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BIOPHYSICAL APPLICATIONS OF AMIDE PROTON HYDROGEN EXCHANGE AND MALDI MASS SPECTROMETRY TO PROTEIN FOLDING
- Abstract:
- The amide hydrogen exchange properties of proteins have been widely exploited in a number of biophysical approaches for studying protein folding and function. SUPREX (Stability of Unpurified Protein from Rates of H/D Exchange) is one such biophysical approach that utilizes matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry to measure thermodynamic parameters associated with protein folding and ligand binding including: protein folding/unfolding free energies, G, protein folding m-values, and the dissociation constants, kd values, of protein ligand complexes.
The use of SUPREX to quantitatively evaluate the above thermodynamic parameters requires that several important assumptions be valid. The use of SUPREX to quantitatively evaluate the above thermodynamic parameters requires that several important assumptions be valid. These assumptions include: 1) that the protein folding/unfolding reaction is at equilibrium in the SUPREX experiment; 2) that there are only two states populated in the folding/unfolding reaction (i.e. the native and denatured state); 3) that the hydrogen exchange reaction should be in the so-called EX2 exchange regime (i.e. the protein folding reaction is faster than the hydrogen exchange reaction of an unprotected amide proton); and 4) that the denatured state of the protein should be well-modeled by a random-coil-like structure.
Described is the behavior of so-called non-ideal proteins in the SUPREX experiments. This work includes the development of a new protocol to handle proteins that do not rapidly equilibrate in the SUPREX experimental and studies of protein folding reactions with relatively stable intermediate states and with relatively unstable intermediate states. Our results on these non-two-state folding proteins reveal that SUPREX is especially useful for the detection of relatively unstable intermediate states that are hard to detect by many other biophysical methods. The importance of the EX2 exchange behavior assumption was also assessed using ubiquitin as a model system and using a series of hypothetical proteins with a wide range of folding/unfolding properties. A series of proteins known to have non-random coil-like denatured states were also investigated, and a new biophysical approach for detecting such non-random-coil-like behavior was developed using a combination of data from SUPREX and conventional spectroscopy-based techniques.
Ph.D. Defense Examination Seminar
Student Exams Seminar