title: Investigation of protein conformational stability employing a multimodal spectrometer.
abstract: The conformational stability of proteins is typically investigated by use of a variety of biophysical measurements as a function of environmental stresses such as pH and temperature. Thus, multiple experiments are required on a variety of instruments, each providing information on a particular aspect of a protein's higher order structural integrity. These measurements typically require large sample quantities and long experimental times. In this study, a new methodology is described to obtain protein conformational stability data simultaneously, including UV absorption, light scattering, and near- and far-UV circular dichroism, by employing a multimodal spectrometer. Fluorescence spectral data are also collected on the same instrument, although not simultaneously. The method was developed by examining the thermal and pH stability of four model proteins. Results showed reproducible and accurate results from this single instrument, and data collection was rapid with minimal protein sample requirements. We illustrate the application of this method to the generation of empirical phase diagrams (EPDs) to better characterize the overall conformational stability of proteins. This new approach facilitates the rapid characterization of protein structure and stability in a single methodology, useful for analysis of unknown proteins as well as screening of solution conditions to optimize stability for protein therapeutic drug candidates.
authors: Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047, United States.
authors: Lei
authors: L
authors: Hu
authors: Christopher
authors: C
authors: Olsen
authors: Nathan
authors: N
authors: Maddux
authors: Sangeeta B
authors: SB
authors: Joshi
authors: David B
authors: DB
authors: Volkin
authors: C Russell
authors: CR
authors: Middaugh
mesh_terms: D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D055550:Protein Stability; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D011506:Proteins / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
chemical_list: D011506:Proteins