Hop is a small, divergent homeodomain protein that lacks certain conserved residues required for DNA binding. Hop gene expression initiates early in cardiogenesis and continues in cardiomyocytes throughout embryonic and postnatal development. Genetic and biochemical data indicate that Hop functions directly downstream of Nkx2-5. Inactivation of Hop in mice by homologous recombination results in a partially penetrant embryonic lethal phenotype with severe developmental cardiac defects involving the myocardium. Inhibition of Hop activity in zebrafish embryos likewise disrupts cardiac development and results in severely impaired cardiac function. Hop physically interacts with serum response factor (SRF) and inhibits activation of SRF-dependent transcription by inhibiting SRF binding to DNA. Hop encodes an unusual homeodomain protein that modulates SRF-dependent cardiac-specific gene expression and cardiac development.

Using X-ray coordinates of antigen-antibody complexes McPC 603, D1.3, and HyHEL-5, we made semiquantitative estimates of Gibbs free energy changes (delta G) accompanying noncovalent complex formation of the McPC 603 Fv fragment with phosphocholine and the D1.3 or HyHEL-5 Fv fragments with hen egg white lysozyme. Our empirical delta G function, which implicitly incorporates solvent effects, has the following components: hydrophobic force, solvent-modified electrostatics, changes in side-chain conformational entropy, translational/overall rotational entropy changes, and the dilutional (cratic) entropy term. The calculated delta G ranges matched the experimentally determined delta G of McPC 603 and D1.3 complexes and overestimated it (i.e., gave a more negative value) in the case of HyHEL-5. Relative delta G contributions of selected antibody residues, calculated for HyHEL-5 complexes, agreed with those determined independently in site-directed mutagenesis experiments. Analysis of delta G attribution in all three complexes indicated that only a small number of amino acids probably contribute actively to binding energetics. These form a subset of the total antigen-antibody contact surface. In the antibodies, the bottom part of the antigen binding cavity dominated the energetics of binding whereas in lysozyme, the energetically most important residues defined small (2.5-3 nm2) "energetic" epitopes. Thus, a concept of protein antigenicity emerges that involves the active, attractive contributions mediated by the energetic antigenic epitopes and the passive surface complementarity contributed by the surrounding contact area. The D1.3 energetic epitope of lysozyme involved Gly 22, Gly 117, and Gln 121; the HyHEL-5 epitope consisted of Arg 45 and Arg 68. These are also the essential antigenic residues determined experimentally. The above positions belong to the most protruding parts of the lysozyme surface, and their backbones are not exceptionally flexible. Least-squares analysis of six different antibody binding regions indicated that the geometry of the VH-VL interface beta-barrel is well conserved, giving no indication of significant changes in domain-domain contacts upon complex formation.

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Limited information is available on inherent stabilities of four-chain coiled-coils. We have developed a model system to study this folding motif using synthetic peptides derived from sequences contained in the tetramerization domain of Lac repressor. These peptides are tetrameric as judged by both gel filtration and sedimentation equilibrium and the tetramers are fully helical as determined by CD. The four-chain coiled-coils are well folded as judged by the cooperativity of thermal unfolding and by the extent of dispersion in aliphatic chemical shifts seen in NMR spectra. In addition, we measured the chain length dependence of this four-chain coiled-coil. To this end, we developed a general procedure for nonlinear curve fitting of denaturation data in oligomeric systems. The dissociation constants for bundles that contain a-helical chains 21, 28, and 35 amino acids in length are 3.1 x 6.7 X and 1.0 x M3, respectively. This corresponds to tetramer stabilities (in terms of the peptide monomer concentration) of 180 pM, 51 nM, and 280 f M , respectively. Finally, we discuss the rules governing coiledcoil formation in light of the work presented here.