COMPARING PROTEIN TERTIARY STRUCTURE BY CHARACTERIZING ENSEMBLE DYNAMICS

Napoleon Tercero; Curt Meuse; Joe Hubbard

The biopharmaceutical industry is currently having problems developing measurements capable of showing that the properties of the biomolecules in a specific dose of medicine are the same as the properties of the biomolecules in the drug that were shown to be safe and effective in clinical trials. Generally, a combination of orthogonal measurements, such as mass spectrometry, chromatography, NMR, etc., are used to compare the characteristics of the biopharmaceuticals. The disadvantage of this approach is, that a larger percentage of the tested batches get rejected with each independent technique used resulting in a significant and costly waste of product.

Here we propose to link a pair of infrared spectroscopic measurements, through the kinetic processes of protein ensemble dynamics, to allow us to assess the similarity between two protein drugs and decrease the number of independent measurements necessary for the acceptance of a given batch. Specifically, we will determine the width of the protein conformational ensemble by using concepts derived from infrared spectroscopic orientation measurements. The presence of a width in the conformational ensemble is an indication that the protein is fluctuating between conformational states. Our model collects conformational ensemble information into a single parameter, for each wavelength, which can then be used to determine the separate spectra of the two limiting conformational states. This two-conformation description is what we plan to evaluate as a method for comparing protein similarity. As a further advancement, we plan to develop time-resolved methods to isolate more conformational intermediates of the ensemble dynamics to more specifically describe the protein tertiary structure.