Pointing the Zinc Finger on Protein Folding: Energetic Investigation into the Role of the Metal-Ion in the Metal-Induced Protein Folding of Zinc Finger Motifs

Abstract

Interactions between inorganic metal-ion cofactors and organic protein scaffolds are important for the proper structure and function of metalloproteins. Zinc finger proteins (ZFPs) are an example of proteins with such crucial metal-protein interactions. Incorporation of the Zn(II)-ion into ZFPs allows for their correct folding into structures that can carry out vital biological functions which include gene expression and tumor suppression. In addition, engineered ZFPs have shown to be promising genetic therapeutics in the clinic. And yet, there is still a gap in a quantitative understanding of the energetic contribution of the metal-protein interactions towards the structure and function of these important metalloproteins. Detailed knowledge of the principles that governs such interactions in natural metalloproteins will provide the much-needed insight for the rational construction of novel engineered zinc proteins for promising corrective therapeutics for genetic disorders. In this thesis, the investigation of the role that a specific metal-ion has on the energetics of metal-induced protein folding events is presented. To compare with Zn(II) related studies, an unstructured peptide scaffold, GGG, with Cys4-xHisx (x=0,1,2) metal-binding coordination motifs is utilized to determine the thermodynamics of Co(II) and Pb(II) binding to the three motifs. Furthermore, the difference between Co(II) and Pb(II) affinities to GGG and their affinities to natural ZFPs that undergo metal- induced protein folding events, provides the energetic cost of folding for these natural ZFPs – values that were hithertofore unknown. The data demonstrate that the Co(II)-ion has no binding preference for cysteine thiolate over histidine imidazole, while Pb(II)-ion prefers cysteine thiolate. Each substitution of Cys by His lower the contribution of Pb(II)-binding towards protein stabilization by approximately 2.0 kcal/mol - similarly to previously observed results for Zn(II). Lastly, both metal-ion studies agreed with previous Zn(II) studies, indicating that the cost for apo-ZFPs folding in natural ZFPs is minimal, less than 4.5 kcal/mol