How Outer Coordination Sphere Modifications Can Impact Metal Structures in Proteins: A Crystallographic Evaluation |
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Authors: | Dr Leela Ruckthong Prof Dr Jeanne A Stuckey Prof Dr Vincent L Pecoraro |
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Institution: | 1. Department of Chemistry, University of Michigan, Ann Arbor, Michigan, 48109 USA;2. Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, 48109 USA |
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Abstract: | A challenging objective of de novo metalloprotein design is to control of the outer coordination spheres of an active site to fine tune metal properties. The well-defined three stranded coiled coils, TRI and CoilSer peptides, are used to address this question. Substitution of Cys for Leu yields a thiophilic site within the core. Metals such as HgII, PbII, and AsIII result in trigonal planar or trigonal pyramidal geometries; however, spectroscopic studies have shown that CdII forms three-, four- or five-coordinate CdIIS3(OH2)x (in which x=0–2) when the outer coordination spheres are perturbed. Unfortunately, there has been little crystallographic examination of these proteins to explain the observations. Here, the high-resolution X-ray structures of apo- and mercurated proteins are compared to explain the modifications that lead to metal coordination number and geometry variation. It reveals that Ala substitution for Leu opens a cavity above the Cys site allowing for water excess, facilitating CdIIS3(OH2). Replacement of Cys by Pen restricts thiol rotation, causing a shift in the metal-binding plane, which displaces water, forming CdIIS3. Residue d -Leu, above the Cys site, reorients the side chain towards the Cys layer, diminishing the space for water accommodation yielding CdIIS3, whereas d -Leu below opens more space, allowing for equal CdIIS3(OH2) and CdIIS3(OH2)2. These studies provide insights into how to control desired metal geometries in metalloproteins by using coded and non-coded amino acids. |
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Keywords: | d-amino acids de novo protein engineering metalloprotein engineering nonnatural amino acids |
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