize protein binding specificities and conformational changes. Protein conformational change induced by ligand binding can cause volume phase transitions (VPTs). Here, we develop a highly selective glucose sensing protein photonic crystal (PC) hydrogel that is fabricated from genetically engineered E. coli glucose/galactose binding protein (GGBP). The resulting 2-D PC-GGBP hydrogel undergoes a VPT in response to glucose. The volume change causes the 2-D PC array particle spacing to decrease, leading to a blue-shifted diffraction which enables our sensors to report on glucose concentrations. This 2-D PC-GGBP responsive hydrogel functions as a selective and sensitive sensor that easily monitors glucose concentrations from ∼0.2 µM to ∼10 mM. This work demonstrates a proof-of-concept for developing responsive, "smart" protein hydrogel materials with VPTs that utilize ligand binding induced protein conformational changes. This innovation may enable the development of other novel chemical sensors and high-throughput screening devices that can monitor protein-drug binding interactions.

A Photonic Crystal Protein Hydrogel Sensor for Candida albicans.

We report two-dimensional (2D) photonic crystal (PC) sensing materials that selectively detect Candida albicans (C. albicans). These sensors utilize Concanavalin A (Con A) protein hydrogels with a 2D PC embedded on the Con A protein hydrogel surface, that multivalently and selectively bind to mannan on the C. albicans cell surface to form crosslinks. The resulting crosslinks shrink the Con A protein hydrogel, reduce the 2D PC particle spacing, and blue-shift the light diffracted from the PC. The diffraction shifts can be visually monitored, measured with a spectrometer, or determined from the Debye diffraction ring diameter. Our unoptimized hydrogel sensor has a detection limit of around 32 CFU/mL for C. albicans. This sensor distinguishes between C. albicans and those microbes devoid of cell-surface mannan such as the gram-negative bacterium E. coli. This sensor provides a proof-of-concept for utilizing recognition between lectins and microbial cell surface carbohydrates to detect microorganisms in aqueous environments.

Glutamine and Asparagine Side Chain Hyperconjugation-Induced Structurally Sensitive Vibrations.

We identified vibrational spectral marker bands that sensitively report on the side chain structures of glutamine (Gln) and asparagine (Asn). Density functional theory (DFT) calculations indicate that the Amide III(P) (AmIII(P)) vibrations of Gln and Asn depend cosinusoidally on their side chain OCCC dihedral angles (the χ3 and χ2 angles of Gln and Asn, respectively). We use UV resonance Raman (UVRR) and visible Raman spectroscopy to experimentally correlate the AmIII(P) Raman band frequency to the primary amide OCCC dihedral angle. The AmIII(P) structural sensitivity derives from the Gln (Asn) Cß-Cγ (Cα-Cß) stretching component of the vibration. The Cß-Cγ (Cα-Cß) bond length inversely correlates with the AmIII(P) band frequency. As the Cß-Cγ (Cα-Cß) bond length decreases, its stretching force constant increases, which results in an upshift in the AmIII(P) frequency. The Cß-Cγ (Cα-Cß) bond length dependence on the χ3 (χ2) dihedral angle results from hyperconjugation between the Cδ═Oϵ (Cγ═Oδ) π* and Cß-Cγ (Cα-Cß) σ orbitals. Using a Protein Data Bank library, we show that the χ3 and χ2 dihedral angles of Gln and Asn depend on the peptide backbone Ramachandran angles. We demonstrate that the inhomogeneously broadened AmIII(P) band line shapes can be used to calculate the χ3 and χ2 angle distributions of peptides. The spectral correlations determined in this study enable important new insights into protein structure in solution, and in Gln- and Asn-rich amyloid-like fibrils and prions.

UV resonance Raman investigation of the aqueous solvation dependence of primary amide vibrations.

We investigated the normal mode composition and the aqueous solvation dependence of the primary amide vibrations of propanamide. Infrared, normal Raman, and UV resonance Raman (UVRR) spectroscopy were applied in conjunction with density functional theory (DFT) to assign the vibrations of crystalline propanamide. We examined the aqueous solvation dependence of the primary amide UVRR bands by measuring spectra in different acetonitrile/water mixtures. As previously observed in the UVRR spectra of N-methylacetamide, all of the resonance enhanced primary amide bands, except for the Amide I (AmI), show increased UVRR cross sections as the solvent becomes water-rich. These spectral trends are rationalized by a model wherein the hydrogen bonding and the high dielectric constant of water stabilizes the ground state dipolar (-)O-C═NH2(+) resonance structure over the neutral O═C-NH2 resonance structure. Thus, vibrations with large C-N stretching show increased UVRR cross sections because the C-N displacement between the electronic ground and excited state increases along the C-N bond. In contrast, vibrations dominated by C═O stretching, such as the AmI, show a decreased displacement between the electronic ground and excited state, which result in a decreased UVRR cross section upon aqueous solvation. The UVRR primary amide vibrations can be used as sensitive spectroscopic markers to study the local dielectric constant and hydrogen bonding environments of the primary amide side chains of glutamine (Gln) and asparagine (Asn).

