Addressing Mechanism of Fibrillization/Aggregation and Its Prevention in Presence of Osmolytes: Spectroscopic and Calorimetric Approach

Understanding the mechanism of protein fibrillization/aggregation and its prevention is the basis of development of therapeutic strategies for amyloidosis. An attempt has been made to understand the nature of interactions of osmolytes L-proline, 4-hydroxy-L-proline, sarcosine and trimethylamine N-oxide with the different stages of fibrillization of hen egg-white lysozyme by using a combination of isothermal titration calorimetry, differential scanning calorimetry, fluorescence spectroscopy, and transmission electron microscopy. Based on thioflavin T fluorescence emission intensities and microscopic images, the nucleation, elongation, and saturation phases of fibrillization have been identified. Isothermal titration calorimetry and differential scanning calorimetry have enabled a quantitative analysis of the nature of interactions of these osmolytes with various conformational states of lysozyme at different stages of fibrillization/aggregation. It is concluded that interaction of the osmolytes with lysozyme fibrils at both the nucleation and elongation stages are important steps in the prevention of fibrillization/aggregation. Identification of the nature of interactions is a key step towards the discovery and synthesis of target oriented potential inhibitors of these associations. This study is a first report in which calorimetry has been used to address interaction of potential inihibitiors with the protein at different stages of fibrillization.


Introduction
The fibrillization or aggregation process in proteins involves several molecules forming higher order of conglomerates which have low solubility in aqueous medium. Depending upon their macroscopic morphology, such aggregates have been classified as ordered or disordered [1,2]. Amorphous aggregates can be formed under physiological conditions at high concentrations in almost all proteins. However, b rich amyloid fibrils have been observed in a smaller set of proteins [2,3]. The ordered aggregation in globular proteins occurs after partial unfolding of the native state into an intermediate state which is amyloidogenic in nature and exposes the aggregation prone regions [4][5][6].
The inability of a protein to adopt or remain in the native state can result into fibrillar and aggregated structures. This forms the basis of some of the most important neurodegenerative and metabolic disorders. Therefore, it is extremely important to discover the methods which lead to prevention of the formation of fibrillar/aggregated structures. This can be achieved by using suitable external agents which can act as potential inhibitors of these association processes. The small molecules which can alter the conformational stability or inhibit the protein fibrillization/ aggregation have helped in the development of potential therapeutic strategies against the diseases which occur due to misfolding. Prevention of misfolding and aggregation of proteins by osmolytes has been reported in literature [7][8][9][10][11]. It is also known that in general the osmolytes enhance the thermal stability of a variety of proteins due to preferential hydration phenomenon [7,8,[12][13][14]. The use of suitable small molecules permit a tremendous scope for their studies as potential therapeutic molecules against protein destabilization or several misfolding related disorders [15,16].
Amongst the proteins which form amyloid fibrils under specific conditions, lysozyme is a suitable model to study the mechanism of amyloid formation and its prevention due to its small size and availability of extensive structural information in literature. For a long time lysozyme has been used as a model protein for understanding the complexity of protein structure and function in physiology and diseases [17,18]. It has also been used as a model molecule for the investigation of enzyme catalysis, and as a disease marker [17].
Although the research over last few years has revealed the morphology and structural features of the amyloid/aggregated forms of the proteins, knowledge about the thermodynamics of amyloid formation and the process of inhibition is scarce. Evaluation of the thermodynamic parameters associated with interaction of potential inhibitors with proteins in the native, unfolded, and various stages of the fibrillization process can reveal the nature of interactions responsible for the inhibition process and hence identification of the functional groups on such molecules for effective inhibition.
In light of this background information, we have carried out calorimetric, spectroscopic and microscopic studies looking into the effect of osmolytes L-proline, 4-hydroxyl-L-proline, sarcosine, and trimethylamine N-oxide on lysozyme amyloid fibrillization. The main objective of this work is to apply quantitative techniques such as calorimetry in combination with spectroscopy and microscopy to unravel the energetics and mode of interaction of such small molecules with the protein which leads to prevention of fibrillization/aggregation. By combining the thermodynamic and structural details, it will be possible to understand the mechanism of interface in the fibrillization/aggregation process and hence suggest further guidelines towards the identification and synthesis of novel potential inhibitors.

