Human acidic fibroblast growth factor (FGF-1) is a member of the β-trefoil superfold, a protein architecture that exhibits a characteristic threefold axis of structural symmetry. FGF-1 contains 11 β-turns, the majority being type I 3:5; however, a type I 4:6 turn is also found at three symmetry-related locations. The relative uniqueness of the type I 4:6 turn in the FGF-1 structure suggests it may play a key role in the stability, folding, or function of the protein. To test this hypothesis a series of deletion mutations were constructed, the aim of which was to convert existing type I 4:6 turns at two locations into type I 3:5 turns. The results show it is possible to successfully substitute the type I 4:6 turn by a type I 3:5 turn with minimal impact upon protein stability or folding. Thus, these different turn structures, even though they differ in length, exhibit similar energetic properties. Additional sequence swapping mutations within the introduced type I 3:5 turns suggests that the turn sequence primarily affects stability but not turn structure (which appears dictated primarily by the local environment). Although the results suggest that a stable, foldable β-trefoil protein may be designed utilizing a single turn type (type I 3:5), a type I 4:6 turn at turn#1 of FGF-1 appears essential for efficient mitogenic function.
Study of the positional frequency of amino acids in different turn types has identified statistical preferences for side chain residues within type I turns; these preferences include Asx (Asp or Asn) at position i , Pro at position i+1, Asx at position i+2, and Gly at position i+3. To elucidate the roles of these amino acids at each position, a series of mutants were designed and characterized in the three symmetry-related type I turns in FGF-1, turn#2, #6 and #10. The results suggest that the statistical preference of Asx residues comes from the unique role of Asx in forming H-bonds inside type I β-turn structures. Asx at i or i+2 positions have a similar stabilizing effect of ∼6kJ/mol. The results also suggest that there is no distinction between Asp and Asn on the contribution to thermodynamics. One exception is found in Asp(i) in turn#2 and it is due to the charge repulsion from adjacent amino acids. Despite a fairly consistent stabilizing effect of Asx residue in those turn regions, each turn exhibits uniquely different effects of Asx upon the protein folding and unfolding kinetics. Asx residues in turn#2 stabilize the protein mainly by acceratating the folding rate. On the other hand, Asx residues in turn#10 provide stabilizing effect by decelerating the unfolding rate.
A preference for Pro at i+1 position was not consistent depending on the turn regions. Only turn#2 exhibits a favorable effect from Pro at the i+1 position whereas the other two turn regions prefer Ala over Pro. The turn structure of Pro(i+1)→Ala mutation in turn#2 shows minimal structural change suggesting a destabilization by Ala at this position caused by stabilizing the denatured state. Therefore, we postulate that the statistical frequency of Pro represents a favorable nucleation role of Pro in the turn in an early stage in the folding pathway. Pro may nucleate the turn formation with its restricted &phis; angle. If a β-turn region tends to fold late, Pro may shows an unfavorable effect from entropic penalty.
The contribution of Gly at the i+3 position to thermostability was studied by substituting Gly (i+3) to Ala in turn#2, #6 and #10. Non-Gly residues located near the left-handed α-helical region (L-α) of the Ramachandran plot are a potential indicator of structural strain. In a previous study, we identified that Gly at the i+3 position of a type I turn stabilizes the protein ∼10kJ/mol. In the current study, Gly→Ala mutations in turn#2 and #10 were 10∼12 kJ/mol less stable showing excellent agreement with previous results. Turn#6 exhibits extra destabilization effect with mutation at i+3 position due to the local environmental difference. Nonetheless, these results suggest that Gly at the i+3 position in type I turn is a key element of protein folding.