Fourth derivative UV-spectroscopy of proteins under high pressure I. Factors affecting the fourth derivative spectrum of the aromatic amino acids

Lange, Reinhard; Frank, Johannes; Saldana, Jean-Louis; Balny, Claude

doi:10.1007/bf00180368

Cited by 66 publications

(75 citation statements)

References 37 publications

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“…1). These changes are caused by alterations in the conformation of rhIFN-␥, because 2D UV spectra of tyrosine and tryptophan are minimally affected by pressure (25). In thermal and urea denaturation experiments with rhIFN-␥, 2D UV spectral changes were concomitant with the loss of secondary structure, as measured by circular dichroism spectroscopy at 222 nm (data not shown).…”

Section: Resultsmentioning

confidence: 94%

Partial molar volume, surface area, and hydration changes for equilibrium unfolding and formation of aggregation transition state: High-pressure and cosolute studies on recombinant human IFN-γ

Webb

Cleland

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

106

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The equilibrium dissociation of recombinant human IFN-␥ was monitored as a function of pressure and sucrose concentration. The partial molar volume change for dissociation was ؊209 ؎ 13 ml/mol of dimer. The specific molar surface area change for dissociation was 12.7 ؎ 1.6 nm 2 /molecule of dimer. The first-order aggregation rate of recombinant human IFN-␥ in 0.45 M guanidine hydrochloride was studied as a function of sucrose concentration and pressure. Aggregation proceeded through a transition-state species, N*. Sucrose reduced aggregation rate by shifting the equilibrium between native state (N) and N* toward the more compact N. Pressure increased aggregation rate through increased solvation of the protein, which exposes more surface area, thus shifting the equilibrium away from N toward N*. The changes in partial molar volume and specific molar surface area between the N* and N were ؊41 ؎ 9 ml/mol of dimer and 3.5 ؎ 0.2 nm 2 / molecule, respectively. Thus, the structural change required for the formation of the transition state for aggregation is small relative to the difference between N and the dissociated state. Changes in waters of hydration were estimated from both specific molar surface area and partial molar volume data. From partial molar volume data, estimates were 25 and 128 mol H2O/mol dimer for formation of the aggregation transition state and for dissociation, respectively. From surface area data, estimates were 27 and 98 mol H2O/mol dimer. Osmotic stress theory yielded values Ϸ4-fold larger for both transitions.A ggregation is of considerable concern to the medical, pharmaceutical, and biotechnology industries, as protein aggregation occurs in human diseases (1-4) and during the production, purification, and storage of protein products (5). Characterization of the conformational state(s) that lead(s) to aggregation is essential for a mechanistic understanding of the aggregation process.Protein aggregation was once viewed as a nonspecific hydrophobically driven process involving the fully unfolded random coil (6-8). However, recent research has shown protein aggregation to follow distinct pathways involving folding intermediates (6, 7, 9). Thus, the folding pathway and aggregation processes are critically linked (7,8). Characterization of the unfolding process, in conjunction with aggregation rate studies, should then provide insight into the mechanisms of protein aggregation and the role of folding intermediates therein. A major challenge in characterizing aggregation pathways is that under conditions that greatly favor the native state (e.g., physiological), the small populations of highly reactive aggregationcompetent species and the transition states leading to these species may be inaccessible to available spectroscopic techniques. To populate putative aggregation-competent species at levels sufficient for spectroscopic measurement, conditions that greatly perturb the native state are typically used (e.g., pH Ͻ4.0). Furthermore, even if the aggregation-competent species populated under nonn...

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Section: Resultsmentioning

confidence: 94%

Partial molar volume, surface area, and hydration changes for equilibrium unfolding and formation of aggregation transition state: High-pressure and cosolute studies on recombinant human IFN-γ

Webb

Cleland

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

106

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“…UV Absorbance Spectroscopy-For all experiments, we used a thermostated high pressure cell, equipped with sapphire windows, allowing experiments from atmospheric pressure (0.1 MPa) to 650 MPa (32). The cell was connected at 180°through focalized optical quartz fibers (200-m-diameter) to a Jobin-Yvon-Spex 30W Deuterium lamp and to a Jobin-Yvon-Spex 270M spectrograph coupled to a UV-coated backthinned Spectrum ONE TM 512/512 array detection CCD 3500 camera (Jobin-Yvon-Spex Inc.) Another fiber served as a reference beam.…”

Section: Methodsmentioning

confidence: 99%

“…The concentration of proteins was 1.5 mg⅐ml Ϫ1 . Fourth derivative spectra were computed as described (32,33).…”

