Abstract
Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopy are analytical techniques employed for the analysis of protein secondary structure. The use of CD spectroscopy is limited to low protein concentrations (<2 mg ml−1), while FTIR spectroscopy is commonly used in a higher concentration range (>5 mg ml−1). Here we introduce a quantum cascade laser (QCL)-based IR transmission setup for analysis of protein and polypeptide secondary structure at concentrations as low as 0.25 mg ml−1 in deuterated buffer solution. We present dynamic QCL-IR spectra of the temperature-induced α-helix to β-sheet transition of poly-L-lysine. The concentration dependence of the α-β transition temperature between 0.25 and 10 mg ml−1 was investigated by QCL-IR, FTIR and CD spectroscopy. By using QCL-IR spectroscopy it is possible to perform IR spectroscopic analysis in the same concentration range as CD spectroscopy, thus enabling a combined analysis of biomolecules secondary structure by CD and IR spectroscopy.
Highlights
Protein secondary structures[8,9]
The experimental setup is composed of an EC-Quantum cascade lasers (QCLs) with a spectral tuning range covering the amide I region of proteins, a peltier-cooled MCT-detector and a temperature-controlled flow cell with a path length of 478 μm
The temporal axis directly corresponds to the wavenumber axis; wavenumber inaccuracies of the external cavity-QCLs (EC-QCLs) as well as time lags introduced during data acquisition are directly translated into wavenumber deviations of the final spectra
Summary
Protein secondary structures[8,9]. CD spectroscopy is only applicable for liquid phase samples and is generally used for optically clear solutions at concentration ranges below 2 mg ml−1 10,11. When working with proteins in aqueous solution, the strong absorbance of the HOH bending band of water near 1,645 cm−1, overlapping with the protein amide I band, requires short path lengths, typically around 8 μm for conventional FTIR spectrometers[3,12]. To overcome this drawback, D2O-based buffers are alternatively used as solvents. We introduce an EC-QCL-based IR transmission setup for secondary structure analysis of proteins and polypeptides in deuterated solutions at concentrations as low as 0.25 mg ml−1. By example of the concentration-dependent temperature-induced α-βtransition of poly-L-lysine (PLL), we demonstrate the low accessible concentration range for the EC-QCL setup, enabling a combined analysis of biomolecules secondary structure by CD and IR spectroscopy
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