title: Structure of very early protein folding intermediates: new insights through a variant of hydrogen exchange labelling.
abstract: BACKGROUND
  Hydrogen exchange labelling has been a key method in characterizing the structure of transient folding intermediates. In studies of several proteins, however, there has been clear spectroscopic evidence for partial folding of some kind at very early times, before any protection from exchange was measurable. These results, presumably a consequence of limited stability of specific backbone interactions, have made it difficult to assess the extent of native-like folding in the very early intermediates. We have used a variant of the labelling method to investigate marginally stable structures formed within the first few milliseconds of refolding of two such proteins, hen lysozyme and ubiquitin.
  
  
  RESULTS
  In lysozyme, population of a subset of native-like secondary structures on this timescale is revealed, thus reconciling the exchange behaviour with circular dichroism measurements and confirming the significance of the rapidly formed embryonic structure as a foundation for the subsequent folding pathway. In the case of ubiquitin, by contrast, no significantly protective structure was detectable, suggesting that here secondary structural elements can be populated only marginally ahead of the major cooperative folding event; this was also supported by stopped-flow circular dichroism measurements.
  
  
  CONCLUSIONS
  The hydrogen exchange approach can be extended to probe the formation of native-like structure formed in very early folding intermediates, even when the stability of specific interactions is marginal. In the case of lysozyme, this has provided a new window on an early stage of organization of the alpha-helical domain.
authors.affiliation: Department of Biochemistry, University of Cambridge, UK.
authors.forename: S T
authors.initials: ST
authors.lastname: Gladwin
authors.forename: P A
authors.initials: PA
authors.lastname: Evans
mesh_terms: D000818:Animals; D002645:Chickens; D006859:Hydrogen / Q000737:chemistry* / Q000378:metabolism; D009113:Muramidase / Q000737:chemistry*; D017510:Protein Folding; D014452:Ubiquitins / Q000737:chemistry*
chemical_list: D014452:Ubiquitins; D006859:Hydrogen; D009113:Muramidase