Stability and Aggregation
Since the RedShiftBio analyzer can directly measure protein secondary structure, it is a powerful tool for monitoring and understanding the mechanisms of protein stability and aggregation. As proteins are subjected to stress and the protein begins to change from it’s native state, details of the process can be easily followed. IR measurements are particularly sensitive to beta sheet structures, which dominate in protein anti-body based drugs. In addition, it is one of the only techniques which can directly monitor the formation of aggregates due to its ability to measure intermolecular beta sheet structures. Below we illustrate the use of the analyzer for two common types of stability studies, thermal and chemical.
A high beta sheet content protein at 1 mg/mL was incubated at an elevated temperature for differing periods of time. The protein series was measured using the MMS analyzer and the second derivative spectra were overlaid and plotted to enhance the spectral changes. The data shown in Figure 1 clearly show the loss of intramolecular beta sheet content as a function of incubation time. Simultaneously, the amount of intermolecular beta sheet structure increases, which is associated with the formation of aggregates. Changes in other regions reflect the state of the protein sub-structure and provide additional details of the denaturation process.
As the protein was incubated, it was noted an insoluble aggregate formed and settled to the bottom of the sample tubes. In this study, only the supernatant fraction was decanted and measured. As a result, the overall concentration of soluble protein decreases at longer incubation times. This is illustrated in Figure 2, which shows the amide I absorbance decrease to less then half of its initial value which corresponds to less then 0.5 mg/mL of protein in solution. Changes in the spectral shape of the amide I band are also clearly evident in the absorption data.
Chemical stress studies are another method of studying protein stability. Alcohols are well known to denature the native state of proteins and also tend to stabilize the alpha-helical conformation in unfolded proteins and peptides . In this study a relatively high concentration of beta lactoglobulin was formulated in phosphate buffer at pH 7.4 at 0, 20, 40 and 60% isopropyl alcohol (IPA) concentration. The IPA/protein series were then measured by both Far UV-CD and MMS to track the structural changes. In the UV-CD data, shown in Figure 3, a clear increase in the alpha helix structure occurs while a general decrease in beta sheet occurs.
In contrast, the RedShiftBio data of Figure 4 shows not only an increase in the alpha helix form at higher IPA concentration, it also shows a dramatic and clear shift in the beta sheet type as noted by the shift of the band from ~1630 cm-1 to 1620 cm-1, again indicating the formation of intermolecular beta sheet, something the CD does not readily show. Not only does RedShiftBio’s MMS analyzer provide greater insight into the denaturation process but it operates over a much wider range of concentrations.
1 Hirota N, Mizuno K, Goto Y, Cooperative alpha helix formation of beta lactoglobulin and melittin induce by hexafluoroisoprpanol. Protein Science (1997) 6:416-421.
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Application Note on Microfluidic Modulation Spectroscopy
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