Materials
Lysozyme (.0.95), L-proline (.0.99), 4-hydroxy-L-proline (. 0.99), sarcosine (.0.98), trimethylamine N-oxide (.0.98) and thioflavin T (dye content: 0.65-0.75) were procured from Sigma-Aldrich Chemical Company USA. The listed purities of these compounds, on mass fraction basis, are given in the parenthesis. The solutions were prepared in deionized water which was double distilled and passed through Cole-Parmer mixed-bed ion exchange column. All the experiments were done in 40 mM phosphate buffer at pH 2.1 containing 100 mM NaCl. The stock solutions of lysozyme were dialyzed overnight at 4uC against the buffer with at least three changes of the latter. The osmolyte solutions were also prepared in the final dialysate buffer. The concentration of the protein and ThT were determined on a Jasco V-550 uv-visible double beam spectrophotometer, using a value of A 1 cm 1% = 26.5 [19] at l = 280 nm and E = 26,620 M 1 cm 21 at l = 412 nm [20], respectively.

Fluorescence Spectroscopy
The steady state fluorescence measurements were done on a Cary Eclipse spectrofluorimeter with excitation and emission slit widths fixed at 2.5 nm. Thioflavin T (ThT), a cationic benzothiazole dye has been widely used to identify the amyloid fibrils having common structural features [21][22][23][24]. The ThT molecules were selectively excited at l ex = 450 nm [25]. The reported fluorescence emission spectra of the complexes have been corrected by subtracting the reference spectra of the control solutions containing same amount of the dye.

Transmission Electron Microscopy
The visualization of the amyloid fibrils was done on a JEOL JEM-100B Transmission Electron Microscope which operates at an accelerating voltage of 80 kV. The Formvar-coated 300 mesh copper grids were used for deposition of the samples. The negative staining of the samples was done with 2% aqueous uranyl acetate solution. After pre-rinsing with large volumes of water, a 0.22 mm filter was used to filter the stains. Uranyl acetate is known to produce high electron density, image contrast, and impart fine grained impression to the image [26].

Isothermal Titration Calorimetry
The interaction of amyloid fibrils with solvent and potential inhibitors was studied by using ultra sensitive isothermal titration calorimeters (VP ITC form Microcal LLC, and Nano ITC from TA Instruments). The fibril solution was titrated into the sample cell containing buffer or appropriate amount of the osmolyte in aliquots using a rotating stirrer-syringe of 250 ml capacity. The reference cell was filled with the respective buffer. The experiments were designed for a total of 25 consecutive injections, each having a volume of 10 ml of 0.606 mM native lysozyme solution or heat induced fibril solution into buffer or osmolyte solution in the cell. The duration between consecutive injections was 10 s with an interval of 4 min between each injection. The same procedure was used to measure the heats of dilutions by titrating buffer with the respective osmolytes at the same concentrations as used in the main experiments. After dilution corrections, the ITC profiles were analyzed to determine the heat of interaction by using Origin 7.0 and Nano Analyzer data analysis softwares supplied by Microcal and TA Instruments, respectively.

Differential Scanning Calorimetry
A Nano DSC from TA Instruments, having capillary cells of volume 300 ml was employed to study the thermal transitions of the protein under different conditions. The sample cell of the DSC contained protein or protein+osmolyte solution and the reference cell was filled with the corresponding buffer or buffer+osmolyte solution. All the DSC experiments were done at a scan rate of 1 K min 21 . The reported excess heat capacity versus temperature scans for the protein transitions have been corrected for the corresponding solvent versus solvent scans heated under the same conditions.