2D photonic crystal protein hydrogel coulometer for sensing serum albumin ligand binding.

Bovine and human serum albumin (BSA and HSA) are globular proteins that function as bloodstream carriers of hydrophobes such as fatty acids and drugs. We fabricated novel photonic crystal protein hydrogels by attaching 2D colloidal arrays onto pure BSA and HSA hydrogels. The wavelengths of the diffracted light sensitively report on the protein hydrogel surface area. The binding of charged species to the protein hydrogel gives rise to Donnan potentials that change the hydrogel volume causing shifts in the diffraction. These photonic crystal protein hydrogels act as sensitive Coulometers that monitor the hydrogel charge state. We find multiple high-affinity BSA and HSA binding sites for salicylate, ibuprofen and picosulfate by using these sensors to monitor binding of charged drugs. We demonstrate proof-of-concept for utilizing protein hydrogel sensors to monitor protein-ionic species binding.

UV resonance Raman spectroscopy monitors polyglutamine backbone and side chain hydrogen bonding and fibrillization.

We utilize 198 and 204 nm excited UV resonance Raman spectroscopy (UVRR) and circular dichroism spectroscopy (CD) to monitor the backbone conformation and the Gln side chain hydrogen bonding (HB) of a short, mainly polyGln peptide with a D(2)Q(10)K(2) sequence (Q10). We measured the UVRR spectra of valeramide to determine the dependence of the primary amide vibrations on amide HB. We observe that a nondisaggregated Q10 (NDQ10) solution (prepared by directly dissolving the original synthesized peptide in pure water) exists in a ß-sheet conformation, where the Gln side chains form hydrogen bonds to either the backbone or other Gln side chains. At 60 °C, these solutions readily form amyloid fibrils. We used the polyGln disaggregation protocol of Wetzel et al. [Wetzel, R., et al. (2006) Methods Enzymol.413, 34-74] to dissolve the Q10 ß-sheet aggregates. We observe that the disaggregated Q10 (DQ10) solutions adopt PPII-like and 2.5(1)-helix conformations where the Gln side chains form hydrogen bonds with water. In contrast, these samples do not form fibrils. The NDQ10 ß-sheet solution structure is essentially identical to that found in the NDQ10 solid formed upon evaporation of the solution. The DQ10 PPII and 2.5(1)-helix solution structure is essentially identical to that in the DQ10 solid. Although the NDQ10 solution readily forms fibrils when heated, the DQ10 solution does not form fibrils unless seeded with the NDQ10 solution. This result demonstrates very high activation barriers between these solution conformations. The NDQ10 fibril secondary structure is essentially identical to that of the NDQ10 solution, except that the NDQ10 fibril backbone conformational distribution is narrower than in the dissolved species. The NDQ10 fibril Gln side chain geometry is more constrained than when NDQ10 is in solution. The NDQ10 fibril structure is identical to that of the DQ10 fibril seeded by the NDQ10 solution.

Characterization of the role of nitric oxide and its clinical applications.

Nitric oxide (NO) has long been known as endothelium-derived relaxing factor. It is a vasodilator, modulating vascular tone, blood pressure and hemodynamics, a role exploited by nitrate donor therapy for angina, heart failure, pulmonary hypertension and erectile dysfunction. In addition, its powerful antioxidant, anti-inflammatory and antithrombotic actions are antiatherogenic with antiatherothrombotic impact. NO signaling modulates skeletal muscle and myocardial contractility and metabolism and is intimately linked with insulin signaling. Vascular and muscle NO signaling coordinate skeletal muscle and myocardial energy demand with supply and are critical for both carbohydrate and fatty acid total-body homeostasis. NO signaling in mitochondria underlies much of NO's metabolic effect, which, at low physiologic levels, links cellular energy demand with mitochondrial energy supply, while beneficially affecting mitochondrial oxidative stress and calcium handling. Mitochondria are also the site for the life-threatening deleterious effects arising from inflammation-related excessive NO levels. NO-deficient states are characterized by cell senescence, oxidative stress, inflammation, endothelial dysfunction, vascular disease, insulin resistance and type 2 diabetes mellitus. NO-enriching therapy would be expected to be of benefit not only for its hemodynamic but also for its metabolic impact. In contrast, strategies are needed to curtail excessive NO in states such as septic shock.