Analysis of the pH-dependent Folding and Stability of Histidine Point Mutants Allows Characterization of the Denatured State and Transition State for Protein Folding

Direct Characterization of the Folded, Unfolded and Urea-denatured States of the C-terminal Domain of the Ribosomal Protein L9

“…20 The stability of the protein is strongly pH-dependent, because unfolding is coupled to the protonation of two buried histidine residues ( Figure 1). 21,22 His106 and His134 have depressed pK a values in the folded state of CTL9, but appear to have normal pK a values in the unfolded state. 22 Below pD 4.2, separate, well-resolved resonances from the folded and unfolded state are observed in the NMR spectrum.…”

“…21,22 His106 and His134 have depressed pK a values in the folded state of CTL9, but appear to have normal pK a values in the unfolded state. 22 Below pD 4.2, separate, well-resolved resonances from the folded and unfolded state are observed in the NMR spectrum. The peaks are in slow exchange on the NMR time-scale.…”

“…The resulting curves were fit to a double-exponential as described. 22 The fast phase (0.34 s K1 ) represents 85% of the amplitude and corresponds to the folding/unfolding transition. The minor slow phase is thought to be due to proline isomerization.…”

Electrostatic interactions in the denatured state ensemble: Their effect upon protein folding and protein stability

“…Not all proteins display significant DSE electrostatic effects. For example the pH dependent stability of the C-terminal domain of L9 can be well accounted for by assuming unperturbed DES pK a values [34]. In addition the ΔΔG 0 values for a range of mutations in a number of proteins have been well predicted by considering only native state electrostatic interactions suggesting that DSE electrostatic interactions may not be critical in these systems [35].…”

Analysis of the pH-dependent stability and millisecond folding kinetics of horse cytochrome c

“…Fersht and coworkers have analyzed the pHdependence of folding and unfolding kinetics of CI-2 and barnase [59]. The pioneering works by Raleigh and coworkers have shown that the comparison of pH-dependent protein stability profiles and folding rates can provide information about the development of electrostatic interactions in transition state of folding [52,56,58].…”