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.

Protein hydrogen exchange mechanism: Local fluctuations

“…Huyghes-Despointes et al (2001) catalog over a dozen proteins plus various destabilizing mutants in which the exchange rate of the very slowest hydrogens, when computed as in equation 4 (including a proline isomer effect; Bai et al 1994), correctly measures the free energy of the global unfolding equilibrium. More limited subglobal unfoldings have been demonstrated in a number of proteins (Bai et al 1995;Chamberlain et al 1996;Chamberlain and Marqusee 1997;Hiller et al 1997;Bhuyan and Udgaonkar 1998;Fuentes and Wand 1998;Llinas et al 1999;Chu et al 2002;Hoang et al 2002). All of these unfolding reactions have a recognizable signature.…”

Increasing Stability Reduces Conformational Heterogeneity in a Protein Folding Intermediate Ensemble

“…It is possible that the Trp53 -Cys25 distance in the N state is heterogeneous due to flexibility in the Cys25 region, and there could exist different populations with slightly different Trp53 -Cys25 distances. Heteronuclear NMR 36 as well as hydrogen exchange experiments, 37 however, have not provided any evidence for flexibility in the Cys25 region.…”

How cooperative are protein folding and unfolding transitions?

“…Site‐specific stabilities determined in the EX2 limit for many proteins, when mapped on to the protein structure, showed a clustering of residues with similar ΔG op (free energy change associated with a structure‐opening event) values delineating cooperative units of structure called “foldons” . The backbone amide sites within a foldon opened in a concerted manner, and the unfolding of each structural unit led to the formation of partially unfolded forms (PUFs) which differed in stability from the N and U states.…”

“…Folding of the protein appeared to occur via “sequential stabilization” of multiple foldons, thus resulting in a modular assembly of the native structure . The delineation of PUFs for many proteins was an example of the reduced thermodynamic cooperativity in protein unfolding reactions. On the other hand, for some proteins, such as T4 lysozyme, a continuous dispersion of the Δ G op values measured in the EX2 limit of exchange, provided evidence for gradual unfolding.…”