title: Glutamine, alanine or glycine repeats inserted into the loop of a protein have minimal effects on stability and folding rates.
abstract: Natural proteins can contain flexible regions in their polypeptide chain. We have investigated the effects of glycine, alanine and glutamine repeats on the stability and folding of a protein by inserting stretches of 7 to 13 residues into a suitable position in a model system, the chymotrypsin inhibitor-2 (CI2). This folds by residues (1-40) docking with residues (41-64) to form a folding nucleus. The peptides GQ4GM, GQ6GM, GQ8GM, GQ10GM, GA2SA4SA2GM and G3SG4SG3M were inserted after residue 40. The stability of the mutant proteins changes only weakly with chain length and nature of insertion, suggesting that the presence of unstructured polypeptide chains in a protein does not have a great energetic penalty. This has implications in catalysis, for example, where floppy regions have been noted in active sites, and in DNA transcription where activators, transcription factors and intermediary proteins all show long repeats of glycine/serine and/or glutamine, which are thought to be important for function. We find that the rate of folding is very insensitive to the length of the linker. The changes in rate are close to those predicted from polymer theory for the loss of configuration entropy on closing a loop. This implies that all the diffusion steps are relatively rapid.
authors.affiliation: MRC Cambridge Centre for Protein Engineering, MRC Centre, UK.
authors.forename: A G
authors.initials: AG
authors.lastname: Ladurner
authors.forename: A R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D019277:Entropy; D019143:Evolution, Molecular; D005973:Glutamine / Q000737:chemistry / Q000378:metabolism; D005998:Glycine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D000596:Amino Acids; D010455:Peptides; D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors; C093336:chymotrypsin inhibitor 2; D005973:Glutamine; D000409:Alanine; D005998:Glycine