Full-sequence computational design and solution structure of a thermostable protein variant.

Computational protein design procedures were applied to the redesign of the entire sequence of a 51 amino acid residue protein, Drosophila melanogaster engrailed homeodomain. Various sequence optimization algorithms were compared and two resulting designed sequences were experimentally evaluated. The two sequences differ by 11 mutations and share 22% and 24% sequence identity with the wild-type protein. Both computationally designed proteins were considerably more stable than the naturally occurring protein, with midpoints of thermal denaturation greater than 99 degrees C. The solution structure was determined for one of the two sequences using multidimensional heteronuclear NMR spectroscopy, and the structure was found to closely match the original design template scaffold.

Prudent modeling of core polar residues in computational protein design.

Hydrogen bond interactions were surveyed in a set of protein structures. Compared to surface positions, polar side-chains at core positions form a greater number of intra-molecular hydrogen bonds. Furthermore, the majority of polar side-chains at core positions form at least one hydrogen bond to main-chain atoms that are not involved in hydrogen bonds to other main-chain atoms. Based on this structural survey, hydrogen bond rules were generated for each polar amino acid for use in protein core design. In the context of protein core design, these prudent polar rules were used to eliminate from consideration polar amino acid rotamers that do not form a minimum number of hydrogen bonds. As an initial test, the core of Escherichia coli thioredoxin was selected as a design target. For this target, the prudent polar strategy resulted in a minor increase in computational complexity compared to a strategy that did not allow polar residues. Dead-end elimination was used to identify global minimum energy conformations for the prudent polar and no polar strategies. The prudent polar strategy identified a protein sequence that was thermodynamically stabilized by 2.5 kcal/mol relative to wild-type thioredoxin and 2.2 kcal/mol relative to a thioredoxin variant whose core was designed without polar residues.

A linear lattice model for polyglutamine in CAG-expansion diseases.

Huntington's disease and several other neurological diseases are caused by expanded polyglutamine [poly(Gln)] tracts in different proteins. Mechanisms for expanded (>36 Gln residues) poly(Gln) toxicity include the formation of aggregates that recruit and sequester essential cellular proteins [Preisinger, E., Jordan, B. M., Kazantsev, A. & Housman, D. (1999) Phil. Trans. R. Soc. London B 354, 1029-1034; Chen, S., Berthelier, V., Yang, W. & Wetzel, R. (2001) J. Mol. Biol. 311, 173-182] and functional alterations, such as improper interactions with other proteins [Cummings, C. J. & Zoghbi, H. Y. (2000) Hum. Mol. Genet. 9, 909-916]. Expansion above the "pathologic threshold" ( approximately 36 Gln) has been proposed to induce a conformational transition in poly(Gln) tracts, which has been suggested as a target for therapeutic intervention. Here we show that structural analyses of soluble huntingtin exon 1 fusion proteins with 16 to 46 glutamine residues reveal extended structures with random coil characteristics and no evidence for a global conformational change above 36 glutamines. An antibody (MW1) Fab fragment, which recognizes full-length huntingtin in mouse brain sections, binds specifically to exon 1 constructs containing normal and expanded poly(Gln) tracts, with affinity and stoichiometry that increase with poly(Gln) length. These data support a "linear lattice" model for poly(Gln), in which expanded poly(Gln) tracts have an increased number of ligand-binding sites as compared with normal poly(Gln). The linear lattice model provides a rationale for pathogenicity of expanded poly(Gln) tracts and a structural framework for drug design.

Using deeply trapped intermediates to map the cytochrome c folding landscape.

Replacement of iron with cobalt(III) selectively introduces a deep trap in the folding-energy landscape of the heme protein cytochrome c. Remarkably, neither the protein structure nor the folding thermodynamics is perturbed by this metal-ion substitution, as shown by data from spectroscopic and x-ray diffraction experiments. Through kinetics measurements, we have found parallel folding pathways involving several different misligated Co(III) species, and, as these folding intermediates persist for several hours under certain conditions, we have been able to elucidate fully their spectroscopic properties. The results, along with an analysis of the fluorescence energy-transfer kinetics during refolding, show that rapidly equilibrating populations of compact and extended polypeptide conformations are present until all molecules have reached the native structure. These measurements provide direct evidence that collapsed denatured structures are not substantially more stable than extended conformations of cytochrome c.

Structure of a beta-alanine-linked polyamide bound to a full helical turn of purine tract DNA in the 1:1 motif.

Polyamides composed of N-methylpyrrole (Py), N-methylimidazole (Im) and N-methylhydroxypyrrole (Hp) amino acids linked by beta-alanine (beta) bind the minor groove of DNA in 1:1 and 2:1 ligand to DNA stoichiometries. Although the energetics and structure of the 2:1 complex has been explored extensively, there is remarkably less understood about 1:1 recognition beyond the initial studies on netropsin and distamycin. We present here the 1:1 solution structure of ImPy-beta-Im-beta-ImPy-beta-Dp bound in a single orientation to its match site within the DNA duplex 5'-CCAAAGAGAAGCG-3'.5'-CGCTTCTCTTTGG-3' (match site in bold), as determined by 2D (1)H NMR methods. The representative ensemble of 12 conformers has no distance constraint violations greater than 0.13 A and a pairwise RMSD over the binding site of 0.80 A. Intermolecular NOEs place the polyamide deep inside the minor groove, and oriented N-C with the 3'-5' direction of the purine-rich strand. Analysis of the high-resolution structure reveals the ligand bound 1:1 completely within the minor groove for a full turn of the DNA helix. The DNA is B-form (average rise=3.3 A, twist=38 degrees ) with a narrow minor groove closing down to 3.0-4.5 A in the binding site. The ligand and DNA are aligned in register, with each polyamide NH group forming bifurcated hydrogen bonds of similar length to purine N3 and pyrimidine O2 atoms on the floor of the minor groove. Each imidazole group is hydrogen bonded via its N3 atom to its proximal guanine's exocyclic amino group. The important roles of beta-alanine and imidazole for 1:1 binding are discussed.