Binding of Thioflavin T with amyloid fibrils
An indepth knowledge of the binding mode of ThT with amyloid fibrils is essential for understanding the structural characteristics of these associated assemblies. An important advantage of ThT compared to other amyloid specific dyes such as Congo Red is its ability to detect the amyloid fibril formation in situ [27]. The main aim of the present study is to understand the energetics and mechanism of interaction of some potential inhibitors of aggregation/fibrillization with the protein in the native and at different stages of fibrillization. For this the changes in fluorescence properties of ThT have been explored upon interaction with these states of the protein. Figure 1A shows the fluorescence emission spectra accompanying the binding of ThT with lysozyme amyloid fibrils. ThT selectively interacts with amyloid fibrils which leads to significant changes in its fluorescence emission at l em = 480 nm when excited at l ex = 450 nm [28]. No fluorescence emission was seen with ThT in buffer and ThT in presence of native lysozyme at 25uC ( Figure 1A). Significant fluorescence emission was seen when ThT was interacted with the heat induced fibrils which were obtained after 72 h of incubation at 57uC ( Figure 1A). The use of ThT as a fibril specific dye permitted monitoring the process of mature fibril formation as a function of time. For this a solution of native lysozyme (690 mM), prepared in 40 mM phosphate buffer containing 0.1 M NaCl at pH 2.1 was incubated at 57uC. A calculated amount of lysozyme solution was taken from this solution and added to ThT solution containing the same buffer, such that the final concentrations of the protein and the dye in the solution were 69 mM and 50 mM, respectively. NaCl was used as it is known to promote fibril extension [29][30][31][32]. A typical sigmoidal curve was obtained which has the characteristics of fibril extension ( Figure 1B). The curve contains three distinct phases: nucleation (region I-II), fibril extension (region II-IV) and saturation (region IV-VI). These three stages contain varying degree of fibrillization in lysozyme ( Figure 1B). As seen in this figure, the fluorescence emission intensity of ThT remains nearly the same from state I (incubation time 10 h) to state II (incubation time 48 h) where the nucleation phase ends and the elongation phase begins sharply. It is seen that the elongation of fibrils rises sharply from 48 h to 61 h of incubation (stage III). After 70 h of incubation, the extent of fibril formation appears to be saturated (region IV to VI). The structural features of the native protein and at these three phases were characterized by TEM. For thermodynamic characterization of different fibrillar states of lysozyme in the absence and presence of different osmolytes, isothermal titration calorimetric studies were performed.

Structural characterization of fibrillar extension by transmission electron microscopy
To characterize different structures formed at various stages of fibrillization, Transmission Electron Microscopy (TEM) was used. The TEM image of native lysozyme at pH 2.1 is shown in Figure 2A. The fibrillar extension in lysozyme solution after incubation at 57uC for 48 h, 60 h and at 90 h are shown in Figure 2B to 2D. Figure 2 shows that at 57uC the morphology of the protein changes from non-aggregated state ( Figure 2A) to a fibril network as the time proceeds. After 48 h it is seen that the oligomeric structures of lysozyme are formed which provide nucleus for further extension, as is observed in Figure 2C which corresponds to incubation for 60 h. After 90 h, a clear mature fibrillar structure is observed as shown in Figure 2D.

