Deep UV Resonance Raman Research Articles

Understanding amyloid fibril formation using protein fragments: structural investigations via vibrational spectroscopy and solid-state NMR.

It is well established that amyloid proteins play a primary role in neurodegenerative diseases. Alzheimer's, Parkinson's, type II diabetes, and Creutzfeldt-Jakob's diseases are part of a wider family encompassing more than 50 human pathologies related to aggregation of proteins. Although this field of research is thoroughly investigated, several aspects of fibrillization remain misunderstood, which in turn slows down, or even impedes, advances in treating and curing amyloidoses. To solve this problem, several research groups have chosen to focus on short fragments of amyloid proteins, sequences that have been found to be of great importance for the amyloid formation process. Studying short peptides allows bypassing the complexity of working with full-length proteins and may provide important information relative to critical segments of amyloid proteins. To this end, efficient biophysical tools are required. In this review, we focus on two essential types of spectroscopic techniques, i.e., vibrational spectroscopy and its derivatives (conventional Raman scattering, deep-UV resonance Raman (DUVRR), Raman optical activity (ROA), surface-enhanced Raman spectroscopy (SERS),tip-enhanced Raman spectroscopy (TERS), infrared (IR) absorption spectroscopy, vibrational circular dichroism (VCD)) and solid-state nuclear magnetic resonance (ssNMR). These techniques revealed powerful to provide a better atomic and molecular comprehension of the amyloidogenic process and fibril structure. This review aims at underlining the information that these techniques can provide and at highlighting their strengths and weaknesses when studying amyloid fragments. Meaningful examples from the literature are provided for each technique, and their complementarity is stressed for the kinetic and structural characterization of amyloid fibril formation.

Hydrogen sulfide inhibits amyloid formation.

Amyloid fibrils are large aggregates of misfolded proteins, which are often associated with various neurodegenerative diseases such as Alzheimer’s, Parkinson’s, Huntington’s, and vascular dementia. The amount of hydrogen sulfide (H2S) is known to be significantly reduced in the brain tissue of people diagnosed with Alzheimer’s disease relative to that of healthy individuals. These findings prompted us to investigate the effects of H2S on the formation of amyloids in vitro using a model fibrillogenic protein hen egg white lysozyme (HEWL). HEWL forms typical β-sheet rich fibrils during the course of 70 min at low pH and high temperatures. The addition of H2S completely inhibits the formation of β-sheet and amyloid fibrils, as revealed by deep UV resonance Raman (DUVRR) spectroscopy and ThT fluorescence. Nonresonance Raman spectroscopy shows that disulfide bonds undergo significant rearrangements in the presence of H2S. Raman bands corresponding to disulfide (RSSR) vibrational modes in the 550–500 cm–1 spectral range decrease in intensity and are accompanied by the appearance of a new 490 cm–1 band assigned to the trisulfide group (RSSSR) based on the comparison with model compounds. The formation of RSSSR was proven further using a reaction with TCEP reduction agent and LC-MS analysis of the products. Intrinsic tryptophan fluorescence study shows a strong denaturation of HEWL containing trisulfide bonds. The presented evidence indicates that H2S causes the formation of trisulfide bridges, which destabilizes HEWL structure, preventing protein fibrillation. As a result, small spherical aggregates of unordered protein form, which exhibit no cytotoxicity by contrast with HEWL fibrils.

Monitoring Intramembrane Proteolytic Cleavage Reactions using Isotope-Assisted Vibrational Interrogation of Membrane Embedded (IVIBE) Proteins

While several intramembrane cleaving proteases (iCliPs), enzymes that carry out proteolysis reactions within the membrane interior, have had their structures solved, basic biochemical questions such as what determines a substrate and how cleavage site is dictated remain. However, our inability to monitor enzyme-substrate interactions in a lipid environment is the dominant factor limiting our understanding of the central questions associated with these enzymes. Specifically, while there are many excellent techniques for the characterization of membrane proteins, many of them are only amenable to steady state measurements, require removal from the membrane, or are too low resolution to offer detailed information about changes in structure and environment. Here we offer a new method by which to observe structural fluctuations of the enzyme and the substrate during cleavage reactions within a membrane environment: isotope-assisted vibrational interrogation of bilayer-embedded systems (iVIBE). Deep UV resonance Raman (DUVRR) spectroscopy has previously been used to observe structural and environmental changes in both soluble and membrane proteins. Now, using an isotopically labeled protease and an unlabeled substrate we can resolve the spectral responses from each protein, allowing us to observe the cleavage reaction over time and determine the binding site, or structural fate of the substrate.

