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.

Improved data visualization techniques for analyzing macromolecule structural changes

“…Figure 2(A) shows an EPD constructed using the previously published data 10 by the current EPD method. The newly developed three-index EPD displayed in Figure 2(B), which is constructed using three structural indices calculated from the same experimental data as described in the Methods section.…”

“…Data obtained for several other proteins including aldolase, 10 chymotrypsin, 10 and SP1650 24 were also used in the construction of the original EPDs as well as the new data visualization methods (threeindex EPDs, the radar diagrams, and the Chernoff face diagrams). The previously obtained data and the newly constructed structural indices for aldolase, chymotrypsin, and SP1650 are shown in Supporting Information Figures S2, S4, and S6, respectively.…”

“…10,26 Thus, the multiple data visualization diagrams of the type described in this work can be obtained rapidly, within a single day is possible, with a minimal amount of protein and effort.…”

“…To demonstrate various visualization techniques, previously published data (e.g., intrinsic Trp and extrinsic ANS fluorescence spectroscopy, CD, and static light scattering) were used in this work including bovine serum albumin (BSA), 10 aldolase, 10 and chymotrypsin 10 as well as serine threonine kinase protein (SP1732) 24 and pneumococcal surface antigen A (SP1650) 24 from S. pneumonia and hemagglutinin from the H1N1 influenza virus (HAC1). 25 The EPDs were constructed using these previously published data and then compared to the three newly proposed data visualization techniques introduced in this work: the three-index EPD, radar diagram, and Chernoff face diagram.…”

“…10,24,25 The combinations of those conditions are aligned to form an m  n input matrix, in which m is the number of experimental techniques and n is the number of condition combinations (e.g., number of pH values  number of temperature measurements). SVD of the input matrix is used to produce a factorization of unitary orthonormal bases matrices and a diagonal matrix composed of singular values.…”