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The focus of Dr. Brooks' research is to identify and understand the function of structural motifs that participate in initiating changes in protein conformation and function. The mechanisms by which molecules rearrange their constituent atoms to modulate their function are not understood. Does each protein have a unique mechanisms to alter its shape, or are there a discrete number of recognizable structural motifs and associated mechanisms that are used in various proteins to alter shape and modulate function? Both post-translational modifications and protein/protein interactions can initiate conformation changes that regulate function. Numerous post-translational modifications affect protein shape and function, including: metal prosthetic groups or phosphorylation. The binding of hormones and receptors are protein/protein interacts that induce conformation changes that modulate function. The amino acids in proteins that directly affect these interactions and pottranslational modifications have not been identified in many proteins and common motifs that contain such residues have not been classified.
The mechanism by which local conformation changes are initiated and transmitted to distal structural elements need to be identified. This information is required in order to engineer regulated enzymes for industrial applications, design peptides and proteins with pharmaceutical applications, understand the basis of diseases that are associated with mutations and loss of functional regulation, and to understand the relationship of conserved protein structures during the evolution of species.
Phosphorylated prolactins and growth hormones have been found in several species and the phosphorylation sites of prolactin have been identified in bovine and rat. We have isolated phosphorylated bovine prolactin (Mol. Cell. Endo. 99: 301, 1994) and have evidence that phosphorylated prolactin is the major form in the bovine pituitary. We have shown bovine prolactin is phosphorylated in vivo at serine 90 and to a lesser extent at serines 26 and 34 (Biochem. J. 296: 41, 1993). Isolated phosphorylated bovine prolactin has reduced biological activities both in vitro (Mol. Cell. Endo. 112: 223, 1995) and in vivo (Life Sciences 63:1281-1287, 1998). We have used site-directed mutagenesis to produce glutamic acid mimics of the individual phosphorylation sites and have identified serine 90 as a site where phosphorylation reduces biological activity (J. Biol. Chem. 270: 27661, 1995). We have shown that prolactin is phosphorylated in secretory vesicles and that this process is zinc-dependant (Molec. Cell. Endo. 147: 125-132, 1999). We also have shown that bovine growth hormone is phosphorylated by the same kinase as prolactin (Endocrine 10: 77-82, 1999). In collaboration with Dr. E. Permyakov of the Institute of Theoretical and Experimental Biophysics in Pushchino, Russia, we have demonstrated that bovine prolactin binds zinc with a :M affinity in a pH-dependant fashion (FEBS Letters 405: 273, 1997). Alanine substitution of the zinc-binding residues in prolactin eliminates the zinc-dependant phosphorylation of prolactin suggesting that zinc-bound prolactin is the most efficient substrate for the secretory vesicle prolactin kinase (Wicks and Brooks, unpublished).
Finally we have tentatively identified the motif surrounding the serine 90 site of phosphorylation as a salt bridge across one turn of putative helix 2 (Arg 89 and Asp 93). This salt bridge stabilizes the Pro 94-induced break in helix 2. We speculate that phosphorylation disrupts the salt bridge and central portion of helix 2 and subsequently shifts the 4-helix bundle so that the receptor-binding episodes are removed from a spatial array that could engage the lactogenic receptor. We have used site-directed mutagenesis to confirm this hypothesis (Molecular and Cellular Endocrinology 204: 117, 2003). A tryptophane is present in this region of helix 2 and may be used to monitor structural changes by absorbance and fluorescence spectroscopy.
Primate growth hormones have both somatotropic and lactogenic actions. We have undertaken studies to identify the structural features that allow human growth hormone to function as a lactogen despite sharing only a modest sequence homology with prolactins. Sequence homology methods were employed to tentatively identify "lactogenic" motifs in human growth hormone. These studies were followed with site-directed mutagenesis studies that identified Phe 44, contained in mini- helix 1 as a key element for expression of lactogenic action. These studies are summarized in J. Biol. Chem. (272 21444, 1997). We have tentatively identified specific residues in the N-terminus of helix 4 (Leu 157, Tyr 160 and Tyr 164) that form a hydrophobic cluster with Phe 44. Removal of either a hydrophobic residue at position 44 or Tyr 164 disrupts the correct articulation of these two structural elements and does not allow the subtle shifts of the surrounding 4-helix bundle that is necessary for effective lactogen receptor binding (Duda and Brooks FEBS letters 449: 120-124, 1999, Duda and Brooks, J. Biol. Chem. 278: 22734-22739, 2003).
Removal of the amino acid sequence lost in the 20K variant of human growth hormone (residues 41-52) created a protein with full somatotrophic activity but with reduced lactogenic activity (Peterson and Brooks Protein Engineering, Design and Selection 17: 417-424, 2004). We have extrapolated this variant to human prolactin by removing residues 32-46. This protein has approximately 10,000 to 15,000-fold less lactogenic activity wild-type human prolactin. Surprisingly, this protein binds the lactogenic receptor with 30-fold greater attraction (Peterson, Duda and Brooks, unpublished). This abbreviated human prolactin functions as a potent prolactin antagonist. In collaboration with Professor Dehua Pei we have identified a lead peptide that will also inhibit the activity human prolactin in cellular assays (Lui et al. Bioorganic & Medicinal Chemistry 17: 1026-1033, 2009).
Finally, we have determined the mechanism by which human prolactin sequentially binds two prolactin receptors (Sivaprasad and Brooks, Biochemistry 43:13755-13765, 2004). This work has provided evidence that receptor binding to site 1 of human prolactin is functionally coupled to the atomic organization and receptor binding to site 2. Recently we have extended this work to all human lactogens (prolactin, growth hormone, and placental lactogens)(Voorhees and Brooks, Journal of Biological Chemistry, in press 2010). This work has provided a mechanistic basis for the design and preparation of antagonists for human prolactin. The Ohio State University has sought patent protection regarding utilization of this mechanism to design and prepare antagonists of human prolactin.