Protein Hyperactive Cysteine Sites Identified by New Quantitative Reactivity Technique

A publication described a new method for determining active regions on enzymes and other proteins by using specific probes in a fashion that discriminated between high and low affinity amino acid sites.

Investigators at the Scripps Research Institute (La Jolla, CA, USA) initially used the new technology to study the reactivity of cysteine residues in enzymes. Cysteine is one of the most reactive of the amino acids and is often critically involved in determining the three-dimensional shape of protein molecules.

Two types of cysteine-specific probes were used. One probe bound to cysteine with high affinity while the other was less avid. This meant that by strictly controlling the concentration of the probe, they could determine whether it was binding to a particularly active or inactive cysteine site.

The investigators reported in the November 17, 2010, online edition of the journal Nature that they tested the technique on proteins found within a human breast cancer-cell line. They were able to identify and locate more than 800 cysteine sites on 522 proteins. For more than 90% percent of these cysteine sites the low and high concentration probes showed correspondingly low and high levels of labeling, indicating that the cysteine residues had ordinary reactivity. The other 10% of cysteine sites demonstrated a constant level of labeling for both low and high concentration probes, indicating that they were hyperreactive.

"What we have needed is a more efficient method to find and quantitatively analyze reactive sites,” said senior author Dr. Benjamin F. Cravatt, professor of chemical biology and physiology at the Scripps Institute, "not just for one protein in a purified sample but for a large set of proteins in their natural setting, such as within a whole cell or tissue.”

"It lets us find functional sites on proteins more efficiently than before, and that is going to be helpful not only for characterizing unknown proteins, but also for finding new sites of importance on already-characterized proteins. This is a relatively precise and straightforward method for screening designed proteins for functional properties,” said Dr. Cravatt. "It could be very useful for creating new enzyme catalysts for basic research and industrial applications.”

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