title: Interaction of arginine with protein during refolding process probed by amide H/D exchange mass spectrometry and isothermal titration calorimetry.
abstract: Arginine has been widely used as low molecular weight additive to promote protein refolding by suppressing aggregate formation. However, methods to investigate the role of arginine in protein refolding are often limited on protein's global conformational properties. Here, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was used to study the effects of arginine on recombinant human granulocyte colony-stimulating factor (rhG-CSF) refolding at the scale of peptide mapping. It was found that deuteration levels of rhG-CSF refolded with arginine was higher than that without arginine during the whole refolding process, but they became almost the same when the refolding reached equilibrium. This phenomenon indicated that arginine could protect some amide deuterium atoms from being exchanged with hydrogen, but the protection diminished gradually along with refolding proceeding. Enzymatic digestion revealed six particular peptides of 16-47, 72-84, 84-93, 114-124, 145-153 and 154-162 were mainly responsible for the deuteration, and all of them dominantly located in protein's α-helix domain. Furthermore, thermodynamics analysis by isothermal titration calorimetry provided direct evidence that arginine could only react with denatured and partially refolded rhG-CSF. Taking all of the results together, we suggest that arginine suppresses protein aggregation by a reversible combination. At the initial refolding stage, arginine could combine with the denatured protein mainly through hydrogen bonding. Subsequently, arginine is gradually excluded from protein with protein's native conformation recovering.
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Dawei
authors.initials: D
authors.lastname: Zhao
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China. Electronic address: ydliu@ipe.ac.cn.
authors.forename: Yongdong
authors.initials: Y
authors.lastname: Liu
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Guifeng
authors.initials: G
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu 610041, China.
authors.forename: Chun
authors.initials: C
authors.lastname: Zhang
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Xiunan
authors.initials: X
authors.lastname: Li
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Qingqing
authors.initials: Q
authors.lastname: Wang
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Shi
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China. Electronic address: zgsu@home.ipe.ac.cn.
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Su
mesh_terms: D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry* / Q000378:metabolism; D002151:Calorimetry / Q000379:methods*; D041961:Deuterium Exchange Measurement; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D013997:Time Factors
chemical_list: D000577:Amides; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D001120:Arginine
keywords: Arginine; HDX-MS; ITC; Refolding; rhG-CSF