Isothermal titration calorimetry of dilution of lysozyme in the native state and heat induced lysozyme fibrils in buffer
There are no reports in literature on the energetics of interaction of inhibitors with proteins at different stages of the aggregation/fibrillization process. Experiments were designed on interaction of the fibrillar protein with different osmolytes. However, prior to that the thermodynamic characterization of the dilution of native lysozyme and at three stages of the fibrillization process in aqueous medium were done by using isothermal titration calorimetry. The protein solution was taken in the syringe of ITC whereas both the reference and sample cells were filled with the buffer. Experiments were conducted at different time intervals of fibrillization and the respective representative ITC profiles are shown in Figure 3. The ITC results suggest that the dilutions of lysozyme into buffer from the native state, denatured state, or aggregated/fibrillar states do not show appreciable heat effects.
Fluorescence kinetic studies of the formation of lysozyme aggregates in presrence of different osmolytes Figure 4 shows a typical sigmoidal path during the fibrillization process having three stages: nucleation, elongation and saturation. The kinetics of the fibrillization process was studied by using 690 mM lysozyme in presence of different osmolytes. The process of fibrillization process was monitored by using 50 mM ThT. The osmolytes chosen for these studies were L-proline, 4-hydroy-Lproline, sarcosine and trimethylamine N-oxide (TMAO). The time course of the fibril extension was examined on fluorescence spectrophotometer at 57uC. Here also a calculated amount of lysozyme solution was taken and added to the ThT solution in the absence and presence of different osmolytes, such that the final concentrations of the protein and the dye in the solution were 69 mM and 50 mM, respectively. The length of the nucleation and elongtation phases under each of these conditions are reproducible within an error of 10%. In Figure Figure 4A shows the progress of the fibril formation as a function of time monitored by the ThT binding assay in the absence and presence of different concentrations of L-proline. The curve I in this Figure represents formation of fibrils in lysozyme in the absence of any additive. In the presence of 0.50 M L-proline (curve V), the duration of the nucleation phase increased substantially as reflected by significant reduction in the fluorescence emission intensity of ThT. This suggests that L-proline at this concentration has acted as an inhibitor of the lysozyme fibrillization process. When the concentration of L-proline was increased to 1.0 M in solution, no binding of ThT with lysozyme was observed as reflected by absence of the fluorescence emission under these conditions (Curve VI). These results suggest that 1.0 M L-proline is able to arrest the fibrillization process in lysozyme yielding a very clear solution. Figure 4B represents the effect of 4-hydroxy-L-proline on fibrillization of lysozyme as a function of concentration and time. It is seen that 0.50 M 4hydroxy-L-proline is able to delay the nucleation and elongation   periods much more than L-proline. It is further observed that the fluorescence emission intensity of ThT also decreased many folds in presence of 0.50 M 4-hydroxy-L-proline compared to that of Lproline. This suggests that 4-hydroxy-L-proline is a stronger inhibitor of fibrillization than L-proline which differs by one -OH group in its chemical structure. Here also 1 M 4-hydroxy-Lproline is able to suppress the fibrillization completely (curve VI). Figure 4C shows the effect of sarcosine on fibrillization of lysozyme as a function of concentration and time. Similar to the effect of 4-hydroxy-L-proline, sarcosine at 0.50 M concentration  level is also able to delay the nucleation and elongation periods significantly. Here again 1.0 M sarcosine is able to suppress the fibrillization completely. Figures 4A to 4C demonstrate that up to a concentration of 0.10 M, proline, 4-hydroxy-L-proline or sarcosine are not effective in the prevention of formation of fibrils. These osmolytes exhibit significant effect at a concentration of 0.25 M and above.
The effect of TMAO on fibrillization process is shown in Figure 4D. It is seen that at all the studied concentrations of TMAO, the fluorescence intensity of ThT totally vanished. The fibrils of lysozyme after an incubation period of 80 h were clearly visible with naked eye. However, in the presence of L-proline, 4hydroxy-L-proline and sarcosine, the solution after the same incubation period was clear. In the case of TMAO, the solution after an incubation period of 80 h contained aggregates which were observed to be amorphous in nature. It is known that ThT does not bind to amorphous aggregates but binds specifically to the fibrillar structures [33,34]. Thus TMAO is observed to follow a different mode of action compared to other osmolytes. It either has a little effect [35] or may induce oligomerization [36] in case of Ab40 proteins. It can induce formation of folded oligomers or enhance fibrillation [37] in a-synuclein. It can also induce formation of amorphous aggregates in polyQ peptides [38].

