Characterization of Single-Tryptophan Mutants of Histidine-Containing Phosphocarrier Protein:  Evidence for Local Rearrangements during Folding from High Concentrations of Denaturant

Abstract

We have used site-directed mutagenesis in combination with a battery of biophysical techniques to probe the stability and folding behavior of a small globular protein, the histidine-containing phosphocarrier protein (HPr). Specifically, the four phenylalanine residues (2, 22, 29, and 48) of the wild-type protein were individually replaced by single tryptophans, thus introducing site-specific probes for monitoring the behavior of the protein. The folding of the tryptophan mutants was investigated by NMR, DSC, CD, intrinsic fluorescence, fluorescence anisotropy, and fluorescence quenching. The heat-induced denaturation of all four mutants, and the GdnHCl-induced unfolding curves of F2W, F29W, and F48W, can be fitted adequately to a two-state model, in agreement with the observations for the wild-type protein. The GdnHCl unfolding transitions of F22W, however, showed the accumulation of an intermediate state at low concentrations of denaturant. Kinetic refolding studies of F2W, F29W, and F48W showed a major single phase, independent of the probe used (CD, fluorescence, and fluorescence anisotropy) and similar to that of the wild-type protein. In contrast, F22W showed two phases in the fluorescence experiments corresponding to the two phases previously observed in ANS binding studies of the wild-type protein [Van Nuland et al. (1998) Biochemistry 37, 622-637]. Residue 22 was found from NMR studies to be part of the binding interface on HPr for ANS. These observations indicate that the second slow phase reflects a local, rather than a global, rearrangement from a well-structured highly nativelike intermediate state to the fully folded native state that has less hydrophobic surface exposed to the solvent. The detection of the second slow phase by the use of selective labeling of different regions of the protein with fluorophores illustrates the need for an integrated approach in order to understand the intricate details of the folding reactions of even the simplest proteins.

Rapid Formation of Non-native Contacts During the Folding of HPr Revealed by Real-time Photo-CIDNP NMR and Stopped-flow Fluorescence Experiments

“…These observations are in agreement with previous results from activity measurements, NMR experiments, and steady-state and time-resolved fluorescence measurements where it was found that the replacement of the phenylalanine residues with tryptophan at the various sites in HPr did not result in any substantial structural changes, but only localized rearrangements around the mutated sites. 31 In the denatured states of all four mutant proteins, the tryptophan residue in each case is fully exposed, consistent with a random coil model for an unfolded state, and competes so efficiently for the flavin dye so that no histidine signals are observed. However, when the tryptophan side-chain is partially (native F29W) or completely buried (native F22W) in the hydrophobic core of the protein, the histidine residues can compete successfully for the flavin dye and their resonances are detectable in the corresponding spectra.…”

“…The replacement of each of the phenylalanine residues in turn with single tryptophan residues, resulting in four single-tryptophan mutants F2W, F22W, F29W, and F48W, does not result in substantial structural changes, but the presence of the bulkier side-chain leads to localized rearrangements around the mutated site. 31 The tryptophan sidechains are in similar environments to the corresponding phenylalanine side-chains in the WT structure, with that of residue 48 completely solvent accessible, those of residues 2 and 29 partially buried, and that of residue 22 completely buried in the core of the protein, inaccessible to the solvent. Replacement of the phenylalanine residues associated with the hydrophobic core of the protein, particularly those at positions 22 and 29, results in lower stability, most likely as a result of the introduction of the larger sized tryptophan side-chain that disrupts slightly the optimal packing of the core.…”

“…Replacement of the phenylalanine residues associated with the hydrophobic core of the protein, particularly those at positions 22 and 29, results in lower stability, most likely as a result of the introduction of the larger sized tryptophan side-chain that disrupts slightly the optimal packing of the core. 31 Figure 1 shows in addition the two histidine residues in HPr; His15 in the active-site is fully exposed to the solvent, while the His76 is less accessible. These residues were not mutated in the present study but act as reporters of the overall exposure of the tryptophan residues as we show below.…”

Multiple Tryptophan Probes Reveal that Ubiquitin Folds via a Late Misfolded Intermediate

“…15,[31][32][33][34][35][36] The strategy described here is more flexible, in that the Trp substitution can be made anywhere on the surface, allowing structural transitions in any region of the protein to be probed with high efficiency, with minimal or no apparent perturbation of refolding pathways. Specifically, we use different Trp probes in order to discriminate between folding transitions that represent conformational changes that occur in the entire protein population versus those that correspond to parallel folding pathways (Figure 1).…”

The role of the local environment of engineered Tyr to Trp substitutions for probing the denaturation mechanism of FIS

“…Trp residues are often engineered into proteins by replacing Tyr or Phe residues because Trp yields higher fluorescence efficiency 3, 5, 32–34. This type of mutation preserves the aromatic ring and usually has minor effects on the overall structure of the protein.…”