Intermolecular interaction of photoexcited Cu(TMpy-P4) with water studied by transient resonance Raman and picosecond absorption spectroscopies

photoinduced complex between Cu(TMpy-P4) and water molecules, reversibly axially coordinated to the central metal, was observed in picosecond transient absorption and nanosecond resonance Raman experiments. This complex is rapidly created (τ1 = 15 ± 5 ps) in the excited triplet (π, π*) state of Cu-porphyrin, and the subsequent relaxation is proposed to proceed via two parallel pathways. One is fast and efficient (≥90% of molecules), and presumably involves a (π, d) charge-transfer state. The second pathway is slow (τ2 >> 1 ns), has a low quantum yield (≤10%) and involves the excited (d, d) state which is responsible for transient Raman features at ≈ 1553 cm−1 (ν2*) and ≈ 1347 cm−1 (ν4*), and for low-intensity long-lived transient absorption features

Ultraviolet resonance Raman spectroscopy for the detection of cocaine in oral fluid

Detecting and quantifying cocaine in oral fluid is of significant importance for practical forensics. Up to date, mainly destructive methods or biochemical tests have been used, while spectroscopic methods were only applied to pretreated samples. In this work, the possibility of using resonance Raman spectroscopy to detect cocaine in oral fluid without pretreating samples was tested. It was found that ultraviolet resonance Raman spectroscopy with 239-nm excitation allows for the detection of cocaine in oral fluid at 10 mu g/mL level. Further method development will be needed for reaching the practically useful levels of cocaine detection. (C) 2017 Elsevier B.V. All rights reserved

Hen Egg White Lysozyme Fibrillation: a Deep-uv Resonance Raman Spectroscopic Study

Amyloid fibrils are associated with numerous degenerative diseases. The molecular mechanism of the structural transformation of native protein to the highly ordered cross-β structure, the key feature of amyloid fibrils, is under active investigation. Conventional biophysical methods have limited application in addressing the problem because of the heterogeneous nature of the system. In this study, we demonstrated that deep-UV resonance Raman (DUVRR) spectroscopy in combination with circular dichroism (CD) and intrinsic tryptophan fluorescence allowed for quantitative characterization of protein structural evolution at all stages of hen egg white lysozyme fibrillation in vitro. DUVRR spectroscopy was found to be complimentary to the far-UV CD because it is (i) more sensitive to β -sheet than to α -helix, and (ii) capable of characterizing quantitatively inhomogeneous and highly light-scattering samples. In addition, phenylalanine, a natural DUVRR spectroscopic biomarker of protein structural rearrangements, exhibited substantial changes in the Raman cross section of the 1000-cm–1 band at various stages of fibrillation. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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. © 2005 Wiley Periodicals, Inc. Biopolymers 79: 58–61, 2005 This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [email protected]

Excited States of Water-Soluble Metal Porphyrins as Microenvironmental Probes for DNA and DNA-Model Compounds: Time-Resolved Transient Absorption and Resonance Raman Studies of Ni(TMpy-P4) in [Poly(dG-dC)]2 and [Poly(dA-dT)]2

