Role of loops in the folding and stability of yeast phosphoglycerate kinase.

Abstract

Yeast phosphoglycerate kinase (yPGK) is a monomeric two domain protein used as folding model representative of large proteins. We inserted short unstructured sequences (four Gly or four Thr) into the connections between secondary structure elements and studied the consequences of these insertions on the folding process and stability of yPGK. All the mutated proteins can refold efficiently. The effect per residue on stability is larger for the first inserted residue. Insertion in two long betaalpha loops (at residue positions 71 and 129) is more destabilizing than an insertion in a short alphabeta loop (at residue position 89) located on the opposite side of the N-terminal domain. The effect on stability is mainly due to a large increase of the unfolding rate rather than a decrease of the folding rate. This suggests that these connections between secondary structure elements do not play an active role in directing the folding process. Insertion into the short alphabeta loop (position 89) has limited effects on stability and results in the detection of a kinetic phase not previously seen with the wild-type protein, suggesting that insertions in this particular loop do qualitatively affect the folding process without a large effect on folding efficiency. For the two long betaalpha loops (positions 71 and 129) located in the inner surface of the N-terminal domain, the effects on stability are possibly associated with decoupling of the two domains as observed by differential scanning calorimetry during thermal unfolding.