Deep Ultraviolet Resonance Raman Spectroscopy Research Articles

Determining the stages of cellular differentiation using deep ultraviolet resonance Raman spectroscopy

Cellular differentiation is a fundamental process in which one cell type changes into one or more specialized cell types. Cellular differentiation starts at the beginning of embryonic development when a simple zygote begins to transform into a complex multicellular organism composed of various cell and tissue types. This process continues into adulthood when adult stem cells differentiate into more specialized cells for normal growth, regeneration, repair, and cellular turnover. Any abnormalities associated with this fundamental process of cellular differentiation are linked to life-threatening conditions, including degenerative diseases and cancers. Detection of undifferentiated and different stages of differentiated cells can be used for disease diagnosis but is often challenging due to the laborious procedures, expensive tools, and specialized technical skills which are required. Here, a novel approach, called deep ultraviolet resonance Raman spectroscopy, is used to study various stages of cellular differentiation using a well-known myoblast cell line as a model system. These cells proliferate in the growth medium and spontaneously differentiate in differentiation medium into myocytes and later into myotubes. The cellular and molecular characteristics of these cells mimic very well actual muscle tissue in vivo. We have found that undifferentiated myoblast cells and myoblast cells differentiated at three different stages are able to be easily separated using deep ultraviolet resonance Raman spectroscopy in combination with chemometric techniques. Our study has a great potential to study cellular differentiation during normal development as well as to detect abnormal cellular differentiation in human pathological conditions in future studies.

Secondary structure assessment of formulated bevacizumab in the presence of SDS by deep ultraviolet resonance Raman (DUVRR) spectroscopy

A deep-ultraviolet resonance Raman (DUVRR) spectroscopic method has been used to study the secondary structural changes of a therapeutic monoclonal antibody (mAb), bevacizumab (Avastin™) under a chemical stress: the presence of sodium dodecyl sulfate (SDS). The results demonstrate that DUVRR spectroscopy can assay the higher order structure of the formulated protein in a sensitive and selective manner. The SDS-induced partially unfolding of the mAb was probed by DUVRR spectroscopy where the amide I, II and III spectral features showed conformational changes between beta-sheet, alpha-helix and random coil forms. A chemometric model was also built to analyze the spectral changes occurring with protein-SDS interactions. The analysis showed there are different stages of mAb-SDS interaction as the SDS concentration increases. In addition, a two-dimensional (2D) correlation analysis was applied to the DUVRR spectra to visualize the secondary structure changes of bevacizumab under stresses. As an addition to the chemometric model, the 2D correlation mapping method suggested different transitions between secondary structure motifs were occurring at different SDS concentrations. Overall, chemometric and 2D analysis provided complimentary information, and show the potential of coupling DUVRR with advanced statistical methods in revealing complex structural information in formulated protein pharmaceuticals.

Unwinding of the Substrate Transmembrane Helix in Intramembrane Proteolysis

Intramembrane-cleaving proteases (I-CLiPs) activate pools of single-pass helical membrane protein signaling precursors that are key in the physiology of prokaryotic and eukaryotic cells. Proteases typically cleave peptide bonds within extended or flexible regions of their substrates, and thus the mechanism underlying the ability of I-CLiPs to hydrolyze the presumably α-helical transmembrane domain (TMD) of these membrane proteins is unclear. Using deep-ultraviolet resonance Raman spectroscopy in combination with isotopic labeling, we show that although predominantly in canonical α-helical conformation, the TMD of the established I-CLiP substrate Gurken displays 310-helical geometry. As measured by microscale thermophoresis, this substrate binds with high affinity to the I-CLiPs GlpG rhomboid and MCMJR1 presenilin homolog in detergent micelles. Binding results in deep-ultraviolet resonance Raman spectra, indicating conformational changes consistent with unwinding of the 310-helical region of the substrate’s TMD. This 310-helical conformation is key for intramembrane proteolysis, as the substitution of a single proline residue in the TMD of Gurken by alanine suppresses 310-helical content in favor of α-helical geometry and abolishes cleavage without affecting binding to the I-CLiP. Complemented by molecular dynamics simulations of the TMD of Gurken, our vibrational spectroscopy data provide biophysical evidence in support of a model in which the transmembrane region of cleavable I-CLiP substrates displays local deviations in canonical α-helical conformation characterized by chain flexibility, and binding to the enzyme results in conformational changes that facilitate local unwinding of the transmembrane helix for cleavage.

