Current Challenges and Future Directions

Present successes in PLIMSTEX rely on a measurable deuterium shift upon li-gand binding accompanying a conformational change or detectable shielding induced by ligand binding region. PLIMSTEX procedures may be extended to proteins that do not significantly change conformation during ligand binding by using competition with a known protein that can serve as an indicator or by em ploying a pulsed-labeling strategy to shorten exchange times and allow focus on the fast exchanging amide hydrogens that may be affected by ligand binding but do not show difference in longer H/D exchange time that are currently used.

Metal ions, small organic molecules, peptides and small proteins are the li-gands tested thus far. PLIMSTEX should be applicable to other ligands including nucleic acids and other proteins. PLIMSTEX should have utility for measuring affinities of proteins in complexes as well as alone, and if this works, it may be one of the few techniques that can probe interaction of a ligand with one protein that is interacting with others.

The current modeling procedure is implemented using Mathcad, which may not be efficient for more complicated protein-ligand binding systems than tested thus far. Other programs (e.g. C or C++) should increase the calculation speed and be more user-friendly. A kinetics factor may be built into the model to accommodate different exchange times used for the titration and to assist the evaluation of a best time-to-quench for a titration study. An example of a more complex system is the binding of two different ligands to one protein or two proteins competing for a single ligand.

A key future direction is expanding PLIMSTEX to provide a higher resolved view than the global view of the whole protein that is currently obtained. Digestion with pepsin followed by peptide analysis by MS and MS/MS should allow kinetics and titrations to be measured for portions of a protein, giving PLIMSTEX a view of the protein that currently emerges from NMR and X-ray methods.

Automation of sample handling and the LC/MS process may give PLIMSTEX a higher throughput character and make it useful for screening the binding of small libraries of drug candidates to target proteins.

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