Multistep aggregation pathway of human interleukin-1 receptor antagonist: kinetic, structural, and morphological characterization.
The complex, multistep aggregation kinetic and structural behavior of human recombinant interleukin-1 receptor antagonist (IL-1ra) was revealed and characterized by spectral probes and techniques. At a certain range of protein concentration (12-27 mg/mL) and temperature (44-48 degrees C), two sequential aggregation kinetic transitions emerge, where the second transition is preceded by a lag phase and is associated with the main portion of the aggregated protein. Each kinetic transition is linked to a different type of aggregate population, referred to as type I and type II. The aggregate populations, isolated at a series of time points and analyzed by Fourier-transform infrared spectroscopy, show consecutive protein structural changes, from intramolecular (type I) to intermolecular (type II) beta-sheet formation. The early type I protein spectral change resembles that seen for IL-1ra in the crystalline state. Moreover, Fourier-transform infrared data demonstrate that type I protein assembly alone can undergo a structural rearrangement and, consequently, convert to the type II aggregate. The aggregated protein structural changes are accompanied by the aggregate morphological changes, leading to a well-defined population of interacting spheres, as detected by scanning electron microscopy. A nucleation-driven IL-1ra aggregation pathway is proposed, and assumes two major activation energy barriers, where the second barrier is associated with the type I --> type II aggregate structural rearrangement that, in turn, serves as a pseudonucleus triggering the second kinetic event.
Krishnan
Sampathkumar
S
Department of Pharmaceutics, Amgen, Thousand Oaks, California 91320, USA.
Raibekas
Andrei A
AA
D005456:Fluorescent Dyes; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein / Q000737:chemistry*; D007700:Kinetics; D008855:Microscopy, Electron, Scanning; D008956:Models, Chemical; D008958:Models, Molecular; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
D005456:Fluorescent Dyes; D053590:Interleukin 1 Receptor Antagonist Protein; D011994:Recombinant Proteins