Structural and Mechanical Properties of Amyloid Beta Fibrils: A Combined Experimental and Theoretical Approach.

In this combined experimental (deep ultraviolet resonance Raman (DUVRR) spectroscopy and atomic force microscopy (AFM)) and theoretical (molecular dynamics (MD) simulations and stress-strain (SS)) study, the structural and mechanical properties of amyloid beta (Aβ40) fibrils have been investigated. The DUVRR spectroscopy and AFM experiments confirmed the formation of linear, unbranched and β-sheet rich fibrils. The fibrils (Aβ40)n, formed using n monomers, were equilibrated using all-atom MD simulations. The structural properties such as β-sheet character, twist, interstrand distance, and periodicity of these fibrils were found to be in agreement with experimental measurements. Furthermore, Young's modulus (Y) = 4.2 GPa computed using SS calculations was supported by measured values of 1.79 ± 0.41 and 3.2 ± 0.8 GPa provided by two separate AFM experiments. These results revealed size dependence of structural and material properties of amyloid fibrils and show the utility of such combined experimental and theoretical studies in the design of precisely engineered biomaterials.

“Parallel factor analysis of multi-excitation ultraviolet resonance Raman spectra for protein secondary structure determination”

Protein secondary structural analysis is important for understanding the relationship between protein structure and function, or more importantly how changes in structure relate to loss of function. The structurally sensitive protein vibrational modes (amide I, II, III and S) in deep-ultraviolet resonance Raman (DUVRR) spectra resulting from the backbone C–O and N–H vibrations make DUVRR a potentially powerful tool for studying secondary structure changes. Experimental studies reveal that the position and intensity of the four amide modes in DUVRR spectra of proteins are largely correlated with the varying fractions of α-helix, β-sheet and disordered structural content of proteins. Employing multivariate calibration methods and DUVRR spectra of globular proteins with varying structural compositions, the secondary structure of a protein with unknown structure can be predicted. A disadvantage of multivariate calibration methods is the requirement of known concentration or spectral profiles. Second-order curve resolution methods, such as parallel factor analysis (PARAFAC), do not have such a requirement due to the “second-order advantage.” An exceptional feature of DUVRR spectroscopy is that DUVRR spectra are linearly dependent on both excitation wavelength and secondary structure composition. Thus, higher order data can be created by combining protein DUVRR spectra of several proteins collected at multiple excitation wavelengths to give multi-excitation ultraviolet resonance Raman data (ME-UVRR). PARAFAC has been used to analyze ME-UVRR data of nine proteins to resolve the pure spectral, excitation and compositional profiles. A three factor model with non-negativity constraints produced three unique factors that were correlated with the relative abundance of helical, β-sheet and poly-proline II dihedral angles. This is the first empirical evidence that the typically resolved “disordered” spectrum represents the better defined poly-proline II type structure.

