Free Energy Change upon Folding of hGH

The sampling of deuterium buildup at time points ranging from 30 s to 100,000 s allows estimation of the free energy change upon folding using Eq. (5). This analysis eliminates effects arising from differences in intrinsic amide hydrogen exchange rates by accurately modeling these rates and allowing for correction among different pH [34]. This becomes important considering the amide hydrogen exchange reaction is primarily base catalyzed near neutral pH. By correcting for these differences this approach is useful for the analysis of pH-dependent structural changes. Fig. 12.5a, b show the localized free energy changes upon folding at pH 7.0 and pH 2.6, respectively. The data clearly show that the overall

Fig. 12.4 H/D exchange analysis of hGH at pH 7.0 and 2.6 [8]. Each block represents a pepsin-generated peptide. Each block consists of eight rows that represent eight distinct on-exchange time points, shown at the right. The level of deuteration in each peptide at each time point is represented by color according to the diagram displayed at the top right. Blocks representing on-exchange at pH 7.0 are on the top row, while blocks representing on-exchange at pH 2.6 are shown on the bottom. Light blue cylinders above the sequence indicate the

Fig. 12.4 H/D exchange analysis of hGH at pH 7.0 and 2.6 [8]. Each block represents a pepsin-generated peptide. Each block consists of eight rows that represent eight distinct on-exchange time points, shown at the right. The level of deuteration in each peptide at each time point is represented by color according to the diagram displayed at the top right. Blocks representing on-exchange at pH 7.0 are on the top row, while blocks representing on-exchange at pH 2.6 are shown on the bottom. Light blue cylinders above the sequence indicate the helices identified from the X-ray crystal structure of hGH (protein data bank ID: 1HGU). Peptides that contain mostly slow exchanging amide hydrogens are represented by blue bars, while red bars represent peptides that contain mostly rapidly exchanging amide hydrogens. Regions of the protein that contain amides that were not observed in the experiments are indicated in white. These gaps are primarily due to the fact that the first two residues of each peptide lose the deuterium during the analysis [34].

hGH structure is significantly more stable at neutral pH. At pH 7.0, interactions between helix B and the central portions of helices A and D were sufficiently stable that several peptide fragments derived from this region showed no deuterium incorporation, even after incubation for 100,000 s. It was estimated that free energy changes upon folding in these regions is greater than 7 kcal mol-1 residue-1 at pH 7.0. Overall, the loop regions are the least stable.

Regions of greatest stabilization in the helix bundle shift as a function of pH (Fig. 12.5a, b). At neutral pH, regions of high stability were located at the central portion of the helix bundle. At pH 2.6, the region of highest stability in the structure had markedly shifted so that most of the stabilizing interactions are located near the end of the bundle containing the N- and C-terminus. In contrast to the

Fig. 12.5 Free energy change (expressed in kcal mol 1 per amino acid) upon folding of hGH: (a) at pH 7.0, and (b) at pH 2.6 [8]. Folding free energies are mapped on the X-ray structure (1HGU) by colored segments according to the key at the right. Gray indicates residues that were not analyzed [34].

stabilization energies of larger than 7 kcal mol 1 residue 1 at pH 7.0, stabilization energies approach only 5 kcal mol-1 residue-1 at pH 2.6.

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