title: Protein folding by the effects of macromolecular crowding.
abstract: Unfolded states of ribonuclease A were used to investigate the effects of macromolecular crowding on macromolecular compactness and protein folding. The extent of protein folding and compactness were measured by circular dichroism spectroscopy, fluorescence correlation spectroscopy, and NMR spectroscopy in the presence of polyethylene glycol (PEG) or Ficoll as the crowding agent. The unfolded state of RNase A in a 2.4 M urea solution at pH 3.0 became native in conformation and compactness by the addition of 35% PEG 20000 or Ficoll 70. In addition, the effects of macromolecular crowding on inert macromolecule compactness were investigated by fluorescence correlation spectroscopy using Fluorescence-labeled PEG as a test macromolecule. The size of Fluorescence-labeled PEG decreased remarkably with an increase in the concentration of PEG 20000 or Ficoll 70. These results show that macromolecules are favored compact conformations in the presence of a high concentration of macromolecules and indicate the importance of a crowded environment for the folding and stabilization of globular proteins. Furthermore, the magnitude of the effects on macromolecular crowding by the different sizes of background molecules was investigated. RNase A and Fluorescence-labeled PEG did not become compact, and had folded conformation by the addition of PEG 200. The effect of the chemical potential on the compaction of a test molecule in relation to the relative sizes of the test and background molecules is also discussed.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Tokuriki
authors.forename: Masataka
authors.initials: M
authors.lastname: Kinjo
authors.forename: Shigeru
authors.initials: S
authors.lastname: Negi
authors.forename: Masaru
authors.initials: M
authors.lastname: Hoshino
authors.forename: Yuji
authors.initials: Y
authors.lastname: Goto
authors.forename: Itaru
authors.initials: I
authors.lastname: Urabe
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Yomo
mesh_terms: D000818:Animals; D002417:Cattle; D002645:Chickens; D002942:Circular Dichroism; D005362:Ficoll / Q000494:pharmacology; D005456:Fluorescent Dyes; D006834:Hydrazines; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011092:Polyethylene Glycols / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000494:pharmacology
chemical_list: C417561:Alexa 488 hydrazide; D005456:Fluorescent Dyes; D006834:Hydrazines; D046911:Macromolecular Substances; D005362:Ficoll; D011092:Polyethylene Glycols; D014508:Urea; D012259:Ribonuclease, Pancreatic; D009113:Muramidase