Miniproteins are adequate models to study various protein-structure modifying effects such as temperature, pH, point mutation(s), H-bonds, salt bridges, molecular packing, etc. Tc5b, a 20-residue Trp-cage protein is one of the smallest of such models with a stable 3D fold (Neidigh J. W. et al. (2002) Nat. Struct. Biol. 9, 425-430). However, Tc5b exhibits considerable heat-sensitivity and is only stable at relatively low temperatures. Here we report a systematic investigation of structural factors influencing the stability of Tc5b by solving its solution structure in different environments, varying temperature, and pH. The key interactions identified are the hydrophobic stacking of the aromatic rings of Tyr3 and Trp6 and the salt bridge formed between Asp9 and Arg18. To verify the importance of these interactions, selected variants (mutated, glycosylated and truncated) of Tc5b were designed, prepared, and investigated by NMR. Indeed, elimination of either of the key interactions highly destabilizes the structure. These observations enabled us to design a new variant, Tc6b, differing only by a methylene group from Tc5b, in which both key interactions are optimized simultaneously. Tc6b exhibits enhanced heat stability and adopts a stable fold at physiological temperature.