Lysozyme encapsulated gold nanoclusters for probing the early stage of lysozyme aggregation under acidic conditions

Protein aggregation can lead to several incurable amyloidosis diseases. The full aggregation pathway is not fully understood, creating the need for new methods of studying this important biological phenomenon. Lysozyme is an amyloidogenic protein which is often used as a model protein for studying amyloidosis. This work explores the potential of employing Lysozyme encapsulated gold nanoclusters (Ly-AuNCs) to study the protein’s aggregation. The fluorescence emission properties of Ly-AuNCs were studied in the presence of increasing concentrations of native lysozyme and as a function of pH, of relevance in macromolecular crowding and inflammation-triggered aggregation. AuNC fluorescence was observed to both redshift and increase in intensity as pH is increased or when native lysozyme is added to a solution of Ly-AuNCs at pH 3. The long (μs) fluorescence lifetime component of AuNC emission was observed to decrease under both conditions. Interestingly it was found via Time Resolved Emission Spectra (TRES) that both AuNC fluorescence components increase in intensity and redshift with increasing pH while only the long lifetime component of AuNC was observed to change when adding native lysozyme to solution; indicating that the underlying mechanisms for the changes observed are fundamentally different for each case. It is possible that the sensitivity of Ly-AuNCs to native lysozyme concentration could be utilized to study early stage aggregation

Detecting Lysozyme unfolding via the fluorescence of Lysozyme encapsulated gold nanoclusters

Protein misfolding plays a critical role in the formation of Amyloidosis type disease. Therefore, understanding and ability to track protein unfolding in a dynamic manner is of considerable interest. Fluorescence-based techniques are powerful tools for gaining real-time information about the local environmental conditions of a probe on the nanoscale. Fluorescent gold nanoclusters (AuNCs) are a new type of fluorescent probes which are <2 nm in diameter, incredibly robust and offer highly sensitive, wavelength tuneable emission. Their small size minimises intrusion and makes AuNCs ideal for studying protein dynamics. Lysozyme has previously been used to encapsulate AuNCs. The unfolding dynamics of Lysozyme under different environmental conditions have been well-studied and being an Amyloid type protein, makes Lysozyme an ideal candidate for encapsulating AuNCs in order to test their sensitivity to protein unfolding. In this study, we tracked the fluorescence characteristics of AuNCs encapsulated in Lysozyme while inducing protein unfolding by Urea, Sodium Dodecyl Sulphate (SDS) and elevated temperature and compared them to complimentary Circular Dichroism spectra. It is found that AuNC fluorescence emission is quenched upon induced protein unfolding either due to a decrease in Forster Resonance Energy Transfer (FRET) efficiency between tryptophan and AuNCs or solvent exposure of the AuNC. Fluorescence lifetime measurements confirmed quenching to be collisional via oxygen dissolved in a solution; increasing as the AuNC was exposed to the solvent during unfolding. Moreover, the longer decay component τ1 was observed to decrease as the protein unfolded, due to the increased collisional quenching. It is suggested that AuNC sensitivity to solvent exposure might be utilised in the future as a new approach to studying and possibly even detecting Amyloidosis type diseases

Lysozyme encapsulated gold nanocluster for studying protein denaturation

Protein denaturation is a change in a protein's structure from its native folded state to a non-native misfolded state. Protein denaturation is the cause of many diseases. Current methods used for protein denaturation studies have provided useful information regarding protein structures, but have limitations, such as their inability to detect early aggregation of protein; hence, a new method for detecting early aggregation is needed. Lysozyme-encapsulated gold nanoclusters (Lyz-AuNCs) have interesting fluorescence properties that can be used in a variety of fluorescence measurement techniques and maybe a promising tool for studying protein denaturation. So far, studies of the fluorescence characteristics of Lyz-AuNCs under protein denaturation conditions, and their correlation to protein unfolding, have been limited. The goal of this research was, therefore, to discover the influence of environmental factors and protein denaturation on the fluorescence properties of Lyz-AuNCs and further explore the potential of Lyz-AuNCs to inhibit human beta-amyloid (1-40) Aβ40 aggregation.;In our study, changes in pH were observed to alter the fluorescence properties of Lyz-AuNCs. At an excitation wavelength 470 nm, the fluorescence intensity of AuNCs increased and redshifted when the pH is increased from pH 7 to 12. This increase correlated to a decrease in its fluorescence lifetime, suggesting a possible mechanism of the enhanced radiative process. Moreover, hen egg-white lysozyme (HEWL) was added to Lyz-AuNCs solutions to study the effect of HEWL concentrations on their fluorescence properties. Especially, the fluorescence lifetime was found to be sensitive to the concentration of HEWL at pH 3, possibly due to the aggregation that changed the local environment. Furthermore, unfolding of Lyz-AuNCs was induced by urea, sodium dodecyl sulphate and elevated temperature. It was found that the fluorescence intensity of Lyz-AuNCs decreased due to increased collisional quenching. Finally, the interaction between Aβ40 and Lyz-AuNCs was studied. The observed decrease in fluorescence intensity was believed to be due to static quenching. Significantly, Lyz-AuNCs was found to inhibit Aβ40 fibre formation. This result suggested Lyz-AuNCs as a promising candidate for Alzheimer's disease (AD) treatment as well as a probe to study Aβ40 accumulation in AD pathology.Protein denaturation is a change in a protein's structure from its native folded state to a non-native misfolded state. Protein denaturation is the cause of many diseases. Current methods used for protein denaturation studies have provided useful information regarding protein structures, but have limitations, such as their inability to detect early aggregation of protein; hence, a new method for detecting early aggregation is needed. Lysozyme-encapsulated gold nanoclusters (Lyz-AuNCs) have interesting fluorescence properties that can be used in a variety of fluorescence measurement techniques and maybe a promising tool for studying protein denaturation. So far, studies of the fluorescence characteristics of Lyz-AuNCs under protein denaturation conditions, and their correlation to protein unfolding, have been limited. The goal of this research was, therefore, to discover the influence of environmental factors and protein denaturation on the fluorescence properties of Lyz-AuNCs and further explore the potential of Lyz-AuNCs to inhibit human beta-amyloid (1-40) Aβ40 aggregation.;In our study, changes in pH were observed to alter the fluorescence properties of Lyz-AuNCs. At an excitation wavelength 470 nm, the fluorescence intensity of AuNCs increased and redshifted when the pH is increased from pH 7 to 12. This increase correlated to a decrease in its fluorescence lifetime, suggesting a possible mechanism of the enhanced radiative process. Moreover, hen egg-white lysozyme (HEWL) was added to Lyz-AuNCs solutions to study the effect of HEWL concentrations on their fluorescence properties. Especially, the fluorescence lifetime was found to be sensitive to the concentration of HEWL at pH 3, possibly due to the aggregation that changed the local environment. Furthermore, unfolding of Lyz-AuNCs was induced by urea, sodium dodecyl sulphate and elevated temperature. It was found that the fluorescence intensity of Lyz-AuNCs decreased due to increased collisional quenching. Finally, the interaction between Aβ40 and Lyz-AuNCs was studied. The observed decrease in fluorescence intensity was believed to be due to static quenching. Significantly, Lyz-AuNCs was found to inhibit Aβ40 fibre formation. This result suggested Lyz-AuNCs as a promising candidate for Alzheimer's disease (AD) treatment as well as a probe to study Aβ40 accumulation in AD pathology