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Updated: Aug 19, 2026

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry
Published on: September 13, 2014
Intramolecular cooperativity in a protein binding site assessed by combinatorial shotgun scanning mutagenesis
Gábor Pál1, Mark H Ultsch, Kevin P Clark
1Department of Biochemistry and Molecular Biology and Institute for Biophysical Dynamics, Cummings Life Sciences Center, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA.
Abstract:
Combinatorial shotgun alanine-scanning was used to assess intramolecular cooperativity in the high affinity site (site 1) of human growth hormone (hGH) for binding to its receptor. A total of 19 side-chains were analyzed and statistically significant data were obtained for 145 of the 171 side-chain pairs. The analysis revealed that 90% of the side-chain pairs exhibited no statistically significant pair interactions, and the remaining 10% of side-chain pairs exhibited only small interactions corresponding to cooperative interaction energies with magnitudes less than 0.4 kcal/mol. The statistical predictions were tested by measuring affinities for purified mutant proteins and were found to be accurate for five of six side-chain pairs tested. The results reveal that hGH site 1 behaves in a highly additive manner and suggest that shotgun scanning should be useful for assessing cooperative effects in other protein-protein interactions.
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