John S. Vrettos and Curtis W. Meuse

National Institute of Standards and Technology
Chemical Science and Technology Laboratory, Biotechnology Division, Biomolecular Materials Group
 

Biotechnology and pharmaceutical companies and regulatory agencies assess the efficacy, quality, and safety of many products by measuring the structural stability of native or recombinant proteins and the integrity of protein-protein or protein-ligand interactions.  There is increasing interest in developing detection methods that can be used with high-throughput devices, such as protein microarrays, to facilitate this process [Nature Biotech. 20, 225 (2002)].  An ideal method would: be adequately sensitive to minimal sample amounts; yield spectra that are simple to evaluate; not require protein modifications, purification, a special supporting matrix or internal standard; and be applicable to soluble and membrane proteins and surfaces (such as protein chips).  Our goal is to develop infrared spectroscopy as a method for measuring protein structural stability and ligand binding affinities.  Infrared spectroscopy satisfies many of the above criteria as a favorable detection method.  We will show that infrared spectroscopy can be used to quantify the thermodynamic structural stability of a protein by measuring the extent of amide hydrogen/deuterium exchange under chemically denaturing conditions.  This approach will be extended to measure ligand binding affinities and the structural stability of a protein in a mixture of proteins or whole cell lysate.