Free energy of burying hydrophobic residues in the interface between protein subunits

Abstract

We have obtained an experimental estimate of the free energy change associated with variations at the interface between protein subunits, a subject that has raised considerable interest since the concept of accessible surface area was introduced by Lee This paper deals with the experimental determination of the free energy change associated with modification of the surface buried in a protein-protein contact, a point of general interest in understanding stability and recognition in proteins. Since the pioneering work of Lee and Richards (1), a wealth of experimental and theoretical investigations have attempted to understand and quantify the forces stabilizing protein oligomers and complexes. After Chothia (2, 3) drew attention to the significance of the surface buried at an interface, other investigators emphasized shape complementarity (4), residue propensities (5), and the relative contribution of polar interactions and hydrophobic effects to the overall ⌬G 0 in proteinprotein association (6-8). Experimental studies have used different proteins to understand the effect of a point mutation on enzyme-inhibitor (9, 10) or antibody-antigen complexes (11-13), as well as subunit interactions in mutants of human Hb (14). These studies led to the conclusion that both types of weak interactions play a role, and that Atom Solvation Parameters derived from partition coefficients can be used to evaluate the stabilization energy at protein interfaces (8,(15)(16)(17).Questions arise whether the theoretical studies can be a guideline in the analysis and design of experiments with a resolution beyond the qualitative prediction of results and how far values obtained from physico-chemical considerations can be useful in protein engineering by mutagenesis. Although it is recognized that buried hydrophobic surface contributes to the stability of a protein-protein contact, theoretical estimates of the free energy gain associated with burial of 1 Å 2 of hydrophobic surface are variable and range between 4 and 32.5 cal͞mol (see ref. 16 for a recent review). We have addressed this point experimentally by using the dimer-tetramer association of human Hb mutants.By analytical ultracentrifuge, we have determined the equilibrium constant for the 2␣␤ 3 (␣␤) 2 association for five single and three double mutants of human HbCO. The mutations are all at the ␣ 1 ␤ 2 interface (Fig. 1) except one at the ␣ 1 ␤ 1 interface, which has a larger number of contacts and is unchanged in the T 3 R allosteric transition (14,(18)(19)(20). The changes in association free energy relative to HbA have been correlated with changes in the buried surface area due to mutations, to dissect the contribution of the polar and hydrophobic contacts in the stabilization of the tetramer. Taking a ϩ1.5 kcal͞mol complementarity cost associated with each mutation, we found that the ⌬(⌬G MATERIALS AND METHODSSite-directed mutants of HbA were produced in Escherichia coli and were purified as described (21,22). Reagents were of analytical grade. Ultracentrifuge ex...

Inhibition of Human Pancreatic Ribonuclease by the Human Ribonuclease Inhibitor Protein

“…The hRI·RNase 1 complex also buries an additional 375 Å 2 of surface area, which could also enhance its stability. 48,45,49 In line with the more intimate complex formed by hRI and RNase 1, RNase 1 dissociates from hRI 150-fold more slowly than does RNase A from hRI ( Figure 5 ; Table 3). 12 The slower dissociation rate (t ½ = 81 days) for the hRI·RNase 1 complex is in the range of that for the hRI·angiogenin complex (t ½ = 62 days).…”

Drying-induced hydrophobic polymer collapse

“…For this reason, a recent study of hydrophobic effects in protein folding requires further analysis (24). Other implicit solvent models are based on estimates of exposed surface area (25,26). This approach can be valid for large enough solutes, provided the time scales of interest are longer than nucleation times (10 Ϫ5 s in the system studied here).…”

The structure at 2 Å resolution of Phycocyanin from Gracilaria chilensis and the energy transfer network in a PC–PC complex

“…The docking models were scored by the program considering desolvation, electrostatic and hydrophobic contributions [30][31][32]. The models generated were then analyzed to select those in agreement with the reported electron micrographs images [4].…”