title: Structural analyses combined with small-angle X-ray scattering reveals that the retention of heme is critical for maintaining the structure of horseradish peroxidase under denaturing conditions.
abstract: We analyzed the structure of horseradish peroxidase (HRP) under denaturing conditions of 9 M urea or 6 M guanidine hydrochloride (GdnHCl). Far-UV circular dichroism (CD) spectra indicated the existence of native-like secondary structure of holo-HRP in 9 M urea. In addition, slight changes in near-UV and Soret region CD spectra of holo-HRP in 9 M urea suggest that the tertiary structure of holo-HRP and the binding of heme remain partially intact in this condition. A transition in the thermal unfolding transition curve of holo-HRP in 9 M urea indicated the existence of a considerable amount of secondary structure. However, no secondary structure, tertiary structure, or interaction between heme and HRP were observed in holo-HRP in 6 M GdnHCl. Small-angle X-ray scattering indicated that although distal and proximal domains of holo-HRP in 9 M urea might be partially unfolded, the central region that contains the heme might maintain its tertiary structure. Our results suggest that retention of the heme is essential for maintenance of the structure of HRP under highly denaturing conditions.
authors.affiliation: Pohang Accelerator Laboratory, Pohang University of Science and Technology (POSTECH), Pohang, Korea.
authors.forename: Hyung Jin
authors.initials: HJ
authors.lastname: Cha
authors.affiliation: Department of Life Sciences, POSTECH, Pohang, Korea.|Huons Co., Ltd., Seongnam, Korea.
authors.forename: Do Soo
authors.initials: DS
authors.lastname: Jang
authors.affiliation: Pohang Accelerator Laboratory, Pohang University of Science and Technology (POSTECH), Pohang, Korea. jinks@postech.ac.kr.
authors.forename: Kyeong Sik
authors.initials: KS
authors.lastname: Jin
authors.affiliation: Department of Life Sciences, POSTECH, Pohang, Korea. kchoi@postech.ac.kr.
authors.forename: Kwan Yong
authors.initials: KY
authors.lastname: Choi
mesh_terms: D002942:Circular Dichroism; D019791:Guanidine / Q000737:chemistry; D006418:Heme / Q000737:chemistry*; D006735:Horseradish Peroxidase / Q000737:chemistry*; D008958:Models, Molecular; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D013696:Temperature; D014508:Urea / Q000737:chemistry; D014961:X-Ray Diffraction
chemical_list: D006418:Heme; D014508:Urea; D006735:Horseradish Peroxidase; D019791:Guanidine
keywords: Conformational stability; Guanidine hydrochloride; Guinier analysis; Porod analysis; Urea