A quantitative description of the relationship between protein expression levels and open reading frame nucleotide sequences (ORFs) is important for understanding natural systems, designing synthetic systems, and optimizing heterologous expression. Codon identity, mRNA secondary structure, and nucleotide composition within ORFs markedly influence expression levels. Bioinformatic analysis of ORF sequences in 816 bacterial genomes revealed that these features show distinct regional trends. To investigate their effects on protein expression, we designed 285 synthetic genes and determined corresponding expression levels in vitro using E. coli extracts. We developed a mathematical function, parameterized using this synthetic gene dataset, which enables computation of protein expression levels from ORF nucleotide sequences. In addition to its practical application in the design of heterologous expression systems, this equation provides mechanistic insight into the factors that control translation efficiency. We found that expression is strongly dependent on the presence of high AU content and low secondary structure in the ORF 5′ region. Choice of high-frequency codons contributes to a lesser extent. The 3′ terminal AU content makes modest, but detectable contributions. We present a model for the effect of these factors on the three phases of ribosomal function: initiation, elongation, and termination.

We report the computational design of soluble protein receptors for pinacolyl methyl phosphonic acid (PMPA), the predominant hydrolytic product of the nerve agent soman. Using recently developed computational protein design techniques, the ligandbinding pockets of two periplasmic binding proteins, glucosebinding protein and ribose-binding protein, were converted to bind PMPA instead of their cognate sugars. The designs introduce 9 -12 mutations in the parent proteins. Twelve of 20 designs tested exhibited PMPA-dependent changes in emission intensity of a fluorescent reporter with affinities between 45 nM and 10 M. The contributions to ligand binding by individual residues were determined in two designs by alanine-scanning mutagenesis, and are consistent with the molecular models. These results demonstrate that designed receptors with radically altered binding specificities and affinities that rival or exceed those of the parent proteins can be successfully predicted. The designs vary in parent scaffold, sequence diversity, and orientation of docked ligand, suggesting that the number of possible solutions to the design problem is large and degenerate. This observation has implications for the genesis of biological function by random processes. The designed receptors reported here may have utility in the development of fluorescent biosensors for monitoring nerve agents.computational protein design ͉ fluorescent biosensor T he most commonly used methods for manipulating ligandbinding specificity are empirical, using either the immune system to generate antibodies or directed evolution of proteins (1). These approaches lose in generality because they either are limited to a particular class of proteins (e.g., antibodies) or are constrained by selection methods, sequence diversity, or library size. Structurebased computational design methods, on the other hand, offer enormous generality for manipulating protein structure and function (2-5). However, limitations in the description of the molecular interactions (6, 7) and the immense combinatorial complexity of the sequence design problem (8) present significant barriers. Nevertheless, recent experiments have shown that, when powerful combinatorial search algorithms, accurate representation of molecular interactions, and state-of-the-art computer hardware are combined, impressive successes are obtained (9-13). Recently, we reported the radical redesign of ligand-binding sites by computational design, converting several sugar-or amino acid-binding proteins into high-affinity, specific receptors for trinitrotoluene (TNT), lactate, or serotonin (11). Here we report a further investigation into the scope of the computational (re-)design of ligand-binding sites, and present the conversion of ribose-and glucose-binding proteins into receptors for pinacolyl methyl phosphonic acid (PMPA), an organophosphate surrogate of the nerve agent soman (Fig.

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