Infrared ATR spectroscopy has recently gained recognition as a viable technique for protein structural analysis due to its
high information content, rapid sampling rate, and minimal sample preparation. Much focus has been placed on studying protein
unfolding, where infrared allows analysis of secondary structural changes on a timescale unachievable by other methods.This
note explores the use of a multiple reflection ATR to analyze small amounts of a protein undergoing secondary structural changes
in response to thermal perturbation.
HarrickConcentratIR2™ (see Figure 1) with its Si ATR crystal and heated liquid cell was installed and aligned in a commercial
FTIR spectrometer with an MCT detector. Spectra were collected at 4 cm-1 resolution and signal averaged over 64 scans. The sample, 750 μg/mL BGG in a 0.9% sodium chloride solution with sodium azide,
was injected into the liquid cell after it had stabilized ata temperature of 100 °C. Immediately after injection, the cell
was capped to prevent evaporation and an initial spectrum was collected. Additional spectra were collected at timed intervals.
Figure 1: ConcentratIR2™ Multiple Reflection ATR with its Heated Flow Cell and Temperature Controller.
Results and Discussion
Figure 2 shows the spectral changes in the amide I band in the 1600–1700 cm-1 region. The most dramatic effect is a decrease in the intensity of the band around 1670 cm-1, which occurs fairly rapidly after heating, and an increase in the intensity of the band at 1620 cm-1.
Figure 2: The ATR spectra of BGG at 100 °C at 0 (lowest peak),1, 2, 3, 4, 5, 6, 7, 8, 9, 20, and 30 (highest peak) min after
injection of sample into cell.
The amide I band consists of overlapping components that reflect secondary structures due to the hydrogen-bonded backbone
of the protein. The most common secondary structures are the α-helices, which have a characteristic band around 1660 cm-1 and the β sheets, which typically show bands around 1620–1640 cm-1. So the observed spectral changes are consistent with the relatively quick thermal degradation of the weaker α-helices followed
by the slower formation of β sheets.
The ConcentratIR2™, a multiple reflection ATR, can be used effectively to examine thermally-induced secondary structural changes
in proteins. Since the active area of the ATR element is only 4-mm in diameter, only a minute quantity of the sample is required
for analysis, making the ConcentratIR2™ extremely useful in situations where only limited quantities of sample are available.
Harrick Scientific Products
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