title: Osmolytes induce structure in an early intermediate on the folding pathway of barstar.
abstract: Osmolytes stabilize proteins against denaturation, but little is known about how their stabilizing effect might affect a protein folding pathway. Here, we report the effects of the osmolytes, trimethylamine-N-oxide, and sarcosine on the stability of the native state of barstar as well as on the structural heterogeneity of an early intermediate ensemble, IE, on its folding pathway. Both osmolytes increase the stability of the native protein to a similar extent, with stability increasing linearly with osmolyte concentration. Both osmolytes also increase the stability of IE but to different extents. Such stabilization leads to an acceleration in the folding rate. Both osmolytes also alter the structure of IE but do so differentially; the fluorescence and circular dichroism properties of IE differ in the presence of the different osmolytes. Because these properties also differ from those of the unfolded form in refolding conditions, different burst phase changes in the optical signals are seen for folding in the presence of the different osmolytes. An analysis of the urea dependence of the burst phase changes in fluorescence and circular dichroism demonstrates that the formation of IE is itself a multistep process during folding and that the two osmolytes act by stabilizing, differentially, different structural components present in the IE ensemble. Thus, osmolytes can alter the basic nature of a protein folding pathway by discriminating, through differential stabilization, between different members of an early intermediate ensemble, and in doing so, they thereby appear to channel folding along one route when many routes are available.
authors.affiliation: National Centre for Biological Sciences, Tata Institute of Fundamental Research, Gandhi Krishi Vigyan Kendra Campus, Bangalore 560 065, India.
authors.forename: Lovy
authors.initials: L
authors.lastname: Pradeep
authors.forename: Jayant B
authors.initials: JB
authors.lastname: Udgaonkar
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008744:Methylamines / Q000494:pharmacology*; D008956:Models, Chemical; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012521:Sarcosine / Q000494:pharmacology*; D013053:Spectrophotometry; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014466:Ultraviolet Rays; D014508:Urea / Q000737:chemistry / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D008744:Methylamines; C057438:barstar protein, Bacillus amyloliquefaciens; D014508:Urea; C005855:trimethyloxamine; D012521:Sarcosine