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 ba loops (at residue positions 71 and 129) is more destabilizing than an insertion in a short ab 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 ab 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 ba 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.

Chimeric Glutathione S-Transferases Containing Inserts of Kininogen Peptides

“…It is known that an increase in loop length usually results in a decrease in protein stability (34,35). Therefore, we decided to shorten the size of the insert, aiming to increase the stability of the chimeric protein without substantially decreasing its activity.…”

“…Similarly, insertions of short unstructured sequences (four glycines and/or four threonines) into the loops of yeast phosphoglycerate kinase did not affect the enzyme folding but led to a decreased stability of the protein (35). Interestingly, the effect per residue on stability was larger for the first inserted residue than for all of the subsequent ones (35). Our GSHKT10 and GSHKT13 crystal structures indicate that insertions of 10 and 13 residues increase the flexibility of a surface loop significantly, providing an explanation for the observed decrease in thermodynamic stability for these chimeras.…”

“…It has been suggested that loops do not actively dictate the final protein fold (25,29,34,35,39). However, an increase in loop length usually leads to a decrease in protein stability (34,35).…”

“…It has been suggested that loops do not actively dictate the final protein fold (25,29,34,35,39). However, an increase in loop length usually leads to a decrease in protein stability (34,35). For example, insertion of a series of glycine linkers (up to 10 residues in length) into a natural two-residue loop in a four-helix bundle protein (Rop) led to a progressive decrease in stability of Rop toward thermal and chemical denaturation (34).…”

“…For example, insertion of a series of glycine linkers (up to 10 residues in length) into a natural two-residue loop in a four-helix bundle protein (Rop) led to a progressive decrease in stability of Rop toward thermal and chemical denaturation (34). Similarly, insertions of short unstructured sequences (four glycines and/or four threonines) into the loops of yeast phosphoglycerate kinase did not affect the enzyme folding but led to a decreased stability of the protein (35). Interestingly, the effect per residue on stability was larger for the first inserted residue than for all of the subsequent ones (35).…”