The dynamics and mechanisms of photoexcitation relaxation of the water-soluble cationic metalloporphyrin nickel(II) 5,10,15,20-tetrakis[4-(N-methylpyridyl)]porphyrin (Ni(TMpy-P4)) bound to DNA-model polynucleotides, i.e. poly(dG-dC)2 and poly(dA-dT)2, and free in a mere phosphate buffer, have been studied in detail by using time-resolved picosecond transient absorption (TA) and nanosecond resonance Raman (RR) spectroscopies. For the Ni(TMpy-P4)−poly(dG-dC)2 complex, double-exponential kinetics of relaxation has been found, with time constants of ≤10 and 350 ± 20 ps, and absolute absorption spectra have been reconstructed from experimentally measured difference spectra. The long-lived transient species has been assigned to the excited intramolecular metal-centered (d,d) state 3B1g of the 4-coordinate Ni porphyrin intercalated between G-C base pairs. Transient RR spectra originating from this state have also been obtained and discussed. A much more complicated process of excitation relaxation has been found for the Ni(TMpy-P4)−poly(dA-dT)2 complex, where at least four relaxation components can be separated with time constants of ≤10, ∼100, ∼450 ps, and ≫1 ns. Our studies support the existence of at least two types of Ni(TMpy-P4) interaction with poly(dA-dT)2, each having its own kinetics of TA decay and transient RR spectra. Both TA and RR sets of data show that a major part of Ni porphyrin molecules yields a photophysical behavior typical for a 4-coordinate species, the excited (d,d) state 3B1g playing the key role in relaxation processes, while a minor part of Ni(TMpy-P4) also participates in axial ligand binding/release photoprocesses. Comparative analysis of transient RR spectra of Ni(TMpy-P4) bound to the A-T sequence and free in a phosphate buffer shows that no 6-coordinate 3B1g(L)2 transient species is photogenerated in the complex with poly(dA-dT)2, and therefore, axial coordination of only one extra-ligand molecule (most probably from the surrounding water solution) to the porphyrin central Ni ion is proposed to explain the experimental results. Possible processes of Ni(TMpy-P4) binding to poly(dA-dT)2 are discussed on the basis of the current photophysical data

Reversible Thermal Denaturation of a 60-kDa Genetically Engineered β-Sheet Polypeptide

A de novo 687-amino-acid residue polypeptide with a regular 32-amino-acid repeat sequence, (GA)(3)GY(GA)(3)GE(GA)(3)GH(GA)(3)GK, forms large β-sheet assemblages that exhibit remarkable folding properties and, as well, form fibrillar structures. This construct is an excellent tool to explore the details of β-sheet formation yielding intimate folding information that is otherwise difficult to obtain and may inform folding studies of naturally occurring materials. The polypeptide assumes a fully folded antiparallel β-sheet/turn structure at room temperature, and yet is completely and reversibly denatured at 125°C, adopting a predominant polyproline II conformation. Deep ultraviolet Raman spectroscopy indicated that 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 the millisecond timescale that could be attributed to nonlocal “nonnative” interactions. The polypeptide folding dynamics agree with a general property of β-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

The first step of hen egg white lysozyme fibrillation, irreversible partial unfolding, is a two-state transition

Amyloid fibril depositions are associated with many neurodegenerative diseases as well as amyloidosis. The detailed molecular mechanism of fibrillation is still far from complete understanding. In our previous study of in vitro fibrillation of hen egg white lysozyme, an irreversible partially unfolded intermediate was characterized. A similarity of unfolding kinetics found for the secondary and tertiary structure of lysozyme using deep UV resonance Raman (DUVRR) and tryptophan fluorescence spectroscopy leads to a hypothesis that the unfolding might be a two-state transition. In this study, chemometric analysis, including abstract factor analysis (AFA), target factor analysis (TFA), evolving factor analysis (EFA), multivariate curve resolution–alternating least squares (ALS), and genetic algorithm, was employed to verify that only two principal components contribute to the DUVRR and fluorescence spectra of soluble fraction of lysozyme during the fibrillation process. However, a definite conclusion on the number of conformers cannot be made based solely on the above spectroscopic data although chemometric analysis suggested the existence of two principal components. Therefore, electrospray ionization mass spectrometry (ESI-MS) was also utilized to address the hypothesis. The protein ion charge state distribution (CSD) envelopes of the incubated lysozyme were well fitted with two principal components. Based on the above analysis, the partial unfolding of lysozyme during in vitro fibrillation was characterized quantitatively and proven to be a two-state transition. The combination of ESI-MS and Raman and fluorescence spectroscopies with advanced statistical analysis was demonstrated to be a powerful methodology for studying protein structural transformations