title: Analysis of site-specific histidine protonation in human prolactin.
abstract: The structural and functional properties of human prolactin (hPRL), a 23 kDa protein hormone and cytokine, are pH-dependent. The dissociation rate constant for binding to the extracellular domain of the hPRL receptor increases nearly 500-fold over the relatively narrow and physiologic range from pH 8 to 6. As the apparent midpoint for this transition occurs around pH 6.5, we have looked toward histidine residues as a potential biophysical origin of the behavior. hPRL has a surprising number of nine histidines, nearly all of which are present on the protein surface. Using NMR spectroscopy, we have monitored site-specific proton binding to eight of these nine residues and derived equilibrium dissociation constants. During this analysis, a thermodynamic interaction between a localized triplet of three histidines (H27, H30, and H180) became apparent, which was subsequently confirmed by site-directed mutagenesis. After consideration of multiple potential models, we present statistical support for the existence of two negative cooperativity constants, one linking protonation of residues H30 and H180 with a magnitude of approximately 0.1 and the other weaker interaction between residues H27 and H30. Additionally, mutation of any of these three histidines to alanine stabilizes the folded protein relative to the chemically denatured state. A detailed understanding of these complex protonation reactions will aid in elucidating the biophysical mechanism of pH-dependent regulation of hPRL's structural and functional properties.
authors.affiliation: Department of Laboratory Medicine, Yale University, New Haven, Connecticut 06520, USA.
authors.forename: M Cristina
authors.initials: MC
authors.lastname: Tettamanzi
authors.forename: Camille
authors.initials: C
authors.lastname: Keeler
authors.forename: Syrus
authors.initials: S
authors.lastname: Meshack
authors.forename: Michael E
authors.initials: ME
authors.lastname: Hodsdon
mesh_terms: D001665:Binding Sites; D005786:Gene Expression Regulation; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D011388:Prolactin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins
chemical_list: D011994:Recombinant Proteins; D006639:Histidine; D011388:Prolactin