Bilayer surface association of the pHLIP peptide promotes extensive backbone desolvation and helically-constrained structures

Despite their presence in many aspects of biology, the study of membrane proteins lags behind that of their soluble counterparts. Improving structural analysis of membrane proteins is essential. Deep-UV resonance Raman (DUVRR) spectroscopy is an emerging technique in this area and has demonstrated sensitivity to subtle structural transitions and changes in protein environment. The pH low insertion peptide (pHLIP) has three distinct structural states: disordered in an aqueous environment, partially folded and associated with a lipid membrane, and inserted into a lipid bilayer as a transmembrane helix. While the soluble and membrane-inserted forms are well characterized, the partially folded membrane-associated state has not yet been clearly described. The amide I mode, known to be sensitive to protein environment, is the same in spectra of membrane-associated and membrane-inserted pHLIP, indicating comparable levels of backbone dehydration. The amide S mode, sensitive to helical structure, indicates less helical character in the membrane-associated form compared to the membrane-inserted state, consistent with previous findings. However, the structurally sensitive amide III region is very similar in both membrane-associated and membrane-inserted pHLIP, suggesting that the membrane-associated form has a large amount of ordered structure. Where before the membrane-associated state was thought to contain mostly unordered structure and reside in a predominantly aqueous environment, we have shown that it contains a significant amount of ordered structure and rests deeper within the lipid membrane.

Évaluation du contrôle de l’asthme en consultation de pneumologie au Cameroun

Despite their presence in many aspects of biology, the study of membrane proteins lags behind that of their soluble counterparts. Improving structural analysis of membrane proteins is essential. Deep-UV resonance Raman (DUVRR) spectroscopy is an emerging technique in this area and has demonstrated sensitivity to subtle structural transitions and changes in protein environment. The pH low insertion peptide (pHLIP) has three distinct structural states: disordered in an aqueous environment, partially folded and associated with a lipid membrane, and inserted into a lipid bilayer as a transmembrane helix. While the soluble and membrane-inserted forms are well characterized, the partially folded membrane-associated state has not yet been clearly described. The amide I mode, known to be sensitive to protein environment, is the same in spectra of membrane-associated and membrane-inserted pHLIP, indicating comparable levels of backbone dehydration. The amide S mode, sensitive to helical structure, indicates less helical character in the membrane-associated form compared to the membrane-inserted state, consistent with previous findings. However, the structurally sensitive amide III region is very similar in both membrane-associated and membrane-inserted pHLIP, suggesting that the membrane-associated form has a large amount of ordered structure. Where before the membrane-associated state was thought to contain mostly unordered structure and reside in a predominantly aqueous environment, we have shown that it contains a significant amount of ordered structure and rests deeper within the lipid membrane.

Deep-UV Resonance Raman Analysis of the Rhodobacter capsulatus Cytochrome bc1 Complex Reveals a Potential Marker for the Transmembrane Peptide Backbone