Section: Methodsmentioning

confidence: 99%

Structural Instability and Fibrillar Aggregation of Non-expanded Human Ataxin-3 Revealed under High Pressure and Temperature

Marchal

Shehi

Harricane

et al. 2003

Journal of Biological Chemistry

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Protein misfolding and formation of structured aggregates are considered to be the earliest events in the development of neurodegenerative diseases, but the mechanism of these biological phenomena remains to be elucidated. Here, we report a study of heat-and pressure-induced unfolding of human Q26 and murine Q6 ataxin-3 using spectroscopic methods. UV absorbance and fluorescence revealed that heat and pressure induced a structural transition of both proteins to a molten globule conformation. The unfolding pathway was partly irreversible and led to a protein conformation where tryptophans were more exposed to water. Furthermore, the use of fluorescent probes (8-anilino-1-naphthalenesulfonate and thioflavin T) allowed the identification of different intermediates during the process of pressure-induced unfolding. At high temperature and pressure, human Q26, but not murine Q6, underwent concentration-dependent aggregation. Fourier transform infrared and circular dichroism spectroscopy revealed that these aggregates are characterized by an increased ␤-sheet content. As revealed by electron microscopy, heat-and pressure-induced aggregates were different; high temperature treatment led to fibrillar microaggregates (8 -10-nm length), whereas high pressure induced oligomeric structures of globular shape (100 nm in diameter), which sometimes aligned to higher order suprastructures. Several intermediate structures were detected in this process. Two factors appear to govern ataxin unfolding and aggregation, the length of the polyglutamine tract and its protein context.

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“…Ultraviolet absorbance in the fourth derivative mode was used as an intrinsic probe for both tryptophan and tyrosine residues. This technique allows one to enhance the generally low resolution of zero-order UV absorbance spectra and to obtain information about structural changes in the local environment of tyrosine and tryptophan residues (12,24). The fourth derivative UV absorbance spectra were characterized by two maxima, reflecting mainly the tyrosine environment at 284 nm and the tryptophan environment at 290.5 nm (Fig.…”

Section: Lithostathine Fibrils Did Not Possess the Classical Propertiesmentioning

confidence: 99%

Lithostathine Quadruple-helical Filaments Form Proteinase K-resistant Deposits in Creutzfeldt-Jakob Disease

Laurine

Grégoire

Fändrich³

et al. 2003

Journal of Biological Chemistry

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Autocatalytic cleavage of lithostathine leads to the formation of quadruple-helical fibrils (QHF-litho) that are present in Alzheimer's disease. Here we show that such fibrils also occur in Creutzfeldt-Jakob and Gerstmann-Strä ussler-Scheinker diseases, where they form protease-K-resistant deposits and co-localize with amyloid plaques formed from prion protein. Lithostathine does not appear to change its native-like, globular structure during fibril formation. However, we obtained evidence that a cluster of six conserved tryptophans, positioned around a surface loop, could act as a mobile structural element that can be swapped between adjacent protein molecules, thereby enabling the formation of higher order fibril bundles. Despite their association with these clinical amyloid deposits, QHF-litho differ from typical amyloid fibrils in several ways, for example they produce a different infrared spectrum and cannot bind Congo Red, suggesting that they may not represent amyloid structures themselves. Instead, we suggest that lithostathine constitutes a novel component decorating disease-associated amyloid fibrils. Interestingly, [6,6 ]bibenzothiazolyl-2,2 -diamine, an agent found previously to disrupt aggregates of huntingtin associated with Huntington's disease, can dissociate lithostathine bundles into individual protofilaments. Disrupting QHF-litho fibrils could therefore represent a novel therapeutic strategy to combat clinical amyloidoses.

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Fourth derivative UV-spectroscopy of proteins under high pressure I. Factors affecting the fourth derivative spectrum of the aromatic amino acids

Cited by 66 publications

References 37 publications

Partial molar volume, surface area, and hydration changes for equilibrium unfolding and formation of aggregation transition state: High-pressure and cosolute studies on recombinant human IFN-γ

Partial molar volume, surface area, and hydration changes for equilibrium unfolding and formation of aggregation transition state: High-pressure and cosolute studies on recombinant human IFN-γ

Structural Instability and Fibrillar Aggregation of Non-expanded Human Ataxin-3 Revealed under High Pressure and Temperature

Lithostathine Quadruple-helical Filaments Form Proteinase K-resistant Deposits in Creutzfeldt-Jakob Disease

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