Deep UV Raman spectroscopy

The irradiation of a molecular system with a monochromatic light always results in two types of light scattering, elastic and inelastic scattering. Elastic scattering, which occurs with no change in photon frequency, is called Rayleigh scattering. Raman scattering is accompanied by the shift in photon frequency due to excitation/deactivation of molecular vibrations. Raman spectrum provides a vibrational signature of the molecular system and, consequently, the information about molecular structure. Normal Raman scattering is relatively inefficient process. The efficiency and selectivity of Raman scattering could be dramatically improved due to the resonance enhancement, which occurs when the excitation wavelength is within an electronic transition of the molecular system. Further increase in Raman spectroscopic efficiency occurs when ultraviolet (UV) excitation is used.

• Characterization of biological systems using DUVRR spectroscopy

Raman spectroscopy provides a NONINVASIVE way to characterize STRUCTURE and DYNAMICS of biological systems under PHYSIOLOGICAL CONDITIONS.

Time-resolved Raman spectroscopy is an effective tool for real-time kinetic studies of protein folding. We have built the first nanosecond time-resolved UV Raman spectrometer combined with a laser induced temperature jump technique. (Lednev et al. JACS 1999, 4076 and 8074, JACS 2001, 2388) We plan to extend further this methodology to initiate protein folding with a pH jump and an ion-concentration jump to study proteins involved in cellular signaling processes.

• Novel bio/chemical sensors utilizing Raman spectroscopic detection

Raman spectroscopy provides unique opportunities for development of remote sensing systems for environmental purposes including nuclear waste testing. This is based upon:
- the striking difference of Raman signatures for different chelating agents
- substantial changes in the chelating agent Raman spectra in the analyte presence
- an enormous increase in sensitivity due to a surface enhanced Raman effect
- modern high throughput fiber optics, inexpensive lasers, and CCD detectors.

A new deep UV Raman spectroscopic apparatus (Lednev et al. Anal Bioanal Chem 2005, 431) has been recently designed, built and characterized at Albany. As a light source, the apparatus utilizes either (i) Indigo S laser system from Coherent allowing for tuning the excitation between 193 and 205 nm, or (ii) Powerlight 9050 laser system from Continuum combined with homebuilt Raman shifter. The apparatus requires only a 100-µl sample with protein concentration as low as 0.1 mg/ml. No special sample preparation is required: the dynamic range has no limitations at the high concentration end, although self-absorption might need to be taken into account for quantitative analysis of Raman spectra. Extending the excitation wavelength deeper into the UV region allows for resonantly enhancing Raman scattering from an amide chromophore, a building block of a polypeptide backbone, that does not exhibit electronic absorption in the near UV and visible spectral range. Amide chromophore Raman spectra provide quantitative information about the secondary structure of proteins. The high sensitivity of deep UV resonance Raman spectroscopy makes this nondestructive method of analysis especially valuable for studying biological systems under physiological conditions. Near UV Raman spectroscopy with excitation at and above 228 nm has already found applications in biology for characterizing protein structure and dynamics.

Amyloid fibril formation

Amyloid fibrils are associated with numerous degenerative diseases. The molecular mechanism of the conformational evolution, i.e., the transformation of native protein to the highly ordered cross-ß structure is still under active investigation. Protein structural transformations on the molecular level during in vitro fibril formation are accompanied by substantial changes in macroscopic properties, such as formation of a gelatinous phase and the formation of insoluble particles. These changes limit the application of conventional methods such as NMR, SAXS, CD, FTIR, intrinsic and ANS fluorescence, etc. for characterizing protein conformational transformations. Raman spectroscopy has been proven to be an efficient technique for characterizing highly-scattering and opaque samples.

Lysozyme fibrillation

We utilize DUVRR spectroscopy, along with other spectroscopic techniques, including tryptophan fluorescence, circular dichroism (CD) spectroscopy, and atomic force microscopy (AFM) to study quantitatively the structural evolution during in vitro fibrillation of hen egg white lysozyme (Xu et al. Biopolymers 2005, 58). Lysozyme formed fibril under prolonged incubation in acidic solution at 65ºC. Unlike fully reversible denaturation of lysozyme caused by brief heating, which resulted in only ~15% of a-helix melting at 65ºC, the prolonged incubation at this temperature caused much larger and irreversible structural changes of the protein. The resulting partially denatured intermediate contained ~8% of a-helix and ~8% of ß-sheet as compared with 32% and 6% of a-helix and ß-sheet, respectively, in the native protein. Both secondary and tertiary structures evolved in a monoexponential fashion with characteristic time of about 30 hours. DUVRR spectroscopy was shown to be superior over the far-UV CD spectroscopy in the quantitative study of fibril formation: DUVRR spectroscopy (i) was capable of characterizing inhomogeneous, highly light scattering samples containing fibrils and (ii) was highly sensitive to the formation of ß-sheet conformation. In addition, phenylalanine was shown to be an informative DUVRR spectroscopic biomarker of protein structural rearrangements during fibril formation.

