title: Denaturation behaviors of two-state and non-two-state proteins examined by an interruption-incubation protocol.
abstract: We established an interruption-incubation protocol to study the denaturation and aggregation behaviors of two-state and non-two-state proteins, represented by hen egg-white lysozyme (lysozyme) and bovine serum albumin (BSA), by differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy. After incubation at selected interrupting temperatures (T(int)), the onset temperature (T(onset)) and denaturation temperature (T(d)) of the reheating scans were found to pose distinct contrasts between the two proteins. BSA shows increasing T(onset) and T(d) as T(int) increases, whereas lysozyme exhibits invariable T(onset) and T(d). After long-time incubation, the reheating scans of BSA still show residual peaks in the DSC curves. Moderate aggregation upon incubation restricts the refolding of the thermodynamically stable unfolding intermediates in the denaturation of BSA. On the contrary, prolonged incubation at selected T(int) causes complete denaturation of lysozyme. The results were also supported by FTIR experiments. It is concluded that the variable T(onset) and T(d) indicate the unfolding of the "trapped" intermediates in the second heating scans, and the invariable T(onset) and T(d) values indicate the nonexistence of such intermediates. The examination of two additional proteins, ribonuclease A and γ-globulin, shows a similar phenomenon. The results may represent the general features of two-state and non-two-state denaturations and the protocol would serve to study protein denaturation cooperativity.
authors.affiliation: Key Laboratory of Bioorganic Phosphorous Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, PR China.
authors.forename: Jun-Jie
authors.initials: JJ
authors.lastname: Luo
authors.forename: Fu-Gen
authors.initials: FG
authors.lastname: Wu
authors.forename: Ji-Sheng
authors.initials: JS
authors.lastname: Yu
authors.forename: Rui
authors.initials: R
authors.lastname: Wang
authors.forename: Zhi-Wu
authors.initials: ZW
authors.lastname: Yu
mesh_terms: D000818:Animals; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D002645:Chickens; D009113:Muramidase / Q000737:chemistry*; D011489:Protein Denaturation; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared; D013816:Thermodynamics
chemical_list: D012710:Serum Albumin, Bovine; C421864:hen egg lysozyme; D009113:Muramidase