title: Two structural subdomains of barstar detected by rapid mixing NMR measurement of amide hydrogen exchange.
abstract: Equilibrium amide hydrogen exchange studies of barstar have been carried out at pH 6.7, 32 degrees C using one- and two-dimensional nuclear magnetic resonance. An unusually large fraction of the backbone amide hydrogens of barstar exchange too fast to be measured, and the exchange rates of only fifteen slow-exchanging amide sites including indole amides of two tryptophans could be measured in the presence of 0 to 1.8 M guanidine hydrochloride (GdnHCl). Measurement of exchange occurring in tens of seconds in the unfolding transition region was possible by the use of a fast stopped-flow mixing method. The observed exchange rates have been simulated in the EX2 limit according to a two-process model that incorporates two exchange-competent states: a transiently unfolded state (U*) in which many amide hydrogens are completely accessible to solvent-exchange, and a near-native locally unfolded state (N*), in which only one or a few amide hydrogens are completely accessible to solvent-exchange. The two-process model appears to account for the observed exchange behavior over the entire range of GdnHCl concentrations studied. For several measurable slow-exchanging amide hydrogens, the free energies of production of exchange-competent states from the exchange-incompetent native state are significantly higher than the free-energy of production of the equilibrium unfolded state from the native state, when the latter is determined from circular dichroism- or fluorescence-monitored equilibrium unfolding curves. The result implies that U*, which forms transiently in the strongly native-like conditions used for the hydrogen exchange studies, is higher in energy than the equilibrium-unfolded state. The higher energy of this transiently unfolded exchange-competent state can be attributed to either proline isomerization or to the presence of residual structure. On the basis of the free energies of production of exchange-competent states, the measured amide sites of barstar appear to define two structural subdomains--a three-helix unit and a two-beta-strand unit in the core of the protein.
authors.affiliation: National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India.
authors.forename: A K
authors.initials: AK
authors.lastname: Bhuyan
authors.forename: J B
authors.initials: JB
authors.lastname: Udgaonkar
mesh_terms: D000577:Amides / Q000737:chemistry / Q000378:metabolism; D001407:Bacillus / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D003903:Deuterium / Q000378:metabolism; D005453:Fluorescence; D019791:Guanidine / Q000494:pharmacology; D006859:Hydrogen / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013816:Thermodynamics
chemical_list: D000577:Amides; D001426:Bacterial Proteins; C057438:barstar protein, Bacillus amyloliquefaciens; D006859:Hydrogen; D003903:Deuterium; D019791:Guanidine