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More general and universally applicable drug discovery assay technologies are needed in order to keep pace with the recent advances in combinatorial chemistry and genomics-based target generation. Ligand-induced conformational stabilization of proteins is a well-understood phenomenon in which substrates, inhibitors, cofactors, and even other proteins provide enhanced stability to proteins on binding. This phenomenon is based on the energetic coupling of the ligand-binding and protein-melting reactions. In an attempt to harness these biophysical properties for drug discovery, fully automated instrumentation was designed and implemented to perform miniaturized fluorescence-based thermal shift assays in a microplate format for the high throughput screening of compound libraries. Validation of this process and instrumentation was achieved by investigating ligand binding to more than 100 protein targets. The general applicability of the thermal shift screening strategy was found to be an important advantage because it circumvents the need to design and retool new assays with each new therapeutic target. Moreover, the miniaturized thermal shift assay methodology does not require any prior knowledge of a therapeutic target's function, making it ideally suited for the quantitative high throughput drug screening and evaluation of targets derived from genomics.

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Sperm whale myoglobin was expressed in Escherichia coli from a totally synthetic gene inserted in the expression vector pUC19. The gene was constructed as 23 overlapping oligonucleotides encoding both strands of the DNA. Gene synthesis provides several advantages over traditional eukaryotic gene-cloning techniques, allowing the incorporation of an efficient ribosome binding site, appropriate initiation and termination sequences, restriction enzyme sites for convenient subcloning and future mutagenesis, and frequently used codons for highly expressed E. coi genes. The sperm whale myoglobin expressed from the synthetic gene constituted o10% of the total soluble protein as holoprotein, indicating that iron-protoporphyrin IX biosynthesis and prosthetic-group incorporation are not limiting in the high-level expression of this heme protein in E. coi. We credit the use of frequently used E. coi codons for the observed high-level expression. The sperm whale myoglobin produced is stable, easily purified to homogeneity, and indistinguishable from commercially available sperm whale myoglobin by optical and magnetic spectroscopic methods.Sperm whale myoglobin (Mb) has been one of the most intensely studied proteins, as attested by the wealth of documented biochemical, biophysical, and spectroscopic data (1). It was the first protein structure determined to high resolution by x-ray crystallographic analyses (2) and, because of its physiological importance and availability, has served as a model system for the study of structure-function relationships in heme proteins. With a desire to use Mb as a model for investigations into the dynamics of protein folding, ligand binding, and conformational transitions, we undertook the complete gene synthesis of sperm whale Mb. We report the successful construction and cloning of this synthetic gene and the high-level expression of authentic sperm whale Mb in Escherichia coli. This provides a crucial starting point for site-directed in vitro mutagenesis studies designed to probe the structure-function relationships of Mb.Successful expression of mammalian genes in a bacterial environment has often proved difficult even when promoters and Shine-Dalgarno sequences (ribosome binding sites) from highly expressed E. coli genes have been utilized (3-7). Numerous factors other than promoters and Shine-Dalgarno sequences may influence the expression of eukaryotic genes in prokaryotic environments, including mRNA stability, appropriate translational initiation sequences, and the efficiency of the translational machinery as reflected in codon frequencies and tRNA pools. Once expressed, resistance to protease digestion and timely synthesis and assembly of necessary prosthetic groups are paramount to the production of a stable and functional holoprotein. Of related concern is the possible accumulation of insoluble protein aggregates and inclusion bodies (8-11) or the toxic effects of an overexpressed protein. Of considerable interest was the work of Nagai, Thogersen, and colleagues (12, 13) i...

Fibroblast growth factors (FGF's) interact on cell surfaces with "low-affinity" heparan sulfate proteoglycans (HSPG) and "high-affinity" FGF receptors (FGFR) to initiate cell proliferation. Previous reports have implicated the binding of heparin, or heparan sulfate, to FGF as essential for FGF-mediated signal transduction and mitogenicity. However, the molecular recognition events which dictate the specificity of this interaction have remained elusive. Amino acid residues on the surface of basic FGF (bFGF) were targeted as potential heparin contacts on the basis of the position of sulfate anions in the X-ray crystal structure of bFGF and of a modeled pentasaccharide heparin-bFGF complex. Each identified amino acid was replaced individually with alanine by site-directed mutagenesis, and the resulting mutant proteins were characterized for differences in binding to a low molecular weight heparin (approximately 3000) by isothermal titrating calorimetry and also for differences in [NaCl] elution from a heparin-Sepharose affinity resin. The combination of site-directed mutagenesis and titrating calorimetry permitted an analysis of the energetic contributions of individual bFGF residues in the binding of heparin to bFGF. The key amino acids which comprise the heparin binding domain on bFGF constitute a discontinuous binding epitope and include K26, N27, R81, K119, R120, T121, Q123, K125, K129, Q134, and K135. Addition of the observed delta delta G degrees of binding for each single site mutant accounts for 8.56 kcal/mol (> 95%) of the free energy of binding. The delta delta G degrees values for N27A, R120A, K125A, and Q134A are all greater than 1 kcal/mol each, and these four amino acids together contribute 4.8 kcal/mol (56%) to the total binding free energy. Amino acid residues K119 through K135 reside in the C-terminal domain of bFGF and collectively contribute 6.6 kcal/mol (76%) of the binding free energy. Although 7 out of the 11 identified amino acids in the heparin binding domain are positively charged, a 7-fold increase in [NaCl] decreases the affinity of wild-type bFGF binding to heparin only 37-fold (Kd at 0.1 M NaCl = 470 nM vs Kd at 0.7 M NaCl = 17.2 microM). This indicates that pure electrostatic interactions contribute only 30% of the binding free energy as analyzed by polyelectrolyte theory and that more specific nonionic interactions, such as hydrogen bonding and van der Waals packing, contribute the majority of the free energy for this binding reaction.