Deep-UV Raman spectrometer tunable between 193 and 205�nm for structural characterization of proteins

Lednev, Igor K.; Ermolenkov, Vladimir V.; He, Wei; Xu, Min

doi:10.1007/s00216-004-2991-5

Cited by 85 publications

(109 citation statements)

References 43 publications

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“…23,26 For atomic force microscopy (AFM) measurements, a drop of the polypeptide solution was placed on highly oriented pyrolytic graphite (HOPG) for 30 min and then removed by a micropipette. The surface was dried with a slow stream of nitrogen before imaging.…”

Section: Methodsmentioning

confidence: 99%

β‐sheet folding of 11‐kDa fibrillogenic polypeptide is completely aggregation driven

Topilina¹,

Ermolenkov²,

Higashiya³

et al. 2007

Biopolymers

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A de novo polypeptide GH(6)[(GA)(3)GY(GA)(3)GE](8)GAH(6) (YE8) was designed and genetically engineered to form antiparallel beta-strands of GAGAGA repeats. Modulation of pH enables control of solubility, folding, and aggregation of YE8 by control of the overall polypeptide charge, a consequence of the protonation or deprotonation of the glutamic acid and histidine residues. YE8 exhibits all the major properties of a fibrillogenic protein providing an excellent model for detailed study of the fibrillation. At neutral pH, YE8 is soluble in disordered form, yet at pH 3.5 folds into a predominantly beta-sheet conformation that is fibrillogenic. Atomic force microscopy and transmission electron microscopy indicated the formation of fibrillar aggregates on well-defined, hydrophobic surfaces. The beta-sheet folding of YE8 exhibited a lag phase that could be eliminated by seeding or stirring. The strong dependence of lag time on polypeptide concentration established the limiting step in aggregation as initiation of beta-sheet folding.

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Section: Methodsmentioning

confidence: 99%

β‐sheet folding of 11‐kDa fibrillogenic polypeptide is completely aggregation driven

Topilina¹,

Ermolenkov²,

Higashiya³

et al. 2007

Biopolymers

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“…Perchlorate was used as the internal standard in this set of ac-phe-ee Raman spectral measurements. No photodegradation was evident for the samples at the laser (197 nm) output power of 0.5 mW, which corresponded to an irradiating power of~0.2 mW at samples [43]. The obtained Raman spectra ( Figure 6) consisted of two sharp acetonitrile bands (918 cm À1 and 1374 cm À1 ) and six prominent bands from ac-phe-ee, which can be assigned to the phenyl ring: n 12 (ring stretching, 1000 cm À1 ), n 18a (inplane C--H bending, 1028 cm À1 ), n 9a (inplane C--H bending, 1182 cm…”

Section: Phenylalanine Is a Natural Duvrr Spectroscopic Probe Of Locamentioning

confidence: 97%

“…197 nm excited DUVRR spectra were measured using a home-built Raman spectrometer as described elsewhere [43]. A spinning NMR tube with a magnetic stirrer inside was used for sampling.…”

Section: Deep-uv Resonance Raman (Duvrr) Spectroscopymentioning

confidence: 99%

“…Among the destabilization conditions are: mutation [7,20,30,31], addition of alcohol [32][33][34][35][36][37], denaturation [18,38], reduction [36,39], incubation of acidic solution at high temperature [40][41][42][43], etc. Biophysical techniques such as NMR [31,39,[44][45][46][47], small angle X-ray/neutron scattering (SAXS/SANS) [34,37,48,49], thioflavin T (ThT) fluorescence [6,15,[50][51][52], circular dichroism (CD) spectroscopy [38,42,47,53], FTIR [37,47,54], static/dynamic light scattering [42,[55][56][57], etc.…”

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Hen egg white lysozyme fibrillation: a deep‐UV resonance Raman spectroscopic study

Ermolenkov

Uversky

et al. 2008

Journal of Biophotonics

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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.

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“…Utilization of UV irradiation at ;200 nm enables selective resonance enhancement of vibrational modes from amide chromophore (i.e., peptide bond), the building block of a polypeptide backbone. The technique, deep UV resonance Raman (DUVRR) spectroscopy, has been shown to be a powerful tool for structural characterization of proteins and polypeptides Asher 2001;Lednev et al 2005;Xu et al 2005;JiJi et al 2006). We have recently demonstrated that DUVRR spectroscopy can be used for probing structural evolution of an amyloidogenic protein at all stages of fibril formation .…”

mentioning

confidence: 99%

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

Shashilov

Ermolenkov

et al. 2007

Protein Science

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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 resolutionalternating 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.

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Deep-UV Raman spectrometer tunable between 193 and 205�nm for structural characterization of proteins

Cited by 85 publications

References 43 publications

β‐sheet folding of 11‐kDa fibrillogenic polypeptide is completely aggregation driven

β‐sheet folding of 11‐kDa fibrillogenic polypeptide is completely aggregation driven

Hen egg white lysozyme fibrillation: a deep‐UV resonance Raman spectroscopic study

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

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