Two structural subdomains of barstar detected by rapid mixing NMR measurement of amide hydrogen exchange.
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.
Bhuyan
A K
AK
National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India.
Udgaonkar
J B
JB
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
D000577:Amides; D001426:Bacterial Proteins; C057438:barstar protein, Bacillus amyloliquefaciens; D006859:Hydrogen; D003903:Deuterium; D019791:Guanidine