Biochemical and structural factors governing metal selectivity in lanthanide-binding proteins
Restricted (Penn State Only)
- Author:
- Featherston, Emily
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 15, 2023
- Committee Members:
- Scott Showalter, Major Field Member
Philip Bevilacqua, Program Head/Chair
Scott Lindner, Outside Unit & Field Member
Joseph Cotruvo, Chair & Dissertation Advisor
Amie Boal, Major Field Member - Keywords:
- Lanthanide
Rare earth element
Protein
Protein structure
Luminescence - Abstract:
- In recent years, lanthanides (LnIII ions) have been recognized as essential metals in biology after the discovery of their specific incorporation into the active sites of certain pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases (ADHs) in methylotrophic bacteria. These enzymes exhibit a highly selective mechanism for LnIII ion incorporation, yet little is understood in terms of LnIII ion selectivity, incorporation, and cofactor insertion. Therefore, the focus of this dissertation is to better understand the principles underlying the utilization of lanthanides for cellular functions; factors influencing the selectivity of lanthanide-utilizing proteins; and protein factors involved in methanol oxidation by the lanthanide-dependent methanol dehydrogenase (MDH), XoxF, in Methylorubrum extorquens AM1. XoxF contains a LnIII ion coordinated to a PQQ prosthetic group in its active site where methanol is oxidized to formaldehyde, and the electrons are sequentially passed to a c-type cytochrome, XoxG. We were motivated to provide a physiological context for understanding the activity of M. extorquens XoxF metallated with different Lns, and by characterizing XoxG and its interaction with XoxF. We demonstrate that in contrast to results obtained via an artificial assay system, assays of XoxFs metallated with La, Ce, and Nd using XoxG displayed Ln-independent activities, but the Km for XoxG markedly increases from La to Nd. Additionally, we determined the x-ray crystal structure of XoxG which revealed the heme moiety enclosed by hydrophobic residues and solvent exposed heme propionates. These observations indicate solvent exposure could be responsible for the relatively low Em of XoxG. We also report discovery of the protein, lanmodulin (LanM), the first natural LnIII-selective chelator and LnIII-binding protein other than an ADH to be isolated and characterized. LanM is an EF-hand containing protein that undergoes a conformational change from disorder to order in a highly LnIII-selective manner, with picomolar affinity for the LnIII series and 108-fold selectivity over the typical metal ion recognized by EF hands, calcium(II). The NMR structure of YIII-bound LanM reveals a three-helix bundle fold with EF hands on the periphery, a unique topology among EF hand-containing proteins. To better understand LanM’s remarkable selectivity for LnIII ions, we probe each EF hand individually by site-specifically incorporating tryptophan residues as photosensitizers for intrinsic luminescence of terbium (Tb). These studies provide insight to several key aspects of LanM’s mechanism of LnIII ion recognition and the structures of its metal-binding sites, including the order and kinetics of LnIII ion binding and the presence of two solvent molecules at each metal-binding site. We show that one of these Trp-substituted LanMs can quantify 3 ppb Tb directly in acid mine drainage at pH 3.2; to the best of our knowledge, this result represents the first time Tb can be quantified at such low levels in such a low-pH environmental sample using a luminescence-based sensor. Finally, we present preliminary structural characterization of CaII-bound LanM, which reveals an unexpectedly disordered protein with low convergence between conformers. Overall, this thesis presents fundamental studies of structure and function of lanthanoproteins and shows how they can inform technology development, such as for luminescence sensing of lanthanides in environmental samples.