Deep-Ultraviolet Resonance Raman (DUVRR) Spectroscopy of Therapeutic Monoclonal Antibodies Subjected to Thermal Stress

The structural assessment of Rituximab, an IgG1 mAb, was investigated with deep-ultraviolet resonance Raman (DUVRR) spectroscopy. DUVRR spectroscopy was used to monitor the changes to the secondary structure of Rituximab under thermal stress. DUVRR spectra showed obvious changes from 22 to 72 °C. Specifically, changes in the amide I vibrational mode were assigned to an increase in unordered structure (random coil). Structural changes in samples heated to 72 °C were related to loss in drug potency via a complement dependent cytotoxicity (CDC) bioassay. The DUVRR spectroscopic method shows promise as a tool for the quality assessment of mAb drug products and would represent an improvement over current methodology in terms of analysis time and sample preparation. To determine the scope of the method, protein pharmaceuticals of different molecular weights (ranging from 4 to 143 kDa) and secondary structure (β-sheet, α-helix and unordered structure) were analyzed. The model illustrated the method's sensitivity for the analysis of protein drug products of different secondary structure. Results show promise for DUVRR spectroscopy as a rapid screening tool of a variety of formulated protein pharmaceuticals.

Effects of fluidity on the ensemble structure of a membrane embedded α-helical peptide.

Melittin, the main hemolytic component of honeybee venom, is unfolded in an aqueous environment and folds into an α-helical conformation in a lipid environment. Membrane fluidity is known to affect the activity and structure of melittin. By combining two structurally sensitive optical methods, circular dichroism (CD) and deep-ultraviolet resonance Raman spectroscopy (dUVRR), we have identified distinct structural fluctuations in melittin correlated with increased and decreased 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer fluidities. CD spectra have reduced intensity at temperatures above 22°C and high concentrations of the cholesterol analog 5α-cholestan-3β-ol indicating distortions in the α-helical structure under these conditions. No increase in the amide S is observed in the temperature-dependent dUVRR spectra, suggesting an increase in 310 -helical structure with increasing temperatures above 22°C. However, incorporation of 25 mol% 5α-cholestan-3β-ol resulted in a small increase in the amide S intensity indicating partial unfolding of melittin.

Evolution of quantitative methods in protein secondary structure determination via deep-ultraviolet resonance Raman spectroscopy

Deep-ultraviolet resonance Raman (DUVRR) spectra is sensitive to secondary structural motifs but, similar to circular dichroism (CD) and infrared spectroscopy, requires the application of multivariate and advanced statistical analysis methods to resolve the pure secondary structure Raman spectra (PSSRS) for determination of secondary structure composition. Secondary structure motifs are selectively enhanced by different excitation wavelengths, a characteristic that inspired the first methods for quantifying secondary structures by DUVRR. This review traces the evolution of multivariate methods and their application to secondary structure composition analyses of proteins by DUVRR spectroscopy from the first experiments using two-wavelengths, and culminating with recent studies utilizing time-resolved DUVRR measurements.

Deep-Uvrr Spectroscopy Studies of Amyloidogenic Transthyretin Fragments

The protein transthyretin (TTR) has been implicated as the pathogen in several amyloid diseases. Normally a transport protein for thyroxine, amyloidosis occurs when the protein aggregates and deposits in organ tissue as β-sheet structured amyloid fibrils. The formation of amyloid fibril deposits associated with TTR diseases is poorly understood. In order to study amyloid fibril formation several amyloidogenic fragments of TTR have been studied in aqueous solutions. It has been suggested that the amyloidogenic fragments TTR(10-20) and TTR(105-115) contain portions of the protein essential to aggregation and amyloid fibril formation, however, neither have been studied in the presence of cell-like lipid membranes. Here, we present the first studies comparing aqueous and model membrane solution studies of TTR(10-20) and TTR(105-115) via deep-ultraviolet resonance Raman spectroscopy. Initial results suggest a change in peptide secondary structure upon interaction with lipid membranes.

