Probing conformational changes of proteins by quantitative second-derivative infrared spectroscopy

Probing protein conformational changes plays a crucial role in protein structure and function Studies. However, the lack of efficient biophysical techniques makes it difficult to obtain the distinct behaviors of different secondary Structure elements in a protein upon perturbation. This paper presents a discussion of the two major problems, the effect of sidelobes and different half-width at half-height (HWHH) values, encountered in quantitative second-derivative infrared (QSD-IR) spectroscopy and introduces the development of two criteria for checking the validity of the results obtained using the QSD-IR method. It was found that neither the sidelobes nor the HWHH significantly affected the quantitative result of protein conformational changes by using poly-L-lysine and hemoglobin as model proteins. A case Study or bovine serum albumin (BSA) thermal aggregation Suggested that the thermal transition of BSA was a process involving sequential events, and the two helical components were found to have a distinct response to heat perturbation. These results were confirmed by two-dimensional infrared correlation spectroscopy and by results in literature. suggesting that the QSD-IR method might be a potentially powerful tool to probe the distinct response of different secondary structures to perturbation. (c) 2005 Elsevier Inc. All rights reserved.