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

Yeast phosphoglycerate kinase (yPGK) is a monomeric two domain protein used as folding model representative of large proteins. We inserted short unstr uctured sequences (four Gly or four Thr) into the connections between secon dary structure elements and studied the consequences of these insertions on the folding process and stability of yPGK. All the mutated proteins can re fold efficiently. The effect per residue on stability is larger for the fir st inserted residue. Insertion in two long beta alpha loops (at residue pos itions 71 and 129) is more destabilizing than an insertion in a short alpha beta loop (at residue position 89) located on the opposite side of the N-t erminal domain. The effect on stability is mainly due to a large increase o f the unfolding rate rather than a decrease of the folding rate. This sugge sts that these connections between secondary structure elements do not play an active role in directing the folding process. Insertion into the short alpha beta 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 qualitativel y affect the folding process without a large effect on folding efficiency. For the two long beta alpha loops (positions 71 and 129) located in the inn er surface of the N-terminal domain, the effects on stability are possibly associated with decoupling of the two domains as observed by differential s canning calorimetry during thermal unfolding.