Isolating Toxic Insulin Amyloid Reactive Species that Lack β-Sheets and Have Wide pH Stability

Amyloid diseases, including Alzheimer's disease, are characterized by aggregation of normally functioning proteins or peptides into ordered, β-sheet rich fibrils. Most of the theories on amyloid toxicity focus on the nuclei or oligomers in the fibril formation process. The nuclei and oligomers are transient species, making their full characterization difficult. We have isolated toxic protein species that act like an oligomer and may provide the first evidence of a stable reactive species created by disaggregation of amyloid fibrils. This reactive species was isolated by dissolving amyloid fibrils at high pH and it has a mass >100 kDa and a diameter of 48 ± 15 nm. It seeds the formation of fibrils in a dose dependent manner, but using circular dichroism and deep ultraviolet resonance Raman spectroscopy, the reactive species was found to not have a β-sheet rich structure. We hypothesize that the reactive species does not decompose at high pH and maintains its structure in solution. The remaining disaggregated insulin, excluding the toxic reactive species that elongated the fibrils, returned to native structured insulin. This is the first time, to our knowledge, that a stable reactive species of an amyloid reaction has been separated and characterized by disaggregation of amyloid fibrils.

Resolution of localized small molecule–Aβ interactions by deep-ultraviolet resonance Raman spectroscopy

The mechanism by which flavonoids prevent formation of amyloid-β (Aβ) fibrils, as well as how they associate with non-fibrillar Aβ is still unclear. Fresh, un-oxidized myricetin exhibited excitation and emission fluorescence maxima at 481 and 531 nm, respectively. Introduction of either Aβ(1–42) or Aβ(25–40) resulted in a fluorescence decrease, when measured at 481 nm, suggesting formation of a myricetin–Aβ complex. Circular dichroism (CD) and ultraviolet resonance Raman (UVRR) studies indicate that the association of myricetin with the Aβ peptide or its hydrophobic fragment, Aβ(25–40), leads to subtle changes in each peptide's conformation. Aβ(25–40) formed amyloid fibrils at a similar rate, when compared to the full-length peptide, Aβ(1–42), using thioflavin T (ThT) fluorescence. Studies also indicated that myricetin was equally effective at preventing the formation of both Aβ(1–42) and Aβ(25–40) fibrils. Although ThT assays indicated that Aβ(1–16) did not form amyloid fibrils, CD studies of the hydrophilic fragment, Aβ(1–16), suggest possible interactions between myricetin and aromatic side chains. UVRR studies of the full-length peptide and Aβ(1–16) showed increases in the intensity of the aromatic modes upon introduction of myricetin. Our findings suggest that myricetin interacts with soluble Aβ via two mechanisms, association with the hydrophobic C-terminal region and interactions with the aromatic side chains.

Deep Ultraviolet Resonance Raman Excitation Enables Explosives Detection

We measured the 229 nm absolute ultraviolet (UV) Raman cross-sections of the explosives trinitrotoluene (TNT), pentaerythritol tetranitrate (PETN), cyclotrimethylene-trinitramine (RDX), the chemically related nitroamine explosive HMX, and ammonium nitrate in solution. The 229 nm Raman cross-sections are 1000-fold greater than those excited in the near-infrared and visible spectral regions. Deep UV resonance Raman spectroscopy enables detection of explosives at parts-per-billion (ppb) concentrations and may prove useful for stand-off spectroscopic detection of explosives.

Near- and Deep-Ultraviolet Resonance Raman Spectroscopy of Pyrazine−Al4 Complex and Al3−Pyrazine−Al3 Junction