Calorimetric studies on the interaction of osmolytes with lysozyme fibrils
The thermodynamic characterization of the mechanism of inhibition of fibrillization by osmolytes was done by ultrasensitve isothermal titration calorimetry. The experiments were performed at different concentrations of osmolytes L-proline, 4-hydroxy Lproline, sarcosine and TMAO. Solutions of lysozyme at different phases of the fibrillization process were titrated with the osmolytes to understand the energetics and nature of interactions operating in these systems. We could not find any studies in literature in which the interaction of potential inhibitors with the protein at different stages of the fibrillization process has been addressed specifically based on heats of interaction. It is understood that since the protein may not have specific binding sites for the inhibitors, the challenge lies in identifying the nature of interactions and hence the properties of the molecules responsible for the inhibition process.
Isothermal titration calorimetry of lysozyme fibrils at the nucleation phase Figure 6 represents the heat evolved when lysozyme, at the nucleation phase of fibrillization after 48 h of the incubation period at 57uC, is interacted with L-proline. The solution of lysozyme chosen for the ITC studies under these conditions corresponds to point II of Figure 1B. In the ITC experiments, different concentrations of osmolytes were taken in the sample cell, and lysozyme solution was taken in the syringe. It is seen in Figure 6A that interaction of L-proline with lysozyme at the nucleation phase is exothermic in nature. The extent of exothermicity decreases with increase in the molar ratio of lysozyme to L-proline. It is known that the propagation of aggregation begins from partially folded or unfolded species that expose a greater backbone surface area than the native protein.
According to Auton and Bolen [39], the unfavorable interactions of L-proline with the peptide backbone cause preferential exclusion of the solute from the protein-water interface of these partially folded intermediates, hence increasing its free energy. It is known that the structural stability of proteins can be enhanced by certain osmolytes through the enhancement of hydrogen bonding in the hydration shell of the macromolecule [40]. This whole mechanism provides exothermic contribution to the overall reaction which is reflected in the ITC measurements. The enthalpy of interaction of lysozyme fibrils with L-proline at nucleation stage is most exothermic with the values changing from 2(141.263.2) kJ mol 21 [41]. Therefore lesser availability of the polar groups of L-proline as a result of supramolecular structure results in reduction of polar interactions between Lproline and protein, and hence decreased exothermicity. It has also been reported [41] that L-proline having amphiphilic nature of its supramolecular assembly, provides hydrophobic surfaces which interact with aggregation prone solvent exposed hydrophobic residues during folding of the protein thereby blocking the protein aggregation effectively. The interaction of lysozyme at the nucleation stage with 4-hydroxyl-L-proline and sarcosine are also observed to be exothermic in nature ( Figure 6B). A comparison of the enthalpy of interaction of lysozyme with L-proline and 4hydroxy-L-proline suggests that additional exothermicity observed with the latter is due to an extra hydroxyl group present in it which can interact with the polar groups of the protein via hydrogen bonding. Similar effect is also observed in the interaction of lysozyme at the nucleation phase with sarcosine ( Figure 6C). Figure 7 represents the heat profiles accompanying the titration of lysozyme fibrils with osmolytes at the elongation phase corresponding to point III in Figure 1B. It is seen in Figure 7 that the titrations of the fibrils with L-proline, 4-hydroxyl-Lproline and sarcosine are mostly exothermic in nature, highest with sarcosine. It is observed that under these conditions, Lproline does not show appreciable heat of interaction or a specific heat pattern ( Figure 7A). The maximum value of heat of interaction of lysozyme fibrils with L-proline under these conditions is -(25.663.3) kJ mol 21 of the protein. The protein species at the nucleation stage will have greater exposed backbone surface area than the native state and are thus preferentially stabilized by L-proline. But with the late aggregates having high molecularity and a significantly smaller exposed surface, the favorable osmolyte interactions would be relatively diminished [42], which is reflected in the less exothermic contribution of the interaction of L-proline with fibrils at the elongated stage.

Isothermal titration calorimetry of interaction of lysozyme fibrils at the elongation phase with different osmolytes
Similarly for 4-hydroxy-L-proline, the heat of interaction is very small ( Figure 7B). For example the enthalpy of interaction of lysozyme at this stage with 0.25 M 4-hydroxy-L-proline is in the range of 3.860.3 to 3.160.3 kJ mol 21 which reaches a maximum range of [2(29.362.2) to 2(20.862.6) kJ mol 21 ) for interaction with 1 M 4-hydroxy-L-proline ( Figure 7B). Interestingly 0.25 M sarcosine and 0.5 M sarcosine show significant heat of interaction with the fibrils at the elongation phase ( Figure 7C). The isothermal titration calorimetry resuls on the interaction of lysozyme in the native, nucleation stage and elongation stage with the osmolytes Lproline, 4-hydroxy-L-proline, and sarcosine suggest that these osmolytes preferentially interact and possibly inhibit the aggregation/fibrillization at both the nucleation stage and elongation stage where the heat of interaction is observed to be higher than that with the native state, and denatured state of the protein.
Interaction of matured fibrils with L-proline   Interaction of matured fibrils with sarcosine Figure 8C shows the integrated heat profile for the interaction of matured fibrils with sarcosine. Here a small trend in the heat of interaction is observed as the concentration of the fibrils increases in 0.25 M sarcosine in solution taken in the cell. This trend is more towards the endothermic nature. However, when the concentration of sarcosine is increased to 1 M in the solution, the heat of interaction of the fibrils do not show a definite trend as the number of injections of the matured fibrils in the sample cell is increased and the overall heat change is very small. Figure 9 represents dilution corrected integrated heat profiles for the titration of fibrils taken after 48 hrs, 61 hrs and 90 hrs of incubation into a solution containing 0.25 M TMAO in the sample cell. It is observed that with 0.25 M TMAO, the protein does not show significant heat of interaction either at the nucleation phase or elongation phase of the fibrillization process. In case of matured fibrils the heat change moves towards exothermic effects as the concentration of the fibrils increases in the solution. Experiments could not be performed at further higher concentration of TMAO due to saturating heat signals.

