We present a study of the role of salt bridges in stabilizing a simplified tertiary structural motif, the coiled-coil. Changes in GCN4 sequence have been engineered that introduce trial patterns of single and multiple salt bridges at solvent exposed sites. At the same sites, a set of alanine mutants was generated to provide a reference for thermodynamic analysis of the salt bridges. Introduction of three alanines stabilizes the dimer by 1.1 kcal/mol relative to the wild-type. An arrangement corresponding to a complex type of salt bridge involving three groups stabilizes the dimer by 1.7 kcall mol, an apparent elevation of the melting temperature relative to wild type of about 22 "C. While identifying local from nonlocal contributions to protein stability is difficult, stabilizing interactions can be identified by use of cycles. Introduction of alanines for side chains of lower helix propensity and complex salt bridges both stabilize the coiled-coil, so that combining the two should yield melting temperatures substantially higher than the starting species, approaching those of thermophilic sequences. Keywords: GCN4; leucine zipper; salt bridge; thermal stabilityInteractions that stabilize the native state of proteins include the hydrophobic effect, van der Waals interactions, hydrogen bonds and ionic effects, including dipole interactions and salt bridges (Creighton, 1993). The question of which of these are most important in protein stabilization has been debated since the review by Kauzmann (1959). One aspect of the problem concerns how to account for the additional stabilization of proteins from thermophiles, which can have very high thermal stabilities (see Hiller et al., 1997). Since the pioneering work of Matthews et al. (1974) on thermolysin, structures of thermophilic and mesophilic proteins have been compared in a search for clues to what accounts for the higher stability of the former (Korndorfer et al., 1995;Yip et al., 1995;Hatanaka et al., 1997;Robb & Maeder, 1998). In 1978, Perutz (1978 observed that the main discernible difference between a thermophilic and mesophilic version of ferrodoxin lay in the greater number of salt bridges on the surface of the thermophile. As more crystal structures of thermophilic proteins have become available, other mechanisms have been proposed to explain their stability (Vogt & Argos, 1997): improved internal packing, burial of a greater hydrophobic area (Chan et al., 1995; Delboni et al., 1995), and networks of complex salt bridges (Yip et al., 1995;Pappenberger et al., 1997 Yip et al., 1995;Robb & Maeder, 1998).Here, we consider the role of complex salt bridges in stabilizing a simplified model protein structure. The strength of a salt bridge can be estimated by different experimental methodologies: changes in the helicity of model peptides (Merutka & Stellwagen, 1990;Lyu et al., 1992;Scholtz et al., 1993), shifts in pK, of interacting side chains (Anderson et al., 1990;, or T, differences in model proteins Dao-Pin et al., 1991). Using the first method, Smi...

Uveal melanoma is the most common primary ocular malignancy, although it is rare in children, and patients presenting with metastatic disease have a median survival of only 2 to 5 months. The tumor is generally unresponsive to systemic chemotherapy, but immunotherapy may be effective in selected patients. This report describes an 8-year-old girl with metastatic uveal melanoma treated with high-dose, bolus interleukin-2 (IL-2) and the antiangiogenic agent thalidomide. She tolerated treatment well and initially responded with stable disease in the liver and pancreas for 23 months. New pulmonary metastases developed and she was re-treated with high-dose IL-2, resulting in regression of her liver lesions and stable pulmonary disease for more than 18 months. These results suggest that IL-2 at high doses, and in combination with thalidomide, may be useful for uveal melanoma with tolerable side effects in children. Further study of this combination in children with immune-responsive tumors is warranted.

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