title: A pH-Induced Switch in Human Glucagon-like Peptide-1 Aggregation Kinetics.
abstract: Aggregation and amyloid fibril formation of peptides and proteins is a widespread phenomenon. It has serious implications in a range of areas from biotechnological and pharmaceutical applications to medical disorders. The aim of this study was to develop a better understanding of the mechanism of aggregation and amyloid fibrillation of an important pharmaceutical, human glucagon-like peptide-1 (GLP-1). GLP-1 is a 31-residue hormone peptide that plays an important role regulating blood glucose levels, analogues of which are used for treatment of type 2 diabetes. Amyloid fibril formation of GLP-1 was monitored using thioflavin T fluorescence as a function of peptide concentration between pH 7.5 and 8.2. Results from these studies establish that there is a highly unusual pH-induced switch in GLP-1 aggregation kinetics. At pH 8.2, the kinetics are consistent with a nucleation-polymerization mechanism for fibril formation. However, at pH 7.5, highly unusual kinetics are observed, where the lag time increases with increasing peptide concentration. We attribute this result to the formation of off-pathway species together with an initial slow, unimolecular step where monomer converts to a different monomeric form that forms on-pathway oligomers and ultimately fibrils. Estimation of the pKavalues of all the ionizable groups in GLP-1 suggest it is the protonation/deprotonation of the N-terminus that is responsible for the switch with pH. In addition, a range of biophysical techniques were used to characterize (1) the start point of the aggregation reaction and (2) the structure and stability of the fibrils formed. These results show that the off-pathway species form under conditions where GLP-1 is most prone to form oligomers.
authors.affiliation: Department of Chemistry, University of Cambridge , Cambridge CB2 1EW, U.K.
authors.forename: Karolina L
authors.initials: KL
authors.lastname: Zapadka
authors.affiliation: Department of Chemistry, University of Cambridge , Cambridge CB2 1EW, U.K.
authors.forename: Frederik J
authors.initials: FJ
authors.lastname: Becher
authors.affiliation: Formulation Sciences, MedImmune Ltd. , Granta Park, Cambridge CB21 6GH, U.K.
authors.forename: Shahid
authors.initials: S
authors.lastname: Uddin
authors.affiliation: Formulation Sciences, MedImmune Ltd. , Granta Park, Cambridge CB21 6GH, U.K.
authors.forename: Paul G
authors.initials: PG
authors.lastname: Varley
authors.affiliation: Formulation Sciences, MedImmune , One MedImmune Way, Gaithersburg, Maryland 20878, United States.
authors.forename: Steve
authors.initials: S
authors.lastname: Bishop
authors.affiliation: Formulation Sciences, MedImmune Ltd. , Granta Park, Cambridge CB21 6GH, U.K.
authors.forename: A L
authors.initials: AL
authors.lastname: Gomes Dos Santos
authors.affiliation: Department of Chemistry, University of Cambridge , Cambridge CB2 1EW, U.K.
authors.forename: Sophie E
authors.initials: SE
authors.lastname: Jackson
mesh_terms: D000595:Amino Acid Sequence; D052216:Glucagon-Like Peptide 1 / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D066329:Protein Aggregates; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary
chemical_list: D066329:Protein Aggregates; D052216:Glucagon-Like Peptide 1