Differential Scanning Calorimetry
The differential scanning calorimetry of native lysozyme and at nucleation, elongation and saturation stages of fibrillization was carried out as shown in Figure 10. A solution of 10 mg ml 21 lysozyme was incubated at 57uC for different time periods to obtain species at different stages of lysozyme fibrillization process. Curve A shows the DSC scan of buffer verses buffer in the temperature range of 40 to 80uC. Curve B shows the DSC profile of thermal unfolding of native lysozyme in buffer only. The native lysozyme unfolds at (57.460.1)uC which is consistent with the transition temperature of the protein reported in literature [9] at pH 2.1. The curve C shows the thermal unfolding of lysozyme at the nucleation stage which corresponds to point II of Figure 1B. The thermal unfolding profile at this stage still shows a significant endothermic transition suggesting presence of significant native like structure in the protein. The DSC scan of lysozyme after 61 h of incubation which corresponds to point III of Figure 1B is shown curve D. This curve belongs to the elongation phase of the fibrillization process. It is seen that the native like properties of lysozyme are largely lost at the elongation stage. Further, after 90 h of incubation corresponding to the saturation phase ( Figure 1B), the endotherm is completely lost as seen in curve E of Figure 10A. These results show that as the course of fibrillization proceeds the native like properties of lysozyme are diminished. Figure 10B shows excess heat capacity over and above the base line for the native state of the protein and at its elongation and  Osmolytes and Prevention of Fibrillization PLOS ONE | www.plosone.org saturation stages of fibrillization. It is seen that the excess heat capacity of the protein in the pre-tansition region varies in the following order. c p (native) vc p (elongation stage) vc p (saturation stage) The extent of aggregation is maximum in the saturation state where matured fibrils have been formed compared to that in the elongation state, where the fibril formation is still in progress. Therefore the excess heat capacity of the protein in the saturation state is observed to be highest ( Figure 10B).

Conclusions
The amyloid fibrils of lysozyme have been prepared and monitored by fluorescence spectroscopy using binding properties of ThT. The ThT fluorescence emission intensities upon binding to lysozyme at nucleation, elongation and saturation phases of fibrillization permitted assessment of these stages and planning the ITC experiments to understand the energetics of interaction of the chosen osmolytes with the fibrils. The transmission electron microscopy permitted visualization of these fibrils at nucleation, elongation and fibrillization stages. It was observed that L-proline, 4-hydroxy-L-proline, sarcosine and TMAO prevent fibrillization. It was also observed that 4-hydroxy-L-proline and sarcosine are able to delay the onset of elongation phase by a substantially longer time. However, with TMAO lysozyme solution showed amorphous aggregates even after 300 minutes. The TEM images support the above mentioned observations. The interaction of lysozyme is observed to be exothermic with L-proline and 4hydroxy-L-proline at the nucleation phase suggesting involvement of polar interactions. These interactions are lesser exothermic at the elongation phase with L-proline and 4-hydroxyl-L-proline compared to that of sarcosine. However, sarcosine undergoes significant polar exothermic interactions with the fibrils at elongation stage. The results suggest that the interaction of the osmolytes with lysozyme fibrils at the nucleation stage involving predominantly polar interactions are major step in the prevention of aggregation/fibrillization, in addition to possibility of inhibition at the elongation phase. In addition to the qualitative information available from the spectroscopic and microscopic measurements, the ITC results have added substantial quantitative understanding on the nature of interactions of these osmolytes with the protein at different stages of the fibrilization process. The DSC results have not only demonstrated the thermal stability of the protein as the fibrilization proceeds, it has also supported the observations on the extent of fibrilization based on the relative changes in the values of heat capacity ( Figure 10).