Near- and deep-ultraviolet (UV) resonance Raman spectroscopy of pyrazine−Al4 complex and Al3−pyrazine−Al3 junction was investigated theoretically with a quantum chemical method. Here, 325 and 244 nm were employed as near- and deep-UV sources in our theoretical study. The intensities of static normal Raman spectra of pyrazine−Al4 complex and Al3−pyrazine−Al3 junction were enhanced on the orders of 10 and 103 by a static chemical mechanism, respectively. The calculated absorption spectra reveal strong 6B2 and 13B2u electronic transitions near 325 nm for pyrazine−Al4 complex and 244 nm for Al3−pyrazine−Al3 junction, respectively. The analyses of orbital transitions in electronic transitions reveal they are the mixture of (metal to molecule) charge transfer excitation and intracluster excitation. The intensity of near-UV resonance Raman spectroscopy of pyrazine−Al4 complex and the intensity of deep-UV resonance Raman spectroscopy of Al3−pyrazine−Al3 junction are strongly enhanced on the order of 105 and 104, respectively, compared to the Raman intensity of isolated pyrazine excited at 325 and 244 nm. The calculations of Mie theory and the three-dimensional finite-difference time domain method reveal strong surface plasmon resonance and strong electromagnetic enhancements at 325 and 244 nm for single and dimer nanoparticles at suitable sizes and gap distance, respectively. The strongest SERS enhancement in the system of junction is on the order of 108 at the incident lights of 325 and 244 nm. The total enhancements, including the chemical and electromagnetic enhancements, can reach up to 1013. So, Al is a suitable material for near- and deep-UV surface-enhanced resonance Raman scattering.

Deep‐UV tip‐enhanced Raman scattering

AbstractWe report for the first time the tip‐enhancement of resonance Raman scattering using deep ultraviolet (DUV) excitation wavelength. The tip‐enhancement was successfully demonstrated with an aluminum‐coated silicon tip that acts as a plasmonic material in DUV wavelengths. Both the crystal violet and adenine molecules, which were used as test samples, show electronic resonance at the 266‐nm excitation used in the experiments. With results demonstrated here, molecular analysis and imaging with nanoscale spatial resolution in DUV resonance Raman spectroscopy can be realized using the tip‐enhancement effect. Copyright © 2009 John Wiley & Sons, Ltd.

Lysozyme fibrillation: Deep UV Raman spectroscopic characterization of protein structural transformation

Deep ultraviolet resonance Raman spectroscopy was demonstrated to be a powerful tool for structural characterization of protein at all stages of fibril formation. The evolution of the protein secondary structure as well as the local environment of phenylalanine, a natural deep ultraviolet Raman marker, was documented for the fibrillation of lysozyme. Concentration-independent irreversible helix melting was quantitatively characterized as the first step of the fibrillation. The native lysozyme composed initially of 32% helix transforms monoexponentially to an unfolded intermediate with 6% helix with a characteristic time of 29 h. The local environment of phenylalanine residues changes concomitantly with the secondary structure transformation. The phenylalanine residues in lysozyme fibrils are accessible to solvent in contrast to those in the native protein.

Hollow cathode ion lasers for deep ultraviolet Raman spectroscopy and fluorescence imaging

This article describes the development of hollow cathode ion lasers and their use in constructing an ultraviolet micro-Raman spectrograph with native fluorescence imaging capability. Excitation at 224.3 nm is provided by a helium–silver hollow cathode metal ion laser and at 248.6 nm by a neon–copper hollow cathode metal ion laser. Refractive microscope objectives focus chopped continuous wave laser light on a sample and collect 180° scattered photons. Imaging is accomplished by broadband visible illumination and by deep ultraviolet laser induced excitation of visible wavelength native fluorescence in untagged micro-organisms. This makes possible a detection strategy employing rapid imaging with laser excitation to locate regions of native fluorescence activity, followed by deep ultraviolet resonance Raman spectroscopy of the identified fluorescent sites. We have employed this probe for in situ detection of micro-organisms on mineral and soil substrates. We present here the deep ultraviolet resonance Raman spectra for the gram negative iron reducing bacterium Shewanella oneidensis obtained while the micro-organism remains in situ on the unpolished surface of the mineral calcite and in a Mars soil analog, JSC1. In the current configuration the in situ mineral surface limit of detection for fluorescence is one organism in 2×104 μm2 field of view and of order 20–30 micro-organisms for Raman spectra. For the Mars soil sample analog fluorescent target selection gives an effective ultraviolet resonance Raman spectral detection limit of 6×104cells/gm or ∼60 ppb.