Studies of the aggregation of RNase Sa.

Thirty-eight mutants of RNase Sa (ribonuclease from Streptomyces aureofaciens) were examined for their structure, thermal sensitivity, and tendency to aggregate. Although a biphasic correlation was seen between the effect of temperature on structure and the free energy of transfer changes in many of the mutants, little correlation was seen between the time at which aggregation is initiated or the rate of aggregation and the thermal sensitivity of the mutants. It is hypothesized that the nature of contacts between protein molecules in the associated (aggregated) phase rather than structural changes dominates the aggregation process for these series of mutants.

Comparative signature diagrams to evaluate biophysical data for differences in protein structure across various formulations.

A solution to the problem of being able to show statistically significant differences in the measurements of various levels of higher-order protein structure has been an elusive one. We propose the use of comparative signature diagrams (CSDs) to this end. CSDs compare datasets from different biophysical techniques that fingerprint the secondary, tertiary, and quaternary structures of a protein molecule and display statistically significant differences in these datasets. In this paper, we explore the differences in the structures of two proteins (Granulocyte Colony Stimulating Factor [GCSF] and a monoclonal antibody [mAb]) in various formulations. These proteins were chosen based on the extent of differences in structure observed in the formulations. As an initial test, we utilize data from circular dichroism, 8-anilino-1-naphthalene-sulfonate and intrinsic fluorescence spectroscopy, and static light scattering measurements to fingerprint protein structure in the different formulations. Several layers of statistics were explored to visualize the regions of significant differences in the protein spectra. This approach provides a rapid, high-resolution methodology to compare various structural levels of proteins using standard biophysical instrumentation.

Investigation of protein conformational stability employing a multimodal spectrometer.

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.