(A) A three-component composition of the solution part of lysozyme incubated at 65 ºC and pH 2. (B) A relative intensity of the 1000-cm-1 phenylalanine band in the Raman spectra of lysozyme solutions incubated for various times. I0 corresponds to the band intensity in the spectrum of a non-incubated solution. I value was normalized to the protein concentration in solutions. (C) The percentage of lysozyme deposited in a gelatinous form. The solid curves represent monoexponential fits.

Gigantic (60-kDa) genetically engineered ß-sheet polypeptide forms fibrils and shows reversible thermal denaturation

A de novo 687-amino acid residue polypeptide (Topilina et al. Biomacromolecules 2006, 1104) with a regular 32 amino acid repeat sequence, (GA)3GY(GA)3GE(GA)3GH(GA)3GK, forms large ß-sheet assemblages which exhibit remarkable folding properties and, as well, form fibrillar structures (Lednev et al. Biophys. J. 2006, 3805). This construct is an excellent tool to explore the details of ß-sheet formation yielding intimate folding information which is otherwise difficult to obtain and may inform folding studies of naturally occurring materials. The polypeptide assumes a fully folded antiparallel ß-sheet/turn structure at room temperature, and yet is completely and reversibly denatured at 125 °C adopting a predominant PPII conformation. Deep UV Raman spectroscopy indicated melting/refolding occurred without any spectroscopically distinct intermediates, yet the relaxation kinetics depend on the initial polypeptide state as would be indicative of a non-two-state process. Thermal denaturation and refolding on cooling appeared to be monoexponential with characteristic times of ~1 and ~60 min, respectively, indicating no detectable formation of hairpin-type nuclei in millisecond timescale that could be attributed to nonlocal “nonnative” interactions. The polypeptide folding dynamics agree with a general property of ß-sheet proteins, i.e., initial collapse precedes secondary structure formation. The observed folding is much faster than expected for a protein of this size and could be attributed to a less-frustrated free energy landscape funnel for folding. The polypeptide sequence suggests an important balance between the absence of strong nonnative contacts (salt bridges or hydrophobic collapse) and limited repulsion of charged side chains.

Statistical analysis of spectroscopic data

DUVRR spectroscopy combined with chemometric analysis was shown to be a powerful tool for quantitative characterization of multiple equilibria between lutetium and a bicyclic diamide (Shashilov et al. Inorg Chem 2006, 3606). Several chemometric methods were utilized for a comparative analysis of Raman spectroscopic data. It was found that a recently developed stepwise maximum angle calculation (SMAC) algorithm followed by alternative least squares (ALS) was more efficient than the commonly used combination of evolving factor analysis (EFA) and ALS methods, especially when little or no information about the system composition and the spectra of individual components was available. Complex formation between a bicyclic diamide, a novel chelating agent for lanthanides and actinides, and lutetium in acetonitrile solution was investigated. A free ligand and its lutetium complexes showed weak, non-characteristic near-UV absorption and no fluorescence that limited the application of absorption and fluorescence spectroscopies for studying this system. A free ligand and 1:1, 1:2 and 1:3 metal:ligand complexes were distinguished in a bicyclic diamide/lutetium solution. The composition evolution of the solution during the course of titration with lutetium was described, and the stepwise stability constants of complex formation, (K1:K2)=0.80±0.15, (K1,2>10ˆ8 Mˆ-1) and K3=(5.5±1)•10ˆ3 Mˆ-1, were estimated.

Latent variable analysis of DUVRR spectra was demonstrated to be a powerful tool for characterizing protein secondary structural composition (Shashilov et al. JQSRT 2006, 46). Non-negative independent component analysis (ICA) and pure variable methods, such as stepwise maximum angle calculation (SMAC) and simple-to-use interactive self-modeling mixture analysis (SIMPLISMA), were employed for examination of ten DUVRR spectra of lysozyme obtained at various stages of its partial denaturation, the first stage of amyloid fibril formation. The non-negative ICA allowed for extracting the spectrum of the ß-sheet from deep UV resonance Raman spectra of lysozyme while principle component analysis (PCA) and multivariate curve resolution (MCR) could not separate the ß-sheet constituent as an individual component. No initial guess about the features of the ß-sheet spectrum was used. Pure variable methods SMAC and SIMPLISMA were found to resolve three independent spectral components assigned to ß-sheet, random coil, and native lysozyme.

Molecular modeling

Molecular modeling, which included structure optimization and calculation of Raman frequencies and resonance intensities, allowed for assigning all strong Raman bands of the bicyclic diamide as well as predicting the band shifts observed due to complex formation with metal ions. A comparative analysis of Raman spectra and the results of the molecular modeling could be used for elucidating the structure of complexes in solution. DUVRR spectroscopy was used for characterizing ligand-metal ion complexes. The obtained results demonstrated a strong intrinsic sensitivity and selectivity of a Raman spectroscopic signature of a bicyclic diamide, a novel chelating agent for lanthanides and actinides recently reported by James Hutchison with coworkers (JACS 2002). We are very grateful to Prof. James E. Hutchison and Ms. Bevin W. Parks from the University of Oregon for providing bicyclic diamides for this study.