An experimental approach to evaluating the role of backbone interactions in proteins using unnatural amino acid mutagenesis.

The contribution of backbone hydrogen bonds in alpha-helices to the overall stability of a protein has been examined experimentally by replacing several backbone amide linkages in alpha-helix 39-50 of T4 lysozyme with ester linkages. T4 lysozyme variants wherein the backbone amide bonds between residues Ser38 and Leu39, Lys43 and Leu44, or Ala49 and Ile50 are replaced with ester bonds were generated by incorporating alpha-hydroxy acids at positions 39, 44, or 50, respectively, using unnatural amino acid mutagenesis. The stabilities of the proteins were determined from their thermal denaturation curves as monitored by circular dichroism. Comparison of the thermal stabilities of the amide- and ester-containing proteins shows that the ester substitution has a similar thermodynamic effect at all three positions. At the N- and C-terminal positions, where only one hydrogen-bonding interaction is perturbed, the ester substitution is destabilizing by 0.9 and 0.7 kcal/mol, respectively. Introduction of the ester linkage in the middle of the helix, which alters two hydrogen-bonding interactions, destabilizes the protein by 1.7 kcal/mol. The values obtained from these ester to amide mutations are compared to the values from similar mutations that have been made in other secondary structures and bimolecular complexes.

Site-directed mutagenesis with an expanded genetic code.

A biosynthetic method has been developed that makes possible the site-specific incorporation of a large number of amino acids and analogues within proteins. In this approach, an amber suppressor tRNA chemically aminoacylated with the desired amino acid incorporates this amino acid site specifically into a protein in response to an amber codon introduced at the corresponding position in the protein's DNA sequence. Using this method, precise changes within a protein can be made to address detailed structure-function questions. A series of fluorinated tyrosine analogues and linear, branched, and cyclic hydrophobic amino acids have been used to determine the impact of hydrogen bonding and hydrophobic packing, respectively, on protein stability. Glutamate analogues and conformationally restricted amino acids have been used to probe the mechanisms of staphylococcal nuclease and ras. In addition, this technique has been used to construct photocaged proteins and proteins containing photoaffinity labels, spin labels, and isotopic labels at specific positions in the protein sequence suitable for biophysical studies.

Stabilizing and destabilizing effects of placing beta-branched amino acids in protein alpha-helices.

In order to gain greater insight into the effects of beta-branched amino acids on protein alpha-helices, hydrophobic amino acids with varying degrees of beta-branching, including the fully beta-substituted L-2-amino-3,3-dimethylbutanoic acid (ADBA), were incorporated into the protein T4 lysozyme. The unnatural and natural amino acids were substituted at two solvent-exposed alpha-helical sites, Ser 44 and Asn 68, in the protein using the technique of unnatural amino acid mutagenesis. The stabilities of the mutant proteins were determined by using a heat of inactivation assay and from their circular dichroism thermal denaturation curves. Surprisingly, while substitution of the amino acid with the greatest degree of beta-branching, ADBA, destabilizes the protein by 2.5 +/- 0.1 degrees C (0.69 +/- 0.03 kcal/mol) relative to Ala at site 44, the same substitution stabilizes the protein by 1.0 +/- 0.1 degree C (0.27 +/- 0.03 kcal/mol) at site 68. The difference observed at these two positions illustrates the extent to which the local context can mediate the impact of a particular mutation. Molecular dynamics simulations were carried out in parallel to model the structures of the mutant proteins and to examine the energetic consequences of incorporating ADBA. Together, these results suggest that the conformationally restricted beta-branched amino acids are destabilizing, in part, because the beta-branched methyl groups can cause distortions in the local helix backbone. In addition, it is proposed that in some contexts the conformational rigidity of beta-branched amino acids may be stabilizing because it lowers the entropic cost of forming favorable side-chain van der Waals interactions.

Site-specific incorporation of biophysical probes into proteins.

Biophysical probes which can detect structural changes in proteins and the interaction of proteins with other macromolecules are important tools in studying protein function. Many difficulties remain, however, in introducing probes into proteins site-specifically. Here we report the successful site-specific incorporation of a spin-labeled, a fluorescent, and a photoactivatible amino acid into a variety of surface and internal sites in bacteriophage T4 lysozyme by using unnatural amino acid mutagenesis. In addition, we report the purification and spectral characterization of T4 lysozyme mutants containing the spin-labeled amino acid and the fluorescent amino acid. The ability to incorporate these probes site-specifically allows for novel studies of protein structure and dynamics. Moreover, this work demonstrates that the Escherichia coli protein biosynthetic machinery can tolerate unnatural amino acids with little resemblance to the natural amino acids.

Probing the role of loop 2 in Ras function with unnatural amino acids.

The YDPT sequence motif (residues 32-35) in loop 2 (residues 32-40) of Ha-Ras p21 protein is conserved in the Ras protein family. X-ray crystal structures have revealed significant conformational differences in this region between the GTP- and GDP-bound forms. Moreover, mutations in this region block neoplastic transformation and prevent interaction with GTPase-activating protein (GAP), suggesting that this region may contribute to the effector function of Ras. To better understand the structural features required for GAP interaction and GTPase activity, the expanded repertoire of unnatural amino acid mutagenesis has been used to investigate the roles of the key residues, Pro-34, Thr-35, and Ile-36. A Pro-34-->methanoproline mutant, in which residue 34 is locked in the trans conformation, was found to retain high levels of intrinsic and GAP-activated GTPase activity, making unlikely conformational isomerization at this position. Deletion of a single methyl group from Ile (Ile-36-->norvaline) abolished GAP activation of Ras, revealing a remarkable specificity in this protein-protein interaction. Finally, replacement of Thr-35 with diastereomeric allo-threonine led to inactivation of Ras, demonstrating the importance of the orientation of this critical residue in Ras function.