Investigation of the Zinc binding region of Prothymosin-alpha : A spectroscopic and thermodynamic approach to study metal binding in Intrinsically disordered proteins

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Sri Ramya Garapati (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

Abstract: The goal of this study is to provide a deeper insight into how metals bind intrinsically disordered proteins (IDPs) by taking zinc binding to prothymosin-alpha as a case study. The involvement of metals in several diseases caused by (IDPs) has been noted long ago (Cu binding of [alpha]-synuclein in Parkinsons disease, prion aggregation due to Cu in madcow disease. etc.). However, there is still a lack of understanding of how metal binds IDPs. A deeper insight into metal binding by IDPs is essential for a full understanding of a proteome and to pave a way for drug development. Prothymosin-alpha (Prot[alpha]) is an acidic, natively unfolded, and highly conserved protein located mostly in the nucleus of eukaryotic cells. It consists of 110 amino acids out of which 53 residues are aspartic (D) and glutamic (E) acids thus it is highly acidic. The exact biological role of the protein is unknown. However, it has been shown to be involved in cell proliferation, chromatin remodeling, antiapoptotic activity etc. One of the most interesting characteristics of Prot[alpha] is its ability to bind metal cations like Ca²?, Mg²?, Mn²?, Zn²?, Al²?. In the background of all these cations, Prot[alpha] selectively interacts with Zn²? and undergoes a structural change plus the interactions of the protein with its binding partners are enhanced in the presence of Zn²?. These features underline the importance of Zn²? binding in Prot[alpha]. Very little information is available about Zn²? binding to Prot[alpha]. A recent NMR study has shown that the 48-110 segment of Prot[alpha] is the Zn²? binding region of the protein. Although the 48-110 region is shown to be the Zn²? binding region, the central 50-89 segment, where most of the Zn²? binding residues are located in the protein, was used for all the studies here. To aid in the synthesis, purification, and characterization of highly negatively charged sequences like Prot[alpha](50-89)N50W new amino acid derivatives, 4-pyridylmethyl protected glutamic and aspartic acids were developed and their application was tested. Subsequently a circular dichroism (CD) spectroscopic study on Prot[alpha](50-89)N50W revealed that the peptide, like the full length protein, undergoes a structural change in the presence of Zn²? and both the full protein and the peptide were saturated at 75 eq of Zn²? indicating that the majority of Zn added binds in this region. The above study had also shown that selective binding of Zn²? by Prot[alpha](50-89)N50W is due to sequence specificity and not solely because of electrostatic interactions between Prot[alpha](50-89)N50W and Zn²?. Further the CD and differential scanning calorimetry (DSC) studies have shown that Prot[alpha](50-89)N50W undergoes a structural change similar to an alpha helix when heated from 20-80°C. To set the stage for determining the specific glutamic and aspartic residues in Prot[alpha] that act as ligands for Zn²?, a proof of principle for spin inversion recovery experiments has been provided using Zn²? binding to EDTA as a model. This idea can further be extended to Prot[alpha](50-89)N50W to understand if Zn²? binds to a specific set of residues or if several degenerate binding sites exist.

Additional Information

Publication

Dissertation

Language: English

Date: 2023

Subjects

Chemistry;Intrinsically disordered proteins;Metal binding

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