title: Intramolecular cooperativity in a protein binding site assessed by combinatorial shotgun scanning mutagenesis.
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.
authors.affiliation: Department 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.
authors.forename: Gábor
authors.initials: G
authors.lastname: Pál
authors.forename: Mark H
authors.initials: MH
authors.lastname: Ultsch
authors.forename: Kevin P
authors.initials: KP
authors.lastname: Clark
authors.forename: Bridget
authors.initials: B
authors.lastname: Currell
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Kossiakoff
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
mesh_terms: D001665:Binding Sites; D019382:Human Growth Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D008565:Membrane Proteins; C405010:delta-hGHR; D019382:Human Growth Hormone