Abstract
Two‒dimensional IR correlation spectroscopy (2D‒IR) is a novel method that provides the analysis of infrared spectra with the capacity to differentiate overlapping peaks and to distinguish between in‒phase and out‒of‒phase spectral responses. Artificial spectra originated from protein amide I band component parameters have been used to study their variation in the correlation maps. Using spectra composed of one, two or three Gaussian peaks, behaviour patterns of the bands in the synchronous and asynchronous maps have been originated, with changes in intensity, band position and bandwidth. Intensity changes produce high‒intensity spots in the synchronous spectra, whereas only noise is observed in the asynchronous spectra. Band shifting originates more complex patterns. In synchronous spectra, several spots are generated at the beginning and at the end of the shifting band. Also, characteristic asynchronous spectra with butterfly‒like shapes are formed showing the trajectory of the shift. Finally, synchronous maps corresponding to band broadening reveal several spots at peak inflection points, related to the zones with higher intensity variation. The asynchronous spectra are very complex but they follow a characteristic symmetric pattern. Furthermore, examples of maps obtained from polypeptides and proteins using temperature as the perturbing factor are interpreted in terms of the patterns obtained from artificial bands.
Highlights
Infrared spectroscopy is a choice method, either as an alternative option or as a complement to the highresolution techniques in protein studies, because of the wealth of information provided and because of the sensitivity of the technique to changes in protein environment
Even if infrared spectroscopy was first applied to proteins as early as in 1952 [14] before any detailed X-ray results were available, its use with proteins in physiological environments has only been possible after the revolution in instrumentation produced by the development of microcomputers that allowed the design of instruments based on the Michelson interferometer and the fast Fourier transform [3]
Left-hand traces correspond to synchronous spectra while right-hand traces are from asynchronous spectra
Summary
Infrared spectroscopy is a choice method, either as an alternative option or as a complement to the highresolution techniques in protein studies, because of the wealth of information provided and because of the sensitivity of the technique to changes in protein environment. Even if infrared spectroscopy was first applied to proteins as early as in 1952 [14] before any detailed X-ray results were available, its use with proteins in physiological environments has only been possible after the revolution in instrumentation produced by the development of microcomputers that allowed the design of instruments based on the Michelson interferometer and the fast Fourier transform [3]. The increase in signal-to-noise ratio obtained with these instruments allows the subtraction of the aqueous buffer, obtaining spectra whose bands contain information on the protein in its native environment, and are free from spectral interference by the solvent.
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