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.
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.
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