title: Stepwise protein folding at near amino acid resolution by hydrogen exchange and mass spectrometry.
abstract: The kinetic folding of ribonuclease H was studied by hydrogen exchange (HX) pulse labeling with analysis by an advanced fragment separation mass spectrometry technology. The results show that folding proceeds through distinct intermediates in a stepwise pathway that sequentially incorporates cooperative native-like structural elements to build the native protein. Each step is seen as a concerted transition of one or more segments from an HX-unprotected to an HX-protected state. Deconvolution of the data to near amino acid resolution shows that each step corresponds to the folding of a secondary structural element of the native protein, termed a "foldon." Each folded segment is retained through subsequent steps of foldon addition, revealing a stepwise buildup of the native structure via a single dominant pathway. Analysis of the pertinent literature suggests that this model is consistent with experimental results for many proteins and some current theoretical results. Two biophysical principles appear to dictate this behavior. The principle of cooperativity determines the central role of native-like foldon units. An interaction principle termed "sequential stabilization" based on native-like interfoldon interactions orders the pathway.
authors.affiliation: Johnson Research Foundation, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Wenbing
authors.initials: W
authors.lastname: Hu
authors.forename: Benjamin T
authors.initials: BT
authors.lastname: Walters
authors.forename: Zhong-Yuan
authors.initials: ZY
authors.lastname: Kan
authors.forename: Leland
authors.initials: L
authors.lastname: Mayne
authors.forename: Laura E
authors.initials: LE
authors.lastname: Rosen
authors.forename: Susan
authors.initials: S
authors.lastname: Marqusee
authors.forename: S Walter
authors.initials: SW
authors.lastname: Englander
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D001703:Biophysics / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology; D006859:Hydrogen / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D016914:Ribonuclease H / Q000737:chemistry*; D012984:Software
chemical_list: D000596:Amino Acids; D010455:Peptides; D006859:Hydrogen; D016914:Ribonuclease H; C078856:ribonuclease HI