Unfolding and aggregation of lysozyme: A thermodynamic and kinetic study by FTIR spectroscopy

Sassi, Paola; Giugliarelli, Alessandra; Paolantoni, Marco; Morresi, Assunta; Onori, G.

doi:10.1016/j.bpc.2011.05.002

Cited by 51 publications

(93 citation statements)

References 44 publications

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“…Moreover, the technique has been extensively used to observe the conformational transition from monomers to cross β-sheet amyloid structures during amyloid formation and to investigate the aggregates structural properties in solution [51, 129, 130]. IR spectroscopy is based on the molecular vibrations produced, under light exposition, by stretching, deformational motions, bending and rotations of chemical bonds.…”

Section: Spectroscopic Bulk Techniques: Measuring Average Propertiesmentioning

confidence: 99%

AFM-Based Single Molecule Techniques: Unraveling the Amyloid Pathogenic Species

Ruggeri

Habchi

Cerreta

et al. 2016

CPD

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BackgroundA wide class of human diseases and neurodegenerative disorders, such as Alzheimer’s disease, is due to the failure of a specific peptide or protein to keep its native functional conformational state and to undergo a conformational change into a misfolded state, triggering the formation of fibrillar cross-β sheet amyloid aggregates. During the fibrillization, several coexisting species are formed, giving rise to a highly heterogeneous mixture. Despite its fundamental role in biological function and malfunction, the mechanism of protein self-assembly and the fundamental origins of the connection between aggregation, cellular toxicity and the biochemistry of neurodegeneration remains challenging to elucidate in molecular detail. In particular, the nature of the specific state of proteins that is most prone to cause cytotoxicity is not established.Methods:In the present review, we present the latest advances obtained by Atomic Force Microscopy (AFM) based techniques to unravel the biophysical properties of amyloid aggregates at the nanoscale. Unraveling amyloid single species biophysical properties still represents a formidable experimental challenge, mainly because of their nanoscale dimensions and heterogeneous nature. Bulk techniques, such as circular dichroism or infrared spectroscopy, are not able to characterize the heterogeneity and inner properties of amyloid aggregates at the single species level, preventing a profound investigation of the correlation between the biophysical properties and toxicity of the individual species.Conclusion:The information delivered by AFM based techniques could be central to study the aggregation pathway of proteins and to design molecules that could interfere with amyloid aggregation delaying the onset of misfolding diseases.

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Section: Spectroscopic Bulk Techniques: Measuring Average Propertiesmentioning

confidence: 99%

AFM-Based Single Molecule Techniques: Unraveling the Amyloid Pathogenic Species

Ruggeri

Habchi

Cerreta

et al. 2016

CPD

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“…[1314] In FTIR, amide I band region between 1700/cm and 1600/cm is very useful to monitor protein's conformational changes. [15] However, generally speaking, conducting FTIR experiments using commercial instrument poses certain difficulties with respect to water subtraction, proper purging, cleaning of sample cells, evaporation of the sample and reproduction of data with minimum errors.…”

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confidence: 99%

“…Even in one of the most recent FTIR based study of high concentration lysozyme, deuterated sample of the enzyme was used probably to overcome the problems with water subtraction. [13] Neither in the food processing industry nor in pharmaceutical applications of lysozyme is the enzyme used in deuterated solvents. [4] This fact highlights the importance of understanding the conformational changes of enzymes in water-based buffers.…”

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confidence: 99%

Thermal stability of high concentration lysozyme across varying pH: A Fourier Transform Infrared study

2013

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AIM:The current work is aimed at understanding the effect of pH on the thermal stability of hen egg white lysozyme (HEWL) at high concentration (200 mg/mL).MATERIALS AND METHODS:Fourier Transform Infrared (FTIR) Spectroscopy with modified hardware and software to overcome some of the traditional challenges like water subtraction, sample evaporation, proper purging etc., are used in this study.RESULTS:HEWL was subjected to thermal stress at pH 3.0-7.0 between 25°C and 95°C and monitored by FTIR spectroscopy. Calculated Tm values showed that the enzyme exhibited maximum thermal stability at pH 5.0. Second derivative plots constructed in the amide I region suggested that at pH 5.0 the enzyme possessed higher amount of α-helix and lower amount of aggregates, when compared to other pHs.CONCLUSIONS:Considering the fact that HEWL has attractive applications in various industries and being processed under different experimental conditions including high temperatures, our work is able to reveal the reason behind the pH dependent thermal stability of HEWL at high concentration, when subjected to heat denaturation. In future, studies should aim at using various excipients that may help to increase the stability and activity of the enzyme at this high concentration.

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“…4). Meersman and Heremans [31] and Sassi et al [32] suggested that the formation of intermolecular b-sheet conformation due to protein aggregation is indicated by the presence of characteristic FTIR absorption at 1618 cm À1 and 1690 cm À1 . Similarly, thermally denatured proteins containing intermolecular b-sheet aggregates also show the FTIR absorption at 1618 and 1690 cm À1 [33].…”

Section: Resultsmentioning

confidence: 98%