A study of protein secondary structure by Fourier transform infrared/photoacoustic spectroscopy and its application for recombinant proteins

Siquan Luo, Chi Ying F. Huang, John F. McClelland, Donald J. Graves*

*此作品的通信作者

研究成果: Article同行評審

30 引文 斯高帕斯(Scopus)

摘要

FT-IR/PAS (Fourier transform infrared/photoacoustic spectroscopy) was used to evaluate the secondary structure of proteins. Four well-studied proteins, concanavalin A, hemoglobin, lysozyme, and trypsin, which have different distributions of secondary structures, were used for assignments of the infrared bands and evaluating the accuracy of FT-IR/PAS methods. Secondary structure contents estimated from FT-IR/PAS and other physical methods (e.g., X-ray diffraction, CD, and traditional FT-IR) show good agreement. In addition, the secondary structure can be evaluated with as little as 0.5 μg of protein (concanavalin A), suggesting that FT-IR/PAS is a sensitive and useful technique that could be applied to studies of the folding of recombinant and mutant proteins where only small amounts of material are available. Recombinant phosphorylase kinase γ1-300 subunit expressed in Escherichia coli was found in the inclusion bodies. We found that renatured phosphorylase kinase γ1-300 subunit has two kinase forms: one has a 10-fold higher activity than the other one. Both fractions, however, are the same as judged from sodium dodecylsulfate-polyacrylamide gel electrophoresis. Differences in conformation were demonstrated by using the FT-IR/PAS method, which showed that the low-activity form has more β-sheet structure than the form with high activity. Analysis of these kinase forms by CD confirms the interpretation made by the FT-IR/PAS method.

原文English
頁(從 - 到)67-76
頁數10
期刊Analytical Biochemistry
216
發行號1
DOIs
出版狀態Published - 1月 1994

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