A major goal of protein engineering is the enhancement of protein stability. Here we demonstrate a rational method for enhancing the stability of globular proteins by targeting glycine residues which adopt conformations with Phi > 0. Replacement of such a glycine by d-alanine can lead to a significant increase in stability. The approach is tested at three sites in two model proteins. NMR and CD indicated that the substitutions do not alter the structure. Replacement of glycine-24 of the N-terminal domain of L9 (NTL9) with d-Ala results in an increase in stability of 1.3 kcal mol-1, while replacement of glycine-34 of NTL9 leads to an increase of 1.9 kcal mol-1. Replacement of glycine-331 of the UBA domain with d-Ala leads to an increase in stability of 0.6 kcal mol-1.

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It is now recognized that the denatured state ensemble (DSE) of proteins can contain significant amounts of structure, particularly under native conditions. Well-studied examples include small units of hydrogen bonded secondary structure, particularly helices or turns as well hydrophobic clusters. Other types of interactions are less well characterized and it has often been assumed that electrostatic interactions play at most a minor role in the DSE. However, recent studies have shown that both favorable and unfavorable electrostatic interactions can be formed in the DSE. These can include surprisingly specific non-native interactions that can even persist in the transition state for protein folding. DSE electrostatic interactions can be energetically significant and their modulation either by mutation or by varying solution conditions can have a major impact upon protein stability. pH dependent stability studies have shown that electrostatic interactions can contribute up to 4 kcal mol −1 to the stability of the DSE.

The p53-binding site of MDM2 holds great promise as a target for therapeutic intervention in MDM2-amplified p53 wild-type forms of cancer. Despite the extensive validation of this strategy, there are relatively few crystallographically determined co-complex structures for small-molecular inhibitors of the MDM2-p53 interaction available in the PDB. Here, a surface-entropy reduction mutant of the N-terminal domain of MDM2 that has been designed to enhance crystallogenesis is presented. This mutant has been validated by comparative ligand-binding studies using differential scanning fluorimetry and fluorescence polarization anisotropy and by cocrystallization with a peptide derived from p53. Using this mutant, the cocrystal structure of MDM2 with the benchmark inhibitor Nutlin-3a has been determined, revealing subtle differences from the previously described co-complex of MDM2 with Nutlin-2.

Interest in the unfolded state of proteins has grown with the realization that this state can have considerable structure in the absence of denaturants. Natively unfolded proteins, mutations that unfold proteins under native conditions, and changes in pH that induce unfolding are attractive models for the unfolded state in the absence of denaturant. The unfolded state of the N-terminal domain of ribosomal protein L9 (NTL9) was previously shown to contain significant non-native electrostatic interactions [Cho, J. H., Sato, S., and Raleigh, D. P. (2004) J. Mol. Biol. 338, 827-837]. NTL9 has a mixed alpha-beta structure and folds via a two-state mechanism. We have generated a model of the unfolded state of NTL9 in the absence of denaturant by substitution of an alanine for phenylalanine 5 located in the core of this protein. The CD spectrum of the variant, denoted as F5A, exhibits significantly less structure than the wild type; however, the mean residue ellipticity of F5A at 222 nm (-8200 deg cm(2) dmol(-)(1)) is considerably larger than expected for a fully unfolded protein, indicating that residual secondary structure is populated. F5A also has more residual structure than the urea-unfolded wild type. The stability of F5A is estimated to be at least 1 kcal/mol unfavorable, showing that the unfolded state is populated to 84% or more. NMR pulsed-field gradient measurements yield a hydrodynamic radius of 16.1 A for wild-type NTL9 and 20.8 A for the F5A variant in native buffer. The physiologically relevant unfolded state of wild-type NTL9 is likely to be even more compact than F5A since the mutation should reduce the level of hydrophobic clustering in the unfolded state in the absence of denaturant. The hydrodynamic radius of F5A increases to 25.9 A in 8 M urea, and a value of 23.5 A is obtained for the wild type under similar conditions. The results show that the unfolded state of F5A in the absence of denaturant is more compact and contains more structure than the urea-unfolded form.

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Inhibition of murine double minute 2 (MDM2)-p53 protein−protein interaction with small molecules has been shown to reactivate p53 and inhibit tumor growth. Here, we describe rational, structure-guided, design of novel isoindolinone-based MDM2 inhibitors. MDM2 X-ray crystallography, quantum mechanics ligand-based design, and metabolite identification all contributed toward the discovery of potent in vitro and in vivo inhibitors of the MDM2-p53 interaction with representative compounds inducing cytostasis in an SJSA-1 osteosarcoma xenograft model following once-daily oral administration.

Stabilization of proteins is a long-sought objective. Targeting the unfolded state interactions of a protein is not a method used for this purpose, although many proteins are known to contain such interactions. The N-terminal domain of ribosomal protein L9 (NTL9) has a lysine residue at position 12, which makes strong non-native interactions in the unfolded state. Substitution of a d-alanine for G34 in NTL9 is known to stabilize the protein by reducing the entropy of the unfolded state. Here we combine these two mutations to design a hyperstable protein. The structure of the variant is the same as that of wild-type as judged by 2D NMR. The variant is hyperstable as judged by denaturation experiments, where complete thermal unfolding of the protein does not occur in native buffer.