title: Using empirical phase diagrams to understand the role of intramolecular dynamics in immunoglobulin G stability.
abstract: Understanding the relationship between protein dynamics and stability is of paramount importance to the fields of biology and pharmaceutics. Clarifying this relationship is complicated by the large amount of experimental data that must be generated and analyzed if motions that exist over the wide range of timescales are to be included. To address this issue, we propose an approach that utilizes a multidimensional vector-based empirical phase diagram (EPD) to analyze a set of dynamic results acquired across a temperature-pH perturbation plane. This approach is applied to a humanized immunoglobulin G1 (IgG1), a protein of major biological and pharmaceutical importance whose dynamic nature is linked to its multiple biological roles. Static and dynamic measurements are used to characterize the IgG and to construct both static and dynamic EPDs. Between pH 5 and 8, a single, pH-dependent transition is observed that corresponds to thermal unfolding of the IgG. Under more acidic conditions, evidence exists for the formation of a more compact, aggregation resistant state of the immunoglobulin, known as A-form. The dynamics-based EPD presents a considerably more detailed pattern of apparent phase transitions over the temperature-pH plane. The utility and potential applications of this approach are discussed.
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas, 2030 Becker Drive, Lawrence, Kansas 66047, USA.
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Ramsey
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Gill
authors.forename: Tim J
authors.initials: TJ
authors.lastname: Kamerzell
authors.forename: E Shane
authors.initials: ES
authors.lastname: Price
authors.forename: Sangeeta B
authors.initials: SB
authors.lastname: Joshi
authors.forename: Steven M
authors.initials: SM
authors.lastname: Bishop
authors.forename: Cynthia N
authors.initials: CN
authors.lastname: Oliver
authors.forename: C Russell
authors.initials: CR
authors.lastname: Middaugh
mesh_terms: D016880:Anisotropy; D002942:Circular Dichroism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry*; D044367:Phase Transition; D011487:Protein Conformation; D055550:Protein Stability; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014461:Ultracentrifugation
chemical_list: D007074:Immunoglobulin G