A mutational study of the peptide corresponding to the second hairpin of the protein GB1 domain (GB1p) provided a series of mutants with significantly increased fold stability. Mutations focused on improvement of the direction-reversing loop and the addition of favorable Coulombic interactions at the sequence termini. The loop optimization was based on a database search for residues that occur with the greatest probability in similar hairpin loops in proteins. This search suggested replacing the native DDATKT sequence with NPATGK, which resulted in a 4.5 kJ/mol stabilization of the hairpin fold. The introduction of positively charged lysines at the N-terminus provided an additional 2.4 kJ/mol of stabilization, affording a GB1p mutant that is 86 +/- 3% folded at 25 degreesC with a melting temperature of 60 +/-2 degreesC. The trpzip version of this peptide, in which three of the hydrophobic core residues were mutated to tryptophan, yielded a sequence that melted at 85 degreesC. Throughout, fold populations and melting temperatures were derived from the mutation and temperature dependence of proton chemical shifts and were corroborated by circular dichroism (CD) melts. The study also suggests that the wild-type GB1p sequence is significantly less stable than reported in some other studies: only 30% folded in water at 25 degreesC.