Engineering Diverse Changes in β-Turn Propensities in the N-Terminal β-Hairpin of Ubiquitin Reveals Significant Effects on Stability and Kinetics but a Robust Folding Transition State

• Published in: Biochemistry (Easton) 2006-04, Vol.45 (13), p.4220-4230
• Main Authors: Simpson, Emma R, Meldrum, Jill K, Searle, Mark S
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Kinetics; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Protein Folding; Protein Structure, Secondary; Saccharomyces cerevisiae - genetics; Ubiquitin - chemistry; Ubiquitin - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi052495g

Using the N-terminal 17-residue β-hairpin of ubiquitin as a “host” for mutational studies, we have investigated the influence of the β-turn sequence on protein stability and folding kinetics by replacing the native G-bulged turn (TLTGK) with more flexible analogues (TG3K and TG5K) and a series of four-residue type I‘ β-turn sequences, commonly found in β-hairpins. Although a statistical analysis of type I‘ turns demonstrates residue preferences at specific sites, the frequency of occurrence appears to only broadly correlate with experimentally determined protein stabilities. The subsequent engineering of context-dependent non-native tertiary contacts involving turn residues is shown to produce large changes in stability. Relatively few point mutations have been described that probe secondary structure formation in ubiquitin in a manner that is independent of tertiary contacts. To this end, we have used the more rigorous rate−equilibrium free energy relationship (Leffler analysis), rather than the two-point φ value analysis, to show for a family of engineered β-turn mutants that stability (range of ∼20 kJ/mol) and folding kinetics (190-fold variation in refolding rate) are linearly correlated (αf = 0.74 ± 0.08). The data are consistent with a transition state that is robust with regard to a wide range of statistically favored and disfavored β-turn mutations and implicate a loosely assembled β-hairpin as a key template in transition state stabilization with the β-turn playing a central role.