Classical strategies for structure analysis of proteins interacting with a lipid phase typically correlate ensemble secondary structure content measurements with changes in the spectroscopic responses of localized aromatic residues or reporter molecules to map regional solvent environments. Deep-UV resonance Raman (DUVRR) spectroscopy probes the vibrational modes of the peptide backbone itself, is very sensitive to the ensemble secondary structures of a protein, and has been shown to be sensitive to the extent of solvent interaction with the peptide backbone [ Wang , Y. , Purrello , R. , Georgiou , S. , and Spiro , T. G. ( 1991 ) J. Am. Chem. Soc. 113 , 6368 - 6377 ]. Here we show that a large detergent solubilized membrane protein, the Rhodobacter capsulatus cytochrome bc(1) complex, has a distinct DUVRR spectrum versus that of an aqueous soluble protein with similar overall secondary structure content. Cross-section calculations of the amide vibrational modes indicate that the peptide backbone carbonyl stretching modes differ dramatically between these two proteins. Deuterium exchange experiments probing solvent accessibility confirm that the contribution of the backbone vibrational mode differences are derived from the lipid solubilized or transmembrane α-helical portion of the protein complex. These findings indicate that DUVRR is sensitive to both the hydration status of a protein's peptide backbone, regardless of primary sequence, and its secondary structure content. Therefore, DUVRR may be capable of simultaneously measuring protein dynamics and relative water/lipid solvation of the protein.

Quantitative methods for structural characterization of proteins based on deep UV resonance Raman spectroscopy

Here we report on novel quantitative approaches for protein structural characterization using deep UV resonance Raman (DUVRR) spectroscopy. Specifically, we propose a new method combining hydrogen–deuterium (HD) exchange and Bayesian source separation for extracting the DUVRR signatures of various structural elements of aggregated proteins including the cross-β core and unordered parts of amyloid fibrils. The proposed method is demonstrated using the set of DUVRR spectra of hen egg white lysozyme acquired at various stages of HD exchange. Prior information about the concentration matrix and the spectral features of the individual components was incorporated into the Bayesian equation to eliminate the ill-conditioning of the problem caused by 100% correlation of the concentration profiles of protonated and deuterated species. Secondary structure fractions obtained by partial least squares (PLS) and least squares support vector machines (LS-SVMs) were used as the initial guess for the Bayessian source separation. Advantages of the PLS and LS-SVMs methods over the classical least squares calibration (CLSC) are discussed and illustrated using the DUVRR data of the prion protein in its native and aggregated forms.

Structural Variations in the Cross-β Core of Amyloid β Fibrils Revealed by Deep UV Resonance Raman Spectroscopy

Understanding fibrillogenesis at a molecular level requires detailed structural characterization of amyloid fibrils. The combination of deep UV resonance Raman (DUVRR) spectroscopy and post mortem hydrogen-deuterium exchange (HX) was utilized for probing parallel vs antiparallel beta-sheets in fibrils prepared from full-length Abeta(1-40) and Abeta(34-42) peptides, respectively. Using previously published structural data based on solid-state NMR analysis, we verified the applicability of Asher's approach for the quantitative characterization of peptide conformation in the Abeta(1-40) fibril core. We found that the conformation of the parallel beta-sheet in the Abeta(1-40) fibril core is atypical for globular proteins, while in contrast, the antiparallel beta-sheet in Abeta(32-42) fibrils is a common structure in globular proteins. In contrast to the case for globular proteins, the conformations of parallel and antiparallel beta-sheets in Abeta fibril cores are substantially different, and their differences can be distinguished by DUVRR spectroscopy.

A de novo designed 11 kDa polypeptide: Model for amyloidogenic intrinsically disordered proteins

A de novo polypeptide GH(6)[(GA)(3)GY(GA)(3)GE](8)GAH(6) (YE8) has a significant number of identical weakly interacting beta-strands with the turns and termini functionalized by charged amino acids to control polypeptide folding and aggregation. YE8 exists in a soluble, disordered form at neutral pH but is responsive to changes in pH and ionic strength. The evolution of YE8 secondary structure has been successfully quantified during all stages of polypeptide fibrillation by deep UV resonance Raman (DUVRR) spectroscopy combined with other morphological, structural, spectral, and tinctorial characterization. The YE8 folding kinetics at pH 3.5 are strongly dependent on polypeptide concentration with a lag phase that can be eliminated by seeding with a solution of folded fibrillar YE8. The lag phase of polypeptide folding is concentration dependent leading to the conclusion that beta-sheet folding of the 11-kDa amyloidogenic polypeptide is completely aggregation driven.

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-beta 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 beta -sheet than to alpha -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.

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.