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Two-Dimensional Infrared Correlation Spectroscopy Study of Sequential Events in the Heat-Induced Unfolding and Aggregation Process of Myoglobin

Yong-Bin Yan ^* , Qi Wang , Hua-Wei He ^*, Xin-Yao Hu , Ri-Qing Zhang ^*  and Hai-Meng Zhou 

^* NMR Laboratory, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China; State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua University, Beijing 100084, China; Department of Chemistry, Tsinghua University, Beijing 100084, China; and Protein Science Laboratory of the Ministry of Education, Tsinghua University, Beijing 100084, China

Correspondence: Address reprint requests to Dr. Yong-Bin Yan, NMR Laboratory, Dept. of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China. Tel.: +86-10-6278-3477; Fax: +86-10-6277-1597; E-mail: ybyan{at}mail.tsinghua.edu.cn.

Unfolding and aggregation are basic problems in protein science with serious biotechnological and medical implications. Probing the sequential events occurring during the unfolding and aggregation process and the relationship between unfolding and aggregation is of particular interest. In this study, two-dimensional infrared (2D IR) correlation spectroscopy was used to study the sequential events and starting temperature dependence of Myoglobin (Mb) thermal transitions. Though a two-state model could be obtained from traditional 1D IR spectra, subtle noncooperative conformational changes were observed at low temperatures. Formation of aggregation was observed at a temperature (50–58°C) that protein was dominated by native structures and accompanied with unfolding of native helical structures when a traditional thermal denaturation condition was used. The time course NMR study of Mb incubated at 55°C for 45 h confirmed that an irreversible aggregation process existed. Aggregation was also observed before fully unfolding of the Mb native structure when a relative high starting temperature was used. These findings demonstrated that 2D IR correlation spectroscopy is a powerful tool to study protein aggregation and the protein aggregation process observed depends on